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122 W.R. Harris

3.8

4

5

M a n g an e se . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Con c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 4

Re f e r e n c e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 5

L i s t o f S y m b o l s a n d A b b r e v i a t i o n s

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a p o t r a n s f e r r i n

c i r c u l a r d i c h r o i s m

ci t r ic ac id

d e s f e r r i o x a m i n e B

e l e c t r o n s p i n r e s o n a n c e

N - ( 2 - h y d r o x y e t h y l ) p i p e r a z i n e - N ' - ( 2 - e t h a n e s u l f o n i c a c i d )l i n e a r f r e e e n e r g y r e l a t i o n s h i p

l o w m o l e c u l a r w e i g h t

m a g n e t i c r e s o n a n c e i m a g i n g

n o r m a l h y d r o g e n e l e c t r o d e

n i t r i l o t r i a c e t i c a c id

p e r t u r b e d a n g u l a r c o r r e l a t i o n s p e c t r o s c o p y

t r a n s f e r r i n

1I n t r o d u c t i o n

1.1Biological Fun ctions of the Transferrins

S e r u m t r a n s f e r r in ( Tf ) is o n e m e m b e r o f a s m a l l f a m i ly o f ir o n - b i n d i n g

p r o t e i n s w h i c h a l s o i n c l u d e s l a c t o f e r r i n a n d o v o t r a n s f e r r i n [ 1 - 6 ] . T h e s e t h r e e

p r o t e i n s h a v e v e r y s i m i l a r s t r u c t u r e s [ 6 ] , b u t a p p e a r t o s e r v e d i f f e r e n t

f u n c t i o n s . T r a n s f e r r i n is r e s p o n s i b l e f o r t h e t r a n s p o r t o f i r o n a s f e rr ic i o n

t h r o u g h t h e b l o o d a m o n g s i te s o f u p t a k e , u t i l iz a t io n , a n d s t o r a g e . S u c h a

c a r r i e r is n e c e s s a r y i n p a r t b e c a u s e t h e s t r o n g t e n d e n c y o f th e f e r ri c i o n t o

f o r m i n s o l u b l e F e ( O H ) 3 a t p h y s i o l o g i c a l p H m a k e s i t i m p o s s i b l e t o t r a n s p o r t

s i g n i fi c a n t a m o u n t s o f u n c h e l a t e d F e 3+ i n b l o o d . T r a n s p o r t o f i r o n a s th e m o r e

s o l u b le F e z+ i o n i s p r e c l u d e d b y t h e o x i d a t i o n o f fr e e d i v a l e n t i r o n b y d i s s o l v e d

oxyge n . T h i s p rod uc e s i nso lub le Fe 3+ a s w e ll a s ge n e ra t i n g tox i c spe c i e s suc h

a s t h e s u p e r o x i d e a n i o n a n d t h e h y d r o x y l r a d i c a l [ 7 , 8 ] .

L a c t o f e r r i n is f o u n d i n t ea r s , m i lk , a n d o t h e r f lu id s . I t b i n d s i r o n m o r e

t i g h t l y t h a n t r a n s f e r r i n [ 9 ] a n d i s t h o u g h t t o a c t a s a b a c t e r i o s t a t i c a g e n t b y

e n s u r i n g t h a t t h e r e i s n o f r e e i r o n a v a i l a b l e t o s u p p o r t t h e g r o w t h o fm i c r o o r g a n i s m s . T h e l a c to f e r r i n i n m i l k m a y a ls o p la y a r o l e i n th e a b s o r p t i o n

o f i r o n b y t h e n u r s i n g i n f a n t. O v o t r a n s f e r r i n is f o u n d i n a v i a n e gg w h i t es , a n d

i t a l s o a p p e a r s t o f u n c t i o n p r i m a r i l y a s a b a c t e r i o s t a t i c a g e n t .

To fa c i li t at e t he de l ive ry o f i ron to spe c i fi c ti s sue s , t he d i fe r r i c - t r a n s fe r r in

c o m p l e x i s r e c o g n i z e d b y t r a n s f e r r i n r e c e p t o r s [ 10 ]. T h e t r a n s f e r r i n r e c e p t o r is



Binding and Transpor t o f Nonfer rous Metals 123

a p r o t e i n b o u n d t o t h e o u t e r s u r f a c e o f t h e c e ll m e m b r a n e w h i c h s e le c t iv e l y

b i n d s t h e d i f e rr i c fo r m o f s e r u m t r a n s f e r r i n . T h e r e c e p t o r - d i f e r r ic t r a n s f e r r i n

complex i s in te rna l ized by a p roces s ca l led endocy tos i s , in which the ce l l

m e m b r a n e i n v a g i n a t e s, a n d t h e r e s u l t i n g c a v i ty e v e n t u a l ly p i n c h e s o f f t o f o r man in t r ace l lu la r ves ic le . Th is ves ic le con ta ins the r ecep to r -d i f e r r ic t r ans fe r r in

c o m p l e x o n i t s i n t e r n a l m e m b r a n e s u r f a c e . A n A T P - d e p e n d e n t p r o t o n p u m p

increases the ac id i ty wi th in the ves ic le to abou t pH 5 .5 . Th is p romotes the

r e l e a s e o f i r o n f r o m t h e t r a n s f e r r i n , b u t a t t h i s l o w p H t h e r e s u l t i n g

a p o t r a n s f e r r i n r e m a i n s b o u n d t o t h e r e c e p t o r . A f t e r t h e i r o n h a s b e e n

r e m o v e d , t h e v e s i c l e f u s e s b a c k i n t o t h e c e l l m e m b r a n e , r e t u r n i n g t h e

r e c e p t o r - a p o t r a n s f e r r i n c o m p l e x t o t h e o u t e r m e m b r a n e s u rf a ce . A t t h e h i g h e r

e x t r a c e ll u l a r p H o f 7 .4 , t h e a p o T f d i s so c i a t e s f r o m t h e r e c e p t o r a n d r e t u r n s t o

t h e s e r u m t o p a r t i c i p a t e i n a d d i t i o n a l c y c l e s o f i r o n t r a n s p o r t a n d u p t a k e .

1.2

Transferr in Structure

T r a n s f e r r i n is a g l y c o p r o t e i n o f a p p r o x i m a t e l y 8 0,0 00 M W . A l t h o u g h i t

cons i s t s o f a s ing le po lypep t ide cha in , the p ro te in fo lds in to two d i s t inc t ,

h o m o l o g o u s l o b es c o n n e c t e d b y a s h o r t p o l y p e p t i d e t e t h e r [1 1, 1 2 ] . E s s e n ti a l ly

t h e s a m e o v e r a l l s t r u c t u r e h a s b e e n r e p o r t e d f o r t h e c l o s e l y r e l a t e d i r o n -

b ind ing p ro te ins l ac to fe r r in [6 , 13-15] and ovo t rans fe r r in [16 , 17] . Each lobe

(usua l ly iden t i f i ed as e i ther N- te rmina l o r C- te rmina l ) i s fu r ther d iv ided in totwo domains by a c le f t . A s ing le , h igh-a f f in i ty meta l b ind ing s i t e i s con ta ined

wi th in each c le f t . The c rys ta l s t ruc tu res fo r apo- and d i f e r r ic l ac to fe r r in show

tha t the two dom ain s p ivo t on a h ing e r eg ion a t the base o f the c le f t, so tha t the

c l e f t i s o p e n w i d e r i n t h e a p o p r o t e i n t h a n i t i s i n t h e f e r r i c - p r o t e i n c o m p l e x

[6 , 18 ]. T h is c o n f o r m a t i o n a l c h a n g e h a s b e e n d e t e c t e d i n s o l u t i o n b y l o w a n g l e

X - r a y s c a t t e r i n g s t u d i e s f o r l a c t o f e r r i n , o v o t r a n s f e r r i n , a n d s e r u m t r a n s f e r r i n

[19].

I t h a s b e e n k n o w n f o r y e a r s t h a t t h e s t r o n g b i n d i n g o f a m e t a l i o n t o a p o T f

r e q u i r e s t h e c o n c o m i t a n t b i n d i n g o f a s y n e r g is t i c a n i o n . U n d e r b i o lo g i c a l

c o n d i t i o n s c a r b o n a t e f u n c t i o n s a s t h i s s y n e r g i s ti c a n i o n , a l t h o u g h a n u m b e r o f

s m a l l c a r b o x y li c a c id s c a n f o r m w e a k e r F e - a n i o n - T f t e r n a r y c o m p l e x e s u n d e r

c a r b o n a t e - f r e e c o n d i t i o n s [ 2 0 ] . T h e c r y s t a l s t r u c t u r e s n o w s h o w t h a t t h e f i r s t

c o o r d i n a t i o n s p h e r e a r o u n d e a c h f e rr ic i o n i n c l u d e s th e p h e n o l i c o x y g e n s o f t w o

t y r o s i n e r e s i d u e s , t h e i m i d a z o l e g r o u p o f a h i s t i d i n e r e s i d u e , a n d a m o n o d e n -

ta te ca rboxy la te s ide cha in o f an asp ar t i c ac id r es id ue (F ig . 1 ) [6 , 11 -14] . The

f if th a n d s i x th p o s i t i o n s i n t h e d i s t o r t e d s i x - c o o r d i n a t e s t r u c t u r e a r e o c c u p i e d

b y t w o o x y g e n s o f t h e b i d e n t a t e c a r b o n a t e s y n e r g i s t i c a n i o n . T h e c a r b o n a t e

a n i o n a l so i n t e r a c t s w i t h t h e p o s i ti v e d i p o l e a s s o c i a t e d w i t h t h e t e r m i n u s o f a n

a l p h a h e l i x a n d h y d r o g e n b o n d s t o a t h r e o n i n e s i d e c h a i n .T h e t r a n s f e r r i n c r y s t a l s t r u c t u r e s d o n o t s h o w a n y s i g n i f i c a n t d i f f e r e n c e

b e t w e e n t h e f er r ic i o n c o o r d i n a t i o n g e o m e t r i e s o f t h e C - t e r m i n a l a n d N -

t e r m i n a l b i n d i n g s i te s . H o w e v e r , sm a l l c h e m i c a l a n d s p e c t r o s c o p i c d i f fe r e n c e s

b e t w e e n t h e C - a n d N - t e r m i n a l b i n d i n g s i t es a r e o b s e r v e d f a ir ly r o u t in e l y . T h e

t w o s i t e s o f t e n h a v e d i f f e r e n t r a t e c o n s t a n t s a n d e q u i l i b r i u m c o n s t a n t s f o r



124 W .R. Harr is

/

Fig. 1. N-terminal i ron b inding s i te of hum an se rum transfer r in

T Y R

m e t a l b i n d i n g , a n d m e t a l s b o u n d a t t h e t w o s it e s o f t e n g iv e s l i g h tl y d i f f e r e n t

E S R o r N M R s p e c t r a [ 1 -3 , 2 1 - 2 3 ] .

2Ge nera l Descr ip t ion o f M eta l B ind ing

T h e t y r o s i n e s i d e c h a i n s i n t h e m e t a l b i n d i n g s i t e a r e s tr o n g , h a r d L e w i s b a s e s ,

s o T f p r e f e r e n t i a l l y b i n d s h a r d m e t a l i o n s s u c h a s F e 3§ G a 3+ , a n d P u 4§ S i m i l a r

s e l e c t i v i t y i s o b s e r v e d f o r l o w m o l e c u l a r w e i g h t l i g a n d s w h i c h c o n t a i n

p h e n o l a t e c o o r d i n a t i n g g r o u p s [ 2 4 -2 7 ] . S i n ce th e r e a r e f ew t r iv a l e n t m e t a l i o n s

o t h e r t h a n i r o n f o u n d i n n o r m a l s e r u m , t r a n s f e r r i n o f te n a p p e a r s t o b e h ig h l y

s e l e c ti v e f o r ir o n . I n f a ct , t h e p r o t e i n w i ll b i n d a w i d e v a r i e t y o f m e t a l i o n s .

T a b l e 1 l is ts a p p r o x i m a t e l y 4 0 m e t a l i o n s t h a t h a v e b e e n r e p o r t e d t o b i n d

( u s u a ll y in v it r o ) t o e it h e r t r a n s f e r r i n o r o v o t r a n s f e r r i n . W h e t h e r t h e s e m e t a l

i o n s b i n d t o t r a n s f e r r i n i n v iv o i s a m o r e d i ff ic u l t q u e s t i o n . T h i s r e v i e w w i l l

f o c u s o n m e t a l s f o r w h i c h t h e r e i s e v i d e n c e t h a t T f i s a c t i v e ly i n v o l v e d i ns e r u m t r a n s p o r t .

O n e g e n e r a l f e a tu r e t o k e e p i n m i n d i s t h a t t h e t r a n s f e r r i n i n n o r m a l s e r u m

i s o n l y a b o u t 3 0 % s a t u r a t e d w i t h f e r r ic i o n [4 , 2 8 ]. G i v e n t h a t t h e s e r u m

t r a n s f e r r i n c o n c e n t r a t i o n r a n g e s f r o m 2 5 t o 5 0 ~ t m o l / 1 [ 4 ] , n o r m a l s e r u m w i l l

c o n t a i n a b o u t 5 0 la mo l/1 v a c a n t t r a n s f e r r i n b i n d i n g s it e s. T h u s w h i l e i t i s t r u e



Binding and Transpor t o f Nonfer rous Meta ls 1 2 5

Table 1. M e t a l i o n s r e p o r t e d t o b i n d t o t h e tr a n s f e r r i n s

T r a n s i t i o n M e t a l I o n s

L a n t h a n i d e s

A c t i n i d e s

P - B l o c k M e t a l I o n s

V 3+, VO +, V O 2+, Cr 3+, M n 2+/3§ Fe 2+/3§ C02+/3+,

Ni 2 . , Cu 2~ , Zn 2~ , Cd 2 . , Ht~*, Sc3+

C e 3 + / 4* , p r 3 + , N d 3+ , S m 3 + , E u 3 + , G d 3 + , T b 3+ , H o 3+ ,E r 3 + , y b 3 + , Lu 3 +

Th 4 + , U4 + , 2 ~ _ 4 + n ^4 + C m 3 +O ~ ,N I . , , r a , P u 4 + , A m 3+ ,T 37+ ,~1 3+ ~-3 +A13+, G a 3 + , I n , t1 , tn

t h a t f e r ri c i o n i s b o u n d m o r e t i g h t l y t h a n o t h e r m e t a l i o n s , i t i s u s u a l l y t h e c a s e

t h a t o t h e r m e t a l i o n s , e v e n a t c o n c e n t r a t i o n s o f s e v e r a l pm o l /1 , n e e d n o t

c o m p e t e d i r e c t l y w i t h f e r ri c i o n fo r b i n d i n g s i t e s o n t h e p r o t e i n . I n s t e a d , t h e

b i n d i n g o f o t h e r m e t a l i o n s t o t r a n s f e r r i n d e p e n d s p r i m a r i l y o n c o m p e t i t i o n

f o r t h e m e t a l i o n f r o m o t h e r l i g a n d s i n s e r u m , i n c l u d i n g p r o t e i n s , s u c h a sa l b u m i n , o r g a n i c l i g a n d s s u c h a s c y s t e i n e , h i s t i d i n e , a n d c i t ra t e , a n d i n o r g a n i c

l i g a n d s s u c h as p h o s p h a t e , c a r b o n a t e , a n d h y d r o x i d e .

F o r m e t a l i o n s t h a t b i n d t o T f v e r y t i g h tl y , e . g. , M n 3§ o r P u 4 +, p r o t e i n

b i n d i n g c a n b e e a s il y d e m o n s t r a t e d b y th e f ra c t i o n a t i o n o f s e r u m c o n t a i n i n g

a n a p p r o p r i a t e r a d i o l a b e l b y m e t h o d s s u c h a s e l e c t r o p h o r e s i s o r c o l u m n

c h r o m a t o g r a p h y . H o w e v e r , fo r m a n y m e t a l io n s o f i n t e re s t , i n c l u d i n g A 13§

G a 3+, a n d Z n 2+, t h e b i n d i n g m a y n o t b e s t r o n g e n o u g h t o s u r v i v e s o m e o f th e

f r a c t i o n a t i o n m e t h o d s . F o r t h e s e m e t a l i o n s , it i s u s e f u l t o e s t i m a t e t h e e x t e n t

o f t r a n s f e r r in b i n d i n g u n d e r b i o l o g i c a l c o n d i t i o n s b y t h e u s e o f e q u i l i b r i u ms p e c i a t i o n c a l c u l a t i o n s . B o t h a p p r o a c h e s t o s p e c i a t i o n a r e d i s c u s s e d i n t h e

f o l l o w i n g s e c t i o n s o n i n d i v i d u a l m e t a l i o n s .

2 .1

Me ta l Co m p l e x a t i o n E q u i l i b r i a

E q u i l i b r i u m c o n s t a n t s h a v e b e e n r e p o r t e d f o r t h e t r a n s f e r r i n c o m p l e x e s o f 15

m e t a l i o n s r a n g i n g i n o x i d a t i o n s t a t e f r o m I I to V : Z n 2+ [2 9 , 30 ] , C u 2+ [3 1 ] ,

Cd 2+ [32 ] , M n 2+ [33 ] , Fe 2§ [34 , 35 ] , N i 2+ [35 ] , Fe 3+ [36 , 37 ] , A13+ [36 , 38 -4 0 ] ,

G a 3+ [ 4 1 - 4 4 ] , I n 3 + [ 4 2 , 4 5 , 4 6 ] , G d 3+ [ 4 7 , 4 8 ] , Y b 3+ [ 4 9 ] , N d 3 + [ 5 0 ] , S m 3+ [ 5 0 ] ,

B i 3+ [ 5 1 ] , V u 4+ [ 4 9 ] , a n d V O + [ 5 2 ]. M o s t s t u d i e s r e p o r t e f f e c ti v e b i n d i n g

c o n s t a n t s f o r a s p ec if ic p H a n d c a r b o n a t e c o n c e n t r a t i o n , u s u a l l y d e f i n e d a s

[ M - C O 3 - T F ]

K ; = [ T f] [M ] ( 1 )

[ M - C O 3 - T F - C O 3 - M ]

K 2 ---- [ M - C O 3 - T f ] [ M ] ( 2 )

w h e r e t h e f re e b i c a r b o n a t e a n i o n i s n o t e x p l i c it l y s h o w n a s a r e a c t a n t i n t h e

c o m p l e x a t i o n e q u i l i b r ia . A s a g e n e r a l r u l e , t h e b i n d i n g c o n s t a n t s f o r t h e tw o T f

b i n d i n g s i te s d if f e r b y a b o u t o n e o r d e r o f m a g n i t u d e [ 21 , 2 2 ]. In m o s t c a s e s i n

w h i c h s it e se l e ct iv i ty h a s b e e n e x a m i n e d , t h e l a r g e r b i n d i n g c o n s t a n t is

a s s o c i a t e d p r i m a r i l y w i t h t h e C - t e r m i n a l b i n d i n g s i t e [ 3 1 , 3 3 , 3 8 , 4 7 , 5 0 , 5 3 , 5 4 ] .



126 W.R. Harris

I n c o n t r a s t , o v o t r a n s f e r r i n t y p i c a l l y s h o w s e i t h e r s t r o n g e r b i n d i n g a t t h e N -

te rmina l s i te [23 , 55 , 56] or no s i te se lec t iv i ty [31 , 57] .

M e t a l a n d a n i o n b i n d i n g t o t r a n s f e r r i n h a s t r a d i t i o n a l l y b e e n r e g a r d e d a s a n

a l l - o r - n o t h i n g e v e n t , w i t h n e i t h e r t h e m e t a l i o n n o r t h e a n i o n b i n d i n g i n t h ea bse nc e o f t he o the r . 13t i s n o w c l ea r f r o m d i f f er e n c e U V [ 5 8 - 6 0 ] a n d C N M R

[23 , 61 , 62 ] s tud i e s t ha t s e ve ra l a n ions , i nc lud ing b i c a rbona te , w i l l b ind to

a p o t r a n s f e r r i n i n th e a b s e n c e o f m e t a l i o ns . A n i o n - a p o T f b i n d i n g c o n s t a n t s

r a n g e f r o m 1 02 "s f o r b i c a r b o n a t e t o a b o u t 10 6 f o r p y r o p h o s p h a t e [ 5 8 -6 0 ] .

F u r t h e r m o r e , k i n e t i c s t u d i e s o n t h e f o r m a t i o n o f f e rr i c t r a n s f e r r i n b y th e

b i n d i n g a n d a i r -o x i d a t io n o f F e 2§ h a v e s h o w n t h a t t h e b i n a r y c a r b o n a t e - T f

s p e c ie s f o r m s f i rs t, f o ll o w e d b y c o m p l e x a t i o n a n d f in a ll y o x i d a t i o n o f t h e

f e r r o u s i o n [ 3 4 ] . S i n c e t h e b i c a r b o n a t e - a p o T f e q u i l i b r i a h a v e b e e n c h a r a c t e r -

i z e d , o n e c a n e a s i l y t r e a t t h e o v e r a l l c o m p l e x a t i o n r e a c t i o n a s t w o s e q u e n t i a l

e qu i l i b r i a

Kc

HCO 3 + apo Tf , ' HCO3-Tf (3)

KM

HCO 3-Tf + M , ' M -CO3-Tf (4)

T h e a d v a n t a g e o f t h is a p p r o a c h is t h a t t h e v a lu e o f K M fo r a g ive n me ta l i on

is i n d e p e n d e n t o f t h e s o l u ti o n b i c a r b o n a t e c o n c e n t r a t io n , a n d t h is p r o v i d e s a

c o m m o n f r a m e o f r e f er e n c e f o r c o m p a r i n g r e s u l ts f r o m e x p e r i m e n t s c o n -

d u c t e d u n d e r d i f f e re n t c o n d i t i o n s . O n e s h o u l d n o t e , h o w e v e r , t h a t b o t h K c a n dK M a re s ti ll c o n d i t i o n a l c o n s t a n t s w i t h r e s p e c t t o th e [ H § a n d a r e v a li d o n l y a t

pH 7 .4 .

F o r t r i v a l e n t m e t a l i o n s s u c h a s F e 3§ a to t a l o f t h r e e p r o t o n s p e r i r o n a r e

re l e a se d by Eqs. (3 ) a nd (4 ). Th e f re e a n io n i s de p ic t e d a s HCO 3 in Eq. (3 )

b e c a u s e t h a t i s t h e p r e d o m i n a n t s p e c ie s i n s o l u t i o n a t p H 7 .4 , b u t b o t h

s t r u c t u r a l a n d s p e c t r o s c o p i c d a t a i n d i c a t e t h a t i n M 3 + - T f c o m p l e x e s , t h e

b o u n d s y n e r g i s ti c a n i o n h a s b e e n d e p r o t o n a t e d t o c a r b o n a t e [1 2, 1 3, 2 3 , 5 5, 6 3 ,

6 4 ]. W h e t h e r t h e d e p r o t o n a t i o n o f b i c a r b o n a t e o c c u r s i n E q . ( 3) o r ( 4) is n o t

k n o w n . T h e o t h e r tw o p r o t o n s w o u l d c o m e f r o m t h e d e p r o t o n a t i o n o f t h e

p h e n o l i c o x y g e n s o f t h e tw o c o o r d i n a t e d t y r o s i n e r e s i d u e s .

C o m p l e x a t i o n o f d i v a l e n t c a t i o n s s u c h a s Z n 2§ r e le a s es o n l y t w o p r o t o n s

[ 65 ], b o t h o f w h i c h c a n b e a c c o u n t e d f o r b y t h e t y r o s i n e r e s i d u e s . T h i s w o u l d

i m p l y t h a t th e a n i o n r e m a i n s p r o t o n a t e d i n c o m p l e x e s o f d i v a le n t c a ti o n s, a n d

t h e c r y s ta l s t r u c t u r e o f t h e C u 2 + - l a ct o f er r in c o m p l e x s h o w s m o n o d e n t a t e

a n i o n c o o r d i n a t i o n a t o n e o f th e t w o s i te s [ 66 ]. H o w e v e r , 1 3 C -N M R s t u d i e s o n

Z n 2 § a r e m o r e c o n s i s t en t w i th c a r b o n a t e t h a n w i t h b i c a r b o n a t e

[ 63 ]. T h u s t h e r e i s st il l s o m e u n c e r t a i n t y a s t o t h e s t a te o f p r o t o n a t i o n o f t h e

s y n e r g i s t i c a n i o n i n c o m p l e x e s w i t h d i v a l e n t a n i o n s .

E x c e p t u n d e r c a r b o n a t e - f r e e c o n d i t i o n s , t h e m e t a l - f r e e si te s o f a p o t r a n s -f e r r in c o n s i s t o f a m i x t u r e o f t r u l y v a c a n t s i te s a n d t h o s e o c c u p i e d b y t h e

car bo na te an ion as(K~)producta of Eq. (3) . Th e m ag ni tu d e o f the e ffec t ive meta l -

b i n d i n g c o n s t a n t is d i r e c t ly p r o p o r t i o n a l t o t h e d e g r e e o f s a t u r a t i o n o f t h e

m e t a l - f r e e s i t e s w i t h b i c a r b o n a t e [ 2 9 ] . T h u s t h e c o n s t a n t s KM and K~ a re

r e l a t e d b y t h e e q u a t i o n



Binding and Transport of N onferrous Meta ls 127

log K~ = log KMq-log ( 5 )

where ~ is defined as

Kc[HCO3]~z = (6 )

1 q-- Kc[H CO 3]

A s imi l a r s e t o f e qu a t ion s c a n be use d fo r K ~. G ive n tha t t he va lue o f l og K c

i s 2 .5 f o r b o t h T f b i n d i n g s it es [ 58 ] , o n e c a n e a s i ly c o n v e r t e x p e r i m e n t a l v a l u e s

o f K* w h i c h h a v e b e e n m e a s u r e d f o r d if f e r e n t s o l u t i o n b i c a r b o n a t e c o n c e n -

t r a t i o n s i n t o c a r b o n a t e - i n d e p e n d e n t K M ' S .

T h e r e i s n o f u n d a m e n t a l r e a s o n t h a t m e t a l s s h o u l d n o t b i n d t o a p o t r a n s -

f e r r in i n t h e a b s e n c e o f c a r b o n a t e . H o w e v e r , in t h e a b s e n c e o f c a r b o n a t e t h e

b i n d i n g o f m a n y m e t a l i o n s , p a r t i c u l a r ly F e 3+, is w e a k e n e d t o t h e p o i n t t h a t

p r o t e i n - b i n d i n g c a n n o l o n g e r c o m p e t e w i th t h e s t r o n g t e n d e n c y o f t h e m e t a l

i o n t o h y d r o l y z e . T h u s , a s a p r a c ti c a l m a t t e r , t h e p r e s e n c e o f c a r b o n a t e u s u a l ly

a c t s a s a n e f fe c t ive t r i gge r t o e na b le me ta l b ind ing . Tw o e xc e p t ions t o t h i s ru l e

i n v o l v e t h e c o m p l e x e s b e t w e e n t r a n s f e r r i n a n d V O + [5 9] a n d b e t w e e n V O 2§

a n d o v o t r a n s f e r r i n [ 67 ]. I n e a c h ca s e, th e m e t a l i o n p r e s u m a b l y b r i n g s i t s o w n

s y n e r g i s ti c a n i o n i n t o t h e b i n d i n g s it e i n t h e f o r m o f a n o x o l ig a n d .

2 .2D i f f e r e n c e U l t r a v i o l e t T i t r a t i o n s

T h e f e r r i c t r a n s f e r r i n c o m p l e x h a s a r e a s o n a b l y s t r o n g c h a r g e t r a n s f e r b a n d a t

4 6 5 n m [ 1 - 3 ] . T h u s o n e c a n m e a s u r e f e r r i c t r a n s f e r r i n b i n d i n g c o n s t a n t s b y

a l lo w i n g t h e p r o t e i n t o e q u i l i b r a te w i t h a c o m p e t i t iv e c h e l a t i n g a g e n t a n d t h e n

m e a s u r i n g t h e d i s t r ib u t i o n o f t h e i ro n b e t w e e n t h e p r o t e i n a n d t h e c o m p e t i ti v e

l i g a n d b a s e d o n t h e v i s i b l e a b s o r b a n c e [ 3 6 , 3 7 ] . H o w e v e r , m a n y m e t a l i o n s o f

i n t e r e s t la c k th i s t y p e o f c h a r g e - t r a n s f e r b a n d , a n d t h e m o s t w i d e l y u s e d

m e t h o d f o r m e a s u r i n g m e t a l - t r a n s f e r r i n b i n d i n g c o n s t a n t s i s d i f f e r e n c e U V

s p e c t r o s c o p y [4 1, 5 0] . I n t h is m e t h o d , a s a m p l e o f th e p r o t e i n is sp l it b e t w e e n

t h e r e f e r e n c e a n d s a m p l e c u v e t te s . A f t e r a b a s e li n e o f p r o t e i n v s p r o t e i n is

r e c o r d e d , a n a l i q u o t o f a m e t a l t i tr a n t is a d d e d t o t h e s a m p l e c u v e t t e, w h i le a n

e q u a l a l i q u o t o f w a t e r i s a d d e d t o th e r e f e r e n c e c u v e t t e to m a i n t a i n t h e s a m e

p r o t e i n c o n c e n t r a t io n . T h e c o o r d i n a t i o n o f t h e m e t a l t o th e p h e n o l i c o x y g e n o f

t h e tw o t y r o s i n e r e s i d u e s p e r t u r b s t h e U V s p e c t r u m o f t h e a r o m a t i c r i n g s . T h i s

r e s u l ts i n a c h a r a c te r i s t ic d i f fe r e n c e s p e c t r u m , w i t h a m a j o r p e a k n e a r 2 4 5 n m

a n d a b r o a d e r , s m a l le r p e a k a r o u n d 2 9 0 n m . T h e s e q u e n ti a l a d d i t i o n o f t i tr a n t

p r o d u c e s a f a m i l y o f s p e c t r a s u c h a s th a t s h o w n i n F ig . 2 f o r t h e ti t r a t io n w i t h

A I3+ [38].T o n o r m a l i z e t h e d a t a f r o m d i f f e r e n t e x p e r i m e n t s , t h e a b s o r b a n c e a t

2 4 5 n m i s c o n v e r t e d t o a n a b s o r p t i v i t y b y d i v i d i n g b y t h e a n a l y t ic a l

c o n c e n t r a t i o n o f t r a n s f e r r i n . T i t r a t i o n c u r v e s a re p r e p a r e d b y p l o t t i n g

a b s o r p t i v i t y v s th e e q u i v a l e n t s o f m e t a l a d d e d . B y r e p e a t i n g t h e t i t r a ti o n

u s i n g a s e r ie s o f t i t r a n t s w h i c h c o n t a i n t h e m e t a l i o n p l u s v a r y i n g



128 W . R . H a r r i s

0 . 2 0

0 . 1 5

0 . 1 0

0rJ l

<

0 . 0 5

0 . 0 0

I I I I I I I I !

2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0

W a v e l e n g t h ( r i m )

Fig. 2. Difference UV spec tra produced by the titration of apotransferrin w ith AI3+. C u r v e B

is a baseline of protein vs protein. Curve 1 - 0.24 eq uiv, of A13+;curve 2 - 0.36; curve 3 -

0.48; curve 4 - 0.60; c u r v e 5 - 0.78; curve 6 - 1.o2; curve 7 - 1.26; curve 8 - 1.5o; curve 9 -

1.86. D ata have been taken from [38]

c o n c e n t r a t i o n s o f a c o m p e t i t i v e l i g a n d , o n e c a n g e n e r a t e a fa m i l y o f t i tr a t io n

c u r v e s a s s h o w n i n F ig . 3 . T h e s e d a t a s h o w t h e t i t r a t i o n o f a p o T f w i t h A 13+

t i t r a n t s c o n t a i n i n g n i t r i li o t r i a c e t ic a c i d ( N T A ) a s t h e c o m p e t i t i v e l i g a n d . T h e

m o l a r a b s o r p t i v i t y o f t h e A 1 - T f c o m p l e x c a n b e c a l c u l a t e d a s t h e i n i ti a l s l o p e o f

t h e t i t ra t i o n c u r v e i n t h e a b s e n c e o f N T A . M e t a l - t r a n s f e r r in e q u i l i b r i u m

c o n s t a n t s c a n b e c a l c u la t e d f r o m n o n - l i n e a r l e a s t s q u a r e s f it s o f t h e t i t r a t io n

c u r v e s u s i n g K~ a n d K~ a s a d j u s t a b l e p a r a m e t e r s [ 21 ] .

T r a n s f e r r i n m e t a l - b i n d i n g c o n s t a n t s (K M 's ) t y p i c a l l y r a n g e f r o m 104 to 1012

f o r t r i v a l e n t l a n t h a n i d e s a n d d i v a l e n t t r a n s i t i o n m e t a l s a n d f r o m 1013 to 1022

f o r o t h e r t r i v a l e n t m e t a l i o n s [ 21 , 2 2 , 3 1 , 5 1 ]. A l t h o u g h t h e s e b i n d i n g c o n s t a n t s

a r e r e a s o n a b l y l a r g e , t h e r e i s o f t e n s i g n i f i c a n t c o m p e t i t i o n f o r t h e m e t a l i o n

f r o m h y d r o x i d e a n d / o r c a r b o n a t e a n i o n s i n t h e b u ff e r. T h i s is i l lu s t r at e d b y

t h e A 13+ d a t a s h o w n i n F i g. 3 . E a r l y in t h e t i t r a t i o n , w h e n t h e o c c u p a n c y o f t h e



Binding and Transpo~ of No n~rrous M eta ls 129

30000 i

25000

20000

~ , 1 5 0 0 0

0 NTA

3:1 NTA :AI 3+

5 : 1

I 0 : I

1 5 : 1

10000

5 0 0 0

0 1 2 3 4

E q u i v . o f A !

Fig. 3. T itrations of apoT f w ith Al3+ titrants w hi ch contain varying concentrations ofnitrilotriacetic acid (NTA ). The curves are identified by the NT A: A13+ ratio . The data havebeen taken from [38]

s t r o n g e r C - t e r m i n a l b i n d i n g s i t e i s r e l a t i v e l y l o w , t h e r e i s a l i n e a r i n c r e a s e i n

t h e a b s o r p t i v i t y . F r o m t h e s l o p e o f t h i s in i t i al l i n e a r s e g m e n t , o n e c a n c a l c u l a te

a m o l a r a b s o r p t i v i t y o f 1 4,8 00 1 / m o l c m -1 f o r t h e A 1 - C O 3 -T f c o m p l e x .

H o w e v e r , a s t h e t i t r a t i o n p r o c e e d s a n d t h e s t r o n g e r C - t e r m i n a l s i t e i s

s a t u r a te d , t h e b i n d i n g o f th e A 1 3+ b y h y d r o x i d e b e g i n s t o c o m p e t e w i t h

b i n d i n g t o t h e r e m a i n i n g , w e a k e r N - t e r m i n a l s i t e . L a t e r i n t h e t i t r a t i o n o n e

o b s e r v e s a c u r v a t u r e i n t h e p l o t , w h i c h e v e n t u a l l y l e v el s o f f a t a b o u t 2 7 ,0 0 0 l /

m o l c m - 1 . T h i s c o r r e s p o n d s t o o n l y 1 .8 A 1 3+ i o n s b o u n d p e r t r a n s f e r r i n

m o l e c u l e . R e s u l t s f r o m ~3C a n d 27A 1 N M R s t u d i e s i n d i c a t e t h a t b o t h s e r u m T fa n d o v o t r a n s f e r r i n b i n d a b o u t 1 .5 A 13 + i o n s p e r m o l e c u l e [ 5 5] .

T h e a p p a r e n t s a t u r a t i o n i n t h e b i n d i n g a t le ss t h a n 2 .0 A1a+ ions per

m o l e c u l e a r i s e s b e c a u s e t h e K s p f o r t h e m e t a l h y d r o x i d e h a s b e e n e x c e e d e d .

E v e n t h o u g h n o p r e c i p i t a ti o n is o b s e r v e d a t lo w m e t a l c o n c e n t r a t i o n s , t h e

f o r m a t i o n o f p o l y m e r i c m e t a l h y d r o x o c o m p l e x e s e s s e n ti a ll y c a p s th e



130 W .R. Harris

c o n c e n t r a t i o n o f f re e m e t a l i o n i n s o l u t i o n a t a l ev e l b e l o w t h a t n e e d e d t o

a c h i e v e s a t u r a t i o n o f b o t h T f b i n d i n g s it es .

S u c h c o m p e t i t i o n f r o m h y d r o x i d e a l so a f fe c ts t h e b i n d i n g o f G a 3+ [4 1 ],

w h i le c o m p e t i t i o n f r o m c a r b o n a t e i s a v e r y s t r o n g f a c t o r in t i t ra t i o n s o f a p o T f

w i t h l a n t h a n i d e s [ 4 7 , 5 0 ] a n d z i n c [ 3 0 ] . T h i s p h e n o m e n o n c a n b e i l l u s t r a t e d

m o r e c l e a r ly b y u s i n g t h e o b s e r v e d a b s o r p t i v i t i e s f r o m d i f f e r e n c e U V t i t r a t io n s

t o c a l c u l a te n, t h e a v e ra g e n u m b e r o f m e t a l io n s b o u n d p e r p r o t e i n m o l e c u l e .

P l o t s o f n v s e q u i v a l e n t o f a d d e d m e t a l f o r se v e r a l m e t a l i o n s a r e s h o w n i n

F ig . 4. It n e e d s t o b e e m p h a s i z e d t h a t t h e v e r y t i g h t b i n d i n g o f t w o e q u i v a l e n t s

o f m e t a l i o n , a s i s o b s e r v e d f o r f e r ri c i o n , i s t h e e x c e p t i o n r a t h e r t h a n t h e r u l e

2 . 0

e~

O

= 1 .5

I. .

r~

~ 1 . 0

o

0

- 0 . 5

F e 3+

~ L u 3+ Ga3+

9 Z n 2 +

Gd 3+

N d 3+

0 . 0 ~ | I I t I

0 1 2 3 4 5

E Q U I V . o f M E T A L

Fig. 4. Plots of n, the average num be r of metal ions bo un d p er transfe rrin molecule, vs theequiv, of metal added to the solution



Binding and Transpo rt of Nonferrous Metals 131

i n t r a n s fe r r i n c h e m i s t ry , a n d t h a t t h e a d d i t i o n o f e x ce s s m e t a l io n t o a p o T f i n

n o w a y g u a r a n t e e s t h e f o r m a t i o n o f t h e s a t u r a t e d M 2 - T f c o m p l e x .

3Binding and Transpor t o f Ind iv idua l Meta l Ions

3.1Gallium

B o t h 67Ga a n d 68Ga are u s e d a s r a d i o p h a r m a c e u t i c a l s [ 68 , 6 9 ]. T h e l a r g e s t

c u r r e n t a p p l i c a t i o n i s t h e u s e o f 6 7 G a f o r s o f t t u m o r s a n d a b s c e s s e s [ 68 ] .

H o w e v e r , 6 8 G a i s o f g r o w i n g i n t e r e s t b e c a u s e i t is s u i t a b le f o r t h r e e

d i m e n s i o n a l i m a g i n g b y p o s i t ro n e m i s s io n t o m o g r a p h y .

G a l l i u m i s o t o p e s a r e u s u a l l y i n j e c te d i n a s o l u t i o n o f c i tr i c a c id , w h i c h i sa d d e d t o p r e v e n t t h e f o r m a t i o n o f i n s o l u b le g a l l i u m h y d r o x i d e c o m p l e x e s .

H o w e v e r , e a r l y s t u d i e s d e m o n s t r a t e d t h a t o n c e in th e c i rc u l a t i o n , t h e m e t a l io n

d i s s o c i a t e s f r o m t h e c i t r a t e a n d b i n d s t o s e r u m t r a n s f e r r i n [ 7 0 - 7 3 ] . T h i s

b i n d i n g is ea s il y u n d e r s t o o d i n t e r m s o f t h e v e r y s tr o n g s i m i la r it ie s b e t w e e nGa 3+ an d 3+e [ 74 ]. The se two t r iva le n t ions a r e a lm os t the sa m e s i ze . I n

a d d i t i o n , t h e e l e c t ro n c o n f i g u r a t i o n s o f t h e h i g h s p i n d 5 F e 3+ i o n a n d t h e

d l~ Ga 3+ ion a r e su c h tha t t he i r c om ple x e s l a c k a ny c r ys t a l f ie ld s t a b i l i z a t ion

e n e r g y . T h u s n e i t h e r m e t a l i o n s h o w s a s t r o n g p r e f e r e n c e f o r s t r i c t o c t a h e d r a l

g e o m e t r y , a n d e a c h c a n e a s il y a d a p t t o th e d i s t o r te d s i x - c o o r d i n a t e g e o m e t r yo f th e T f b i n d i n g s it e.

I t is n o t s u r p r i s i n g t h a t G a 3+ b i n d s n o t o n l y t o t r a n s f e r r i n a n d l a c t o f e r r in

[ 7 5 - 7 8 ] , b u t a l s o t o t h e i r o n s t o r a g e p r o t e i n f e r r i t i n [ 7 7 , 79- 82] , s inc e f e r r i t i n

a l s o b i n d s i r o n i n i t s + 3 o x i d a t i o n s t a t e . T h e m a j o r c h e m i c a l d i f f e r e n c e

b e t w e e n F e 3+ a n d G a 3+ i s t h e a b s e n c e o f a s t a b l e d i v a l e n t o x i d a t i o n s t a t e f o r

G a . T h u s o n e d o e s n o t o b s e r v e i n c o r p o r a t i o n o f G a i n t o h e m o g l o b i n a n d o t h e r

h e m e p r o t e i n s t h a t u t i l i z e d i v a l e n t i r o n .

A v e r y w i d e r a n g e o f G a - T f b i n d i n g c o n s t a n t s h a v e b e e n r e p o r t e d [ 4 1 - 4 4 ].

T h e i n i ti a l a t te m p t a t m e a s u r i n g t h e G a - T f b i n d i n g c o n s t a n t s u s e d a n e x c es s o f

G a in a c o m p e t i t i v e a s s a y v s s e p h a d e x G - 1 0 0 [ 4 4] . A n a l y s i s o f t h e d a t a b y a

S c a t c h a r d ' s p l o t i n d i c a t e d t h a t t h e r e w e r e 1 4 b i n d i n g s i t es w i t h a b i n d i n g

c o n s t a n t o f l o g K = 0 . 2 5 . L a r s o n e t a l. [4 3] m e a s u r e d t h e G a b i n d i n g c o n s t a n t s

o f T f u s i n g e q u i l i b r i u m d i a ly s i s w i th a la r g e ex c es s o f T f a n d r e p o r t e d a

b i n d i n g c o n s t a n t o f l o g K = 5. 4.

N e i t h e r o f t h e s e s t u d i e s t o o k i n t o c o n s i d e r a t i o n t h e h y d r o l y s i s o f t h e f re e

g a l l i u m i o n . T h e s m a l l , h i g h l y c h a r g e d G a 3§ i o n h y d r o l y z e s e x t e n s iv e l y , e v e n i n

a c i d i c s o l u t io n s [ 83 ] . T h e h y d r o l y s i s c o n s t a n t s f o r G a a r e d e f in e d a s

fl~ = [Ga(O H)3n-"][H+ ]~[Ga3+] (7)

with log [3n va lues o f - 2 .9 , -6 .6 , -11 .0 , and -16 .5 for n = l- 4 [83]. Thus the G a 3+ ion

acts as a poly protic acid with fou r stepwise pKfls ranging from 2.9 to 5.5 . At

physiolog ical pH, mo no m eric Ga 3+ exists in solution as 98% Ga(OH)4 and 2%



132 W.R. Harris

G a (O H ) 3. A t h ig h e r c o n c e n t r a t i o n s o f t h e m e ta l i o n , p o ly n u c l e a r h y d r o x o c o m p le x e s

b e g in t o f o r m , a n d a n a c c u r a t e d e s c r ip t i o n o f t h e h y d r o ly s i s e q u i l i b ri a b e c o m e s

v i r t u a ll y im p o s s ib l e . B e c a u s e o f th i s e x t e n s iv e h y d r o ly s is , p r o t e in b in d in g s tu d i e s

such as tha t o f C lause n e t a l. [ 44 ], wh ich invo lve a la rge excess o f the m eta l over thep r o t e in , s h o u ld b e a v o id e d .

H a r r i s a n d P e c o r a r o u s e d d i f fe r e n c e U V s p e c t r o s c o p y t o m e a s u r e G a [ T f

b i n d i n g c o n s t a n t s [4 1] . T h e t i t r a t io n o f a p o T f w i th G a 3+ p r o d u c e s a f a m i ly o f

s p e c t r a t h a t a r e v e r y s im i l a r t o t h e a l u m i n u m - T f s p e c t r a s h o w n i n F ig . 2 .

N i t r il o t ri a c e ti c a c i d a n d e t h y l e n e d i a m i n e d i a c e t i c a c i d w e r e i n c l u d e d i n t h e G a

t i t r a t i o n s a s c o m p e t i t i v e c h e l a t i n g a g e n t s . T h i s w a s t h e f i r s t s t u d y o n g a l l i u m

t r a n s f e r r i n t o a c c o u n t e x p l i c it l y f o r h y d r o l y s i s o f t h e f r e e G a 3+ i o n . T h e

e f f e c ti v e b i n d i n g c o n s t a n t s ( d e f i n e d i n E q s . ( 1 ) a n d ( 2 ) ) w e r e l o g K ~ ---- 1 9 .5

a n d l o g K~ = 1 8. 6 f o r p H 7 .4 a n d 5 m m o l / 1 b i c a r b o n a t e .

T h e a c t u a l c h e m i c a l e q u i l i b r i u m b e t w e e n T f a n d G a at p h y s i o l o g ic a l p Hc o r r e s p o n d s e s s e n ti a ll y to

Kobs

G a ( O H ) 4 + a p o T f , ' G a - T f (8 )

T h u s t h e b i n d i n g c o n s t a n t o f 10 5.4 r e p o r t e d b y L a r s o n e t a l . [ 4 3 ] w o u l d

c o r r e s p o n d t o E q. ( 8) . I f t h i s b i n d i n g c o n s t a n t i s c o r r e c t e d f o r h y d r o l y s i s o f

t h e G a a n d a d j u s t e d t o 5 m m o l / 1 b i c a r b o n a t e , t h e r e s u l t i n g v a l u e i s l o g

K ~ = 1 9 .6 , i n e x c e l le n t a g r e e m e n t w i t h t h e d i f f e r e n c e U V r e s u l ts .

O n e o t h e r G a - T f b i n d i n g c o n s t a n t o f l o g K = 2 3.7 h a s b e e n r e p o r t e d [ 42 ],b u t t h i s v a l u e i s c l e a r l y t o o h i g h . T h e a c c e p t e d f e r r i c t r a n s f e r r i n b i n d i n g

c o n s t a n t s a r e o n l y l o g K ~ - - 2 0 .7 a n d l o g K * - - 1 9 . 4 [3 7 ] , B e c a u s e o f t h e s t r o n g2 - -

c h e m i c a l s i m i l a r i t y b e t w e e n F e 3+ a n d G a 3+, t h e r e i s a v e r y s t r o n g l i n e a r

c o r re l a t i o n b e t w e e n t he b i n d i n g c o n s t a n t s o f i r o n a n d g a l l i u m f o r a c o m m o n

s e t o f l i g a n d s . S u c h a l in e a r f r e e e n e r g y r e l a t i o n s h i p ( L F E R ) i s s h o w n i n F ig . 5 .

T h i s p l o t i s t y p i c a l o f s e v e r a l s u c h L F E R s t h a t h a v e b e e n r e p o r t e d f o r t h e

c o m p l e x a t i o n o f F e 3+ a n d G a 3+ [ 4 1 - 4 4 , 7 5 , 8 4 ]. T h e l o g K ~ v a l u e s f o r F e 3+ [ 37 ]

a n d G a 3+ [4 1] h a v e b e e n c o n v e r t e d t o K M v a l u e s t o a c c o u n t f o r d i f f e r e n t

e x p e r i m e n t a l c o n d i t i o n s , a n d t h e T f v a l u e s a r e p l o t t e d a s t h e s o l id t r ia n g l e s i n

F i g . 5 . T h e s e p o i n t s a r e i n e x c e l l e n t a g r e e m e n t w i t h t h e L F E R . C o n v e r s e l y , t h e

h i g h e r G a - T f b i n d i n g c o n s t a n t s f r o m K u l p r a t h i p a n j a e t a l. [4 2] w o u l d m o v e

t h e s e p o i n t s t o a b o u t t h r e e l o g u n i ts a b o v e t h e L F E R a n d t h u s w o u l d b e

i n c o n s i s t e n t w i t h t h e n o r m a l r e l a t i o n s h i p b e t w e e n t h e r e l a t i v e m e t a l - l i g a n d

b i n d i n g c o n s t a n t s f o r F e 3+ a n d G a 3+.

S e r u m f r a c t i o n a t i o n s t u d i e s i n d i c a t e t h a t a t l o w g a l l i u m c o n c e n t r a t i o n s ,

b e t w e e n 8 5 a n d 1 0 0% o f s e r u m g a l l i u m b i n d s t o p r o t e i n [ 70 , 7 1 , 8 5 - 9 0 ] . T h e r e

i s n o e v i d e n c e t h a t p r o t e i n s o t h e r t h a n t r a n s f e r r i n a r e i n v o l v e d in g a l l iu m

b i n d i n g , a n d t h e r e a r e s p e c i f i c d a t a w h i c h i n d i c a t e t h a t s e r u m a l b u m i n d o e s

n o t b i n d G a [ 7 1 , 7 8 , 9 0 ] . I t i s d i f f i c u l t t o g e t a m o r e p r e c i s e m e a s u r e f o r t h ep e r c e n ta g e o f g a l li u m b o u n d t o T f i n s e r u m . T h e c o m p l e x a t io n e q u i l i b r i u m i n

s e r u m b e t w e e n t h e g a ll a t e a n i o n a n d a p o T f is l a b il e a n d e a s i l y s h i f t e d b y t h e

p h y s i c a l m e t h o d s u s e d t o s e p a r a t e m a c r o m o l e c u l e s f r o m lo w - m o l e c u l a r -

w e i g h t c o m p l e x e s . I n a d d i t io n , t h e e ff ec ti ve G a - T f b i n d i n g c o n s t a n t d r o p s o f f

r a t h e r s h a r p ly w h e n t h e b i c a r b o n a t e c o n c e n t r a t i o n d r o p s b e l o w - 1 0 m m o l/ 1



Binding and Transpor t o f Nonfer rous Meta ls 133

i o l

4 0

~ , 3 0+

O 2 0

10

Oo

O,

oo

~ TransferrinKM'so

0

0 1 0 2 0 3 0 4 0

L O G K ( F e 3+)

F i g . 5. Linear free ene rgy relationship between Fe3+ an d Ga3+. Ea ch data poin t represents aligand. The x-coordinate is equal to the log K value of that ligand w ith F e 3 + , and the y-coordinate is the log K value of that ligand with G a 3 +. The log KM, and log KM2 values for

serum transferrin are shown as the f i l l e d t r i a n g l e s . Data on the LMW ligands used toconstruct the plot were taken from [lo9]

[2 2, 8 0, 87 ], so t h a t t h e p r o b l e m o f d i s s o c i a t i o n o f G a - T f d u r i n g t h e p h y s i c a l

s e p a r a t i o n o f t r a n s f e r r in f r o m t h e o t h e r s e r u m p r o t e i n s c a n b e c o m e q u i t e

s e r i o u s u n l e s s o n e t a k e s c a r e t o m a i n t a i n t h e b i c a r b o n a t e c o n c e n t r a t i o n

t h r o u g h o u t t h e s e p a r a t i o n p r o c e s s [ 7 3 , 8 5 , 8 7 , 8 9 , 9 1 ] . I t h a s e v e n b e e n

s u g g e s te d t h a t t h e lo w e r i n t ra c e l l u la r b i c a r b o n a t e c o n c e n t r a t i o n ( 1 - 5 m m o l / l )

c o u l d b e a f a c to r i n t h e i n t r a c e l l u l a r r e l e a se o f g a l l i u m f r o m t r a n s f e r r i n [ 80 ].

A l t h o u g h g a l l iu m b i n d s t o t r a n s f e r r i n r a t h e r t i g h t l y i n s e r u m , G a c l e ar sf r o m t h e b l o o d m o r e q u i c k l y t h a n e i t h e r F e 3| o r I - la b e l e d a l b u m i n [ 8 5 - 8 7 ].

T h i s h a s b e e n a t t r i b u t e d t o a s m a l l b u t k i n e t i c a l l y s i g n i fi c a n t la b i le p o o l o f l o w

m o l e c u l a r w e i g h t ( L M W ) g a ll iu m . T h e c h e m i c a l c o m p o s i t i o n o f t h is L M W

p o o l i s s t il l n o t k n o w n . C i t r a t e i s a n o b v i o u s p o s s i b i l i t y a s a G a l ig a n d .

H o w e v er , s p ik in g s e r u m w i th 5 0 0 ~ tmo l/1 [8 9 ] o r ev en 1 0 0 0 ~tmo l/1 c i t r a t e [ 92 ]



134 W.R. Ha rris

removes very l i t t l e Ga f rom t r ans fe r r in . Thus i t s eems un l ike ly tha t the

100 l, mol/1 c i t r a te in no rm al s e ru m cou ld b ind a s ign i f ican t fr ac t ion o f s e rum

gal l ium. C om pu ter m odels o f ga l l ium spec ia t ion in s e rum [22 , 93] ind ica te tha t

t h e L M W p o o l c o n s i s t s p r i m a r i l y o f G a (O H ) 4 - a n d G a (O H ) 3 , w i t h o n l y t r a c e

a m o u n t s o f G a - c it ra t e c o m p le x e s. A n u m b e r o f p h o s p h a t e c o m p o u n d s a r e

m o r e e ff ec ti ve t h a n c i t ra t e a s m e d i a t o r s o f g a l l iu m e x c h a n g e b e t w e e n

t rans fe r r in and e i ther l ac to fe r r in o r f e r r i t in [77 , 79 , 80] . Thus i t i s poss ib le

t h a t p h o s p h a t e s m a y a l s o c o n t r i b u t e t o t h e p o o l o f k i n e t i c a l ly ac t iv e G a in

s e r u m .

T h e c l e a r a n c e o f g a ll i u m f r o m b l o o d i s b i e x p o n e n t i a l , c o n s i s t i n g o f tw o

r o u g h l y e q u a l c o m p o n e n t s w i t h c l e a r a n c e h a l f- li v e s o f a r o u n d 10 a n d 15 0 m i n

[85] . Organ up take i s h ighes t in bone , fo l lowed by l iver , k idney , sp leen , and

l y m p h n o d e s [9 4, 9 5]. T h e b o n e u p t a k e i s n o t t r a n s f e r r i n - d e p e n d e n t , a n d t h e

m e t a l a p p e a r s t o b e b o u n d t o t h e b o n e m i n e r a l s u r f a c e , n o t t o e r y t h r o i d c e l l sin the ma r ro w [96]. W he n re la t ive ly low doses o f Ga a re in jec ted , abou t 25% of

t h e i n j e c t e d d o s e i s e x c r e t e d t h r o u g h t h e k i d n e y s w i t h i n a f e w d a y s , b u t

i n j e c t i o n o f d o s e s t h a t e x c e e d t h e t r a n s f e r r i n b i n d i n g c a p a c i t y l e a d s t o h i g h e r

u r i n a r y e x c r e t i o n a n d k i d n e y d a m a g e [ 9 4 ] .

T h e r e h a s b e e n s o m e c o n t r o v e r s y r e g a r d i n g t h e r o le o f T f i n t h e u p t a k e o f

Ga in to so f t t i s sues and tumors . Lar son and co-worker s [72 , 97] o r ig ina l ly

p r o p o s e d t h e " t r a n s f e r r i n r e c e p t o r h y p o t h e s i s " , i n w h i c h G a f o l l o w s t h e

r e c e p t o r - m e d i a t e d p a t h w a y d e s c r i b e d i n S e c t . 1 . 1 f o r t h e c e l l u l a r u p t a k e o f

i r o n . R e c e p t o r - m e d i a t e d u p t a k e o f G a in a v a r i e t y o f t u m o r c e ll l in e s h a s b e e n

repor ted [72, 82 , 97-99] .

D a t a o n t u m o r u p t a k e o f G a i n w h o l e a n i m a l s h a s b e e n s o m e w h a t d i f f i c u l t

t o in t e r p r e t . T i s s u e a n d t u m o r u p t a k e t e n d t o d e c r e a s e i f s e r u m t r a n s f e r r i n

l ev e ls a r e lo w o r i f t h e t r a n s f e r r i n i s s a t u r a t e d w i t h i r o n , w h i c h i s c o n s i s t e n t

w i t h r e c e p t o r - m e d i a t e d u p t a k e [96 , 98 , 100] . However , i t has been a rgued tha t

i t i s a c t u a l l y L M W G a w h i c h i s t a k e n u p , a n d a p o T f e n h a n c e s t i s s u e u p t a k e

s i m p l y b y b i n d i n g G a , w h i c h r e t a r d s G a e x c r e t i o n a n d m a i n t a i n s a b u f f e r e d

f ree Ga conc en t ra t io n in the s e rum [73 , 101 , 102]. P ro po ne n ts o f th i s v iew

p o i n t o u t t h a t l o w e r i n g th e p H , w h i c h i n c r e a s e s t h e fr e e G a c o n c e n t r a t i o n [ 92 ],

t ends to enhance the ce l lu la r up take o f Ga [73 , 103] . I t has a l so been shownt h a t i n t r a c e l l u l a r G a c a n t r a v e r s e b a c k a c r o s s t h e c e l l m e m b r a n e i n t o t h e

extracel lu lar f lu id [82, 99] .

R e c e p t o r - m e d i a t e d u p t a k e o f G a h a s n o w b e e n d e m o n s t r a t e d c o n c lu s iv e l y

b y s t u d i e s w h i c h s h o w t h a t a n t i b o d i e s t o t h e t r a n s f e r r i n r e c e p t o r b l o c k G a

up take , even in the p resence o f normal l eve l s o f apoTf [82 , 99] . Bu t the re a re

c e l l l i n e s f o r w h i c h G a u p t a k e i s n e i t h e r e n h a n c e d b y a p o T f n o r b l o c k e d b y

an t ibo d ies to the t r an s fe r r in r e cep to r [82 , 101] . In add i t ion , Soh n e t al. [96]

i m p l a n t e d t w o t u m o r t y p e s i n t o h y p o t r a n s f e r r i n e m i c m i c e a n d f o u n d t h a t t h e

r e d u c e d t r a n s f e r r i n l e v el s m a r k e d l y r e d u c e d G a u p t a k e i n o n e t y p e o f t u m o r ,b u t h a d v e r y l it tl e e ff ec t o n t h e o t h e r t y p e . T h u s i t n o w s e e m s c l e a r t h a t t h e r e

a r e t w o p a t h w a y s f o r G a u p t a k e , o n e w h i c h i s t r a n s f e r r i n - d e p e n d e n t a n d o n e

w h i c h i s t r a n s f e r r i n - i n d e p e n d e n t [ 8 2 , 9 6 , 9 8 ] . W e i n e r [ 9 8 ] h a s e s t i m a t e d t h a t

i n m o s t c e l l l i n e s , t h e t r a n s f e r r i n - d e p e n d e n t p a t h w a y a c c o u n t s f o r 7 5 - 9 0 % o f

G a u p t a k e . T h e r e l a ti v e im p o r t a n c e o f t h e t w o m e c h a n i s m s f o r a n y p a r t i c u l a r




c el l t y p e m ay " d e p e n d o n f a c to r s s u c h a s t h e d e n s i t y o f t r a n s f e r r i n r e c e p t o r s i n

t h a t c e ll l in e . W e i n e r [9 8] h a s s p e c u l a t e d t h a t t h e i m p o r t a n c e o f t h e r e c e p t o r -

m e d i a t e d p a t h w a y m i g h t a l s o d e p e n d o n t h e e x t e n t t o w h i c h t h e c e l l r e l i e s o n

re d uc t io n o f Fe 3+ to Fe 2+ to re l e a se i ron f ro m Tf i n t ra c e l lu l a r ly , s i nc e G a 3+ -Tfc a n n o t b e r e d u c e d .

R e c e p t o r - m e d i a t e d c e ll u la r u p t a k e o f G a - T f a p p e a r s t o b e le s s i m p o r t a n t i n

the l oc a l i z a t ion o f G a in pu r u l e n t a bsc e s se s [98 , 104]. Ins t e a d , t h i s p ro c e ss

a p p e a r s t o d e p e n d m o r e o n t h e tr a n s f e r o f G a f r o m s e r u m t r a n s f e r ri n t o

l a c to fe r r in , w h ic h i s r e l e a se d in to t he e x t ra c e l lu l a r f l u id i n i n f l a mma tory

l e s ions [76 , 78, 104, 105]. Suc h a tr a ns fe r i s t he rm od yn a m ic a l ly fa vora b le . Th i s

w a s o r i g i n a l l y d e m o n s t r a t e d b y d i r e c t c o m p e t i t i o n s t u d i e s b e t w e e n t h e t w o

p r o t e i n s [ 78 ]. T h e h i g h e r b i n d i n g a f f i ni ty f o r l a c t o f e r ri n h a s b e e n c o n f i r m e d b y

i n d e p e n d e n t d e t e r m i n a t i o n s o f t h e b i n d i n g c o n s t a n t s o f G a w i t h b o t h

t r a n s f e r r i n a n d l a c t o f e r r i n b y d i f f e r e n c e U V s p e c t r o s c o p y [ 4 1 , 7 5 ] . T h e s es tud ie s r e por t e d l og K ~ va lue s o f 19 . 4 fo r t r a ns fe r r in a nd 21 . 4 fo r l a c to fe r r in .

T h e T f b i n d i n g o f G a 3+ i s al so m o r e s e n s it i v e t o p H t h a n t h a t o f la c t o f e rr i n , s o

t h a t l o w e r i n g t h e p H s h i f t s t h e r e l a t i v e g a l l i u m b i n d i n g a f f i n i t i e s e v e n f u r t h e r

in fa vor o f la c to fe r r in [78] .

I t h a s a l s o b e e n s u g g e s t e d t h a t g a l l i u m l o c a l i z a t i o n i n abscesses m i g h t

i n v ol v e th e b i n d i n g o f G a b y s i d e r o p h o r e s, w h i c h a r e p o w e r f u l L M W i r o n

c he la to r s e xc re t e d by the i nva d ing mic roorga n i sm [104 , 105] . I t i s c l e a r t ha t

t h e s i d e r o p h o r e s a r e c a p a b l e o f b i n d i n g G a 3+. T h e s t a b il it y c o n s t a n t o f G a w i th

t h e t r i h y d r o x a m a t e d e s f e r r i o x a m i n e B i s 1 02 8.5 [ 10 6 ], w h i c h is m o r e t h a ne n o u g h t o r e m o v e G a f r o m t r a n s f e r r i n . T h i s G a b i n d i n g c o n s t a n t is s ti ll

s u b s t a n t i a l l y s m a l l e r t h a n t h e F e 3 + - d e s f e r r i o x a m i n e s t a b i l i t y c o n s t a n t o f

1 0 3 0 " 9 9 , a nd i t is ve ry un l ike ly t ha t G a 3+ c a n d i sp l a c e Fe 3+ f ro m s ide r oph ore s .

H o w e v e r , E m e r y h a s s h o w n t h a t G a 3+ c a n e f fe c ti v el y r e p la c e i r o n u n d e r

r e d u c i n g c o n d i t i o n s , w h e r e G a - b i n d i n g t o t h e s i d e r o p h o r e f a c i l i t a t e s r e d u c t i o n

of t he Fe 3+ to t he ve ry w e a k ly bo un d Fe 1+ [107] . E m e ry a n d H o f fe r h a ve

c o n f i r m e d t h a t t h e G a c o m p l e x o f t h e s i d e r o p h o r e f e r r ic h r o m e is ta k e n u p b y

Ustilago sphaerogena a t t he sa me ra t e a s t he fe r r i c c omple x [108] .

3.2

Indium

Like G a 3+ , t he In 3+ ion ha s be e n inve s t iga t e d in t e ns ive ly be c a u se o f t he

w i d e s p r e a d i n t e r e s t i n i t s u s e i n r a d i o p h a r m a c e u t i c a l s . T w o ? - e m i t t i n g

isotop es , 11~In ( tl /2 = 2 .8 d) an d 113mln ( t1 /2 = 1 .7 h) a re of in te r es t [69] .

Th e re a re a n um be r o f c he m ic a l s im i l a r i ti e s b e tw e e n G a 3+ a nd In 3+ [74]. Bo th

are f i l led she l l d ~~ ions lack ing a ny c rys ta l f ie ld s tabi l iza t io n energ ies , a nd for

e a c h m e t a l o n l y t h e + 3 o x i d a t i o n s t a t e i s f o u n d u n d e r b i o l o g i c a l c o n d i t i o n s .

B o t h a r e e x t e n s i v e l y h y d r o l y z e d a t p h y s i o l o g i c a l p H . H o w e v e r , i t i s i m p o r t a n tt h a t I n 3+ i s m u c h l es s a m p h o t e r i c . T h e f o u r t h p K a d e s c r i b i n g t h e h y d r o l y s i s o f

In (O H )3 to In (O H ) 4 - i s 9 .7 , c om pa re d to 5 .5 fo r G a 3+ [109]. T hus a t ne u t ra l

pH , t he In 3+ ion e x i s t s i n a qu e ou s so lu t io n a lm os t e x c lus ive ly a s t he i nso lub le

In (O H )3 (K sp = 10-36"9). In th i s resp ec t In 3+ mo re c lose ly rese m bles Fe 3+ th an

G a 3+. Th e b in d in g o f u nc h e l a t e d In 3+ o r Fe 3+ to a p oT f a t ne u t ra l pH i s ve ry



136 W .R. Harris

s lo w , p r e s u m a b l y b e c a u s e o f t h e v e r y r a p i d f o r m a t i o n o f u n r e a c t i v e h y d r o x y

p o l y m e r s o f t h e m e t a l h y d r o x i d e s [ 45 ]. I n c o n t r a s t, t h e a d d i t i o n o f u n c h e l a t e d

G a 3§ to a p o T f p r o c e e d s s m o o t h l y a n d r a t h e r q u i c k l y b e c a u s e o f t h e

p r e d o m i n a n c e o f t h e s o lu b l e, m o n o m e r i c G a ( O H ) 4 a n i o n a t n e u t r a l p H [ 41 ].S t u d ie s o n t h e I n - T f b i n d i n g c o n s t a n t s h a v e l a g g e d b e h i n d t h o s e o n A1 a n d

G a . T h e i n i t i a l v a l u e s f o r t h e I n - T f b i n d i n g c o n s t a n t s o f a l m o s t 1 0 30 w e r e

r e p o r t e d b y K u l p r a t h i p a n j a e t a l. [4 2 ]. H o w e v e r , a s d i s c u s s e d a b o v e f o r th e G a -

T f b i n d i n g c o n s t a n t s , t h e s e I n - T f c o n s t a n t s a r e m u c h t o o h ig h a n d s h o u l d b e

d i s r e g a r d e d . L u r i e e t a l. [4 6] r e p o r t e d a n I n - T f b i n d i n g c o n s t a n t o f 1 0 2 4 , w h i c h

w a s d e t e r m i n e d b y u s i n g p e r t u r b e d a n g u l a r c o r r e la t i o n s p e c t r o s c o p y t o

m e a s u r e t h e d i s t r ib u t i o n o f I n b e tw e e n T f a n d a s er ie s o f b i d e n t a t e c o m p e t i ti v e

c h e l a t i n g a g e n t s .

M o r e r e c e n t l y , t h e I n - T f s y s t e m h a s b e e n s t u d i e d b y u s e o f d i f f e re n c e U V

s p e c t r o s c o p y [ 45 ]. T h e b i n d i n g o f I n 3+ t o a p o T f w a s m u c h s l o w e r t h a na n t ic i p a te d , a n d a n e w b a t c h w i s e t i t r a ti o n m e t h o d w a s d e v e l o p e d t o a ll o w ti m e

f o r e q u i l ib r a t io n . I n - T f b i n d i n g c o n s t a n t s d e t e r m i n e d b y t h es e d i f fe r en c e U V

stud ies t i t r a t ion s a re log K~ = 18 .52 an d log K~ = 16 .64 in 5 m m ol/1 HCO~-.

T h e e v a l u a t io n o f t h e se i n d i u m t r a n s f e r ri n b i n d i n g c o n s t a n t s b y u s e o f a L F E R

b e t w e e n F e 3§ a n d I n 3+ is m o r e c o m p l i c a t e d t h a n w a s t h e c a s e f o r G a 3§ T h e l o w

m o l e c u l a r w e i g h t d a t a f o r a m i n o c a r b o x y l i c a c i d s s u c h a s E D T A fa ll o n a

s e p a r at e l i n e f r o m t h e b i n d i n g c o n s t a n t s f o r l i g an d s w h i c h c o o r d i n a t e v i a

p h e n o l i c o x y g e n s [ 2 5] . T h e I n - T f v a l u e s a r e c o n s i s t e n t w i t h t h e L F E R b a s e d o n

t h e p h e n o l a t e l i g a n d s . B a s e d o n t h e c u r r e n t l y a c c e p t e d b i n d i n g c o n s t a n t f o rF e - T f a n d t h e p h e n o l a t e L F E R , o n e o b t a i n s a n e s t i m a t e o f 1 8.8 f o r th e l o g K ~

v a l u e f o r In - T f , w h i c h i s i n g o o d a g r e e m e n t w i t h t h e r e s u l ts f r o m t h e d i f fe r e n c e

U V s t u d i e s [ 4 5] .

T r a n s f e r r i n a p p e a r s t o b e t h e o n l y s ig n i fi c an t I n - b i n d i n g p r o t e i n i n s e ru m .

I f I n i s i n j e c te d e i the r a s a n a c id ic so lu t ion o r a s a w e a k c h e la t e , e .g . , I n - c i t r a t e ,

t h e n > 9 5 % b i n d s t o m a c r o m o l e c u l a r l i g a n d s , a n d e s s e n ti a ll y al l o f t h is a p p e a r s

t o b e a s s o c i a t e d w i t h t r a n s f e r r i n [ 8 8 - 9 0 , 1 1 0 - 1 1 2 ] . I n c o n t r a s t t o t h e s l o w

b i n d i n g o f m a c r o s c o p i c a m o u n t s o f In 3+ t o a p o T f w h i c h w a s o b s e r v e d i n

d i f fe r e n c e U V s t u d i e s [ 4 5] , t h e b i n d i n g o f t r a c e a m o u n t s o f I n 3§ i n p l a s m a

a p p e a r s t o b e q u i t e r a p i d [ 8 6 , 1 1 2 ] . E i t h e r t h e h i g h e r d i l u t i o n r e t a r d s

p o l y m e r i z a t io n , o r s e r u m l i g a n d s s u c h a s c it ra t e s e rv e t o d e l iv e r m o n o m e r i c I n

to Tf .

C o m p a r e d w i t h G a , t h e c l e a r a n c e o f I n - T f f r o m t h e b l o o d is r e l a ti v e l y s lo w ,

wi th a ha l f - l if e in the r a nge o f 4 - 1 0 h [ 86 , 88 , 113]. The s low r e l e a se o f I n f r o m

T f a n d s l o w b l o o d c l e a r a n c e r a t e s f o r I n m a k e i t p o s s i b l e t o u s e I n - T f a s a

m a r k e r f o r v a s c u l a r p e r m e a b i l i t y [ 86 , 8 8 , 11 0]. I f t h e p H o f t h e i n d i u m s t o c k

s o l u t i o n i s ra i s e d t o n e u t r a l o r b a s ic v a l u e s p r i o r t o i n j e c t io n , c o l l o id a l i n d i u m -

h y d r o x i d e p o l y m e r s f o r m w h i c h d o n o t d o n a t e I n to T f a n d a r e c l e ar e d f r o m

t h e s e r u m w i t h i n a b o u t 5 m i n [ 69 , 9 0 ].T h e r e a r e s e v e r a l r e p o r t s t h a t I n - T f i s m o r e s t a b l e i n s e r u m t h a n i s G a - T f

[ 86 , 8 8 - 9 0 , 1 14 ]. I n a d d i t i o n , I n - T f is m o r e s t a b l e t h a n G a - T f t o d i s s o c i a t i o n

d u r i n g u r e a / p o l y a c r y a m i d e e l e c t r o p h o r e s i s [ 11 5 ]. T h i s at f ir s t s e e m s i n c o m -

p a t i b l e w i t h t h e r e p o r t e d s t a b i l it y c o n s t a n t s o f l o g K ~ = 1 9.4 f o r G a - T f a n d

o n l y 1 8 . 5 f o r I n - T f [ 4 1 , 4 5 ] . T o u n d e r s t a n d t h e b e h a v i o r i n s e r u m , o n e m u s t




a ls o c o n s i d e r t h e i m p a c t o f m e t a l h y d r o l y s i s , w h i c h l o w e r s t h e e f f e c t i v eb in d in g c on s t a n t s a t pH 7 .4 t o l og K~ = 7 fo r G a a nd a r ou nd 10 fo r In [45 ]. I t

is th e s t r o n g e r c o m p e t i t i o n f r o m G a ( O H ) 4 r e la t iv e t o I n ( O H ) 3 w hic h l e a ds t o a

h i g h e r d e g r e e o f d i s s o c i a t i o n o f G a f r o m t r a n s f e r r i n i n v iv o .T h e c l e a r a n c e o f I n - T f f r o m b l o o d i s e v e n s lo w e r t h a n t h e c l e a ra n c e o f i r o n ,

w h i c h s u g g e s t s t h a t t h e r e c e p t o r m e d i a t e d p r o c e s s e s i n v o l v e d i n ir o n c l e a r a n c e

a re l es s e f fe c t ive fo r In [86 ] . Th e b in d in g a f f in i ti e s o f In -T f a nd Fe -T f t o t he T f

re c e p to r s on re t i c u loc y te s a re ve ry s imi l a r [116] . H ow e ve r , w he re a s i ron i s

s u c c e s s f u l l y i n t e r n a l i z e d a n d s u b s e q u e n t l y a p p e a r s i n h e m o g l o b i n i n c i r c u -

l a ti n g e r y th r o c y te s , th e I n - T f r e m a i n s m e m b r a n e b o u n d a n d a l m o s t n o I n

a p p e a r s w i t h t h e m a t u r e e r y t h r o c y t e s . I n s t e a d , t h e r e i s a s l o w t r a n s l o c a t i o n o f

I n f r o m t h e r e ti c u lo c y te m e m b r a n e t o t h e m i n e r a l s u r fa c e o f t h e n e a r b y b o n e .

T h e l a c k o f e ff e ct iv e r e c e p t o r m e d i a t e d u p t a k e o f I n m a y r e fl ec t a d i f fi c u lt y i n

d i s s o c i a t i n g I n 3 + f r o m t h e c o m p l e x b e t w e e n I n - T f a n d t h e t r a n s f e r r i n r e c e p t o r.

O t h e r s t u d i e s h a v e s h o w n t h a t I n - T f c a n b i n d t o t h e t r a n s f e r r i n r e c e p t o r s a t

t h e p l a c e n ta , b u t a g a i n i t a p p e a r s t h a t t h e r e i s n o a c t u a l tr a n s p o r t o f I n a c r o s s

t h e m e m b r a n e [ 9 4 ] . T h u s a s i d e f r o m t h e b i n d i n g t o T f , I n 3 + doe s no t c lose ly

m i m i c t h e g e n e r a l b e h a v i o r o f F e 3+ [ 1 1 7 ] .

I t h a s a l s o b e e n s u g g e s t e d t h a t t h e l a r g e r r a d i u s o f I n c h a n g e s t h e

c o n f o r m a t i o n o f I n - T f i n s u c h a w a y a s to l o w e r t h e b i n d i n g a f f in i ty o f I n - T f

w i t h t h e t r a n s f e r r i n r e c e p t o r [ 86 ]. S p e c t r o s c o p i c d a t a o n t h e e f fe c t o f t h e m e t a l

i o n i c r a d i u s o n t h e t r a n s f e r r i n c o n f o r m a t i o n a r e i n c o n s i s t e n t . S m a l l a n g l e X -

r a y s c a t t e r i n g s t u d i e s h a v e s h o w n t h a t F e 3 + a n d I n 3+ c a u s e e sse n t i a l l y t he sa med e g r e e o f c l o s u r e o f t h e i n t e r d o m a i n c le ft i n t h e p r o t e i n i n w h i c h t h e m e t a l

b i n d i n g s i t e r e s i d e s [ 1 1 8 ] . I n c o n t r a s t , c i r c u l a r d i c h r o i s m s p e c t r a i n t h e

a r o m a t i c r e g i o n s u g g e s t t h a t i n d i u m i n d u c e s a c o n f o r m a t i o n d i f f e r e n t f r o m

tha t for Ga 3+ an d A13+ [ 1 1 9 ] .

D e s p i te t h e a p p a r e n t p r o b l e m s a s s o c ia t e d w i t h r e c e p to r m e d i a t e d u p t a k e o f

I n , I n s ti ll t e n d s t o l o c a li z e in t i s s u e s w h i c h h a v e l a r g e n u m b e r s o f tr a n s f e r r i n

r e c e p t o r s [ 12 0 ]. H o w e v e r , I n - T f h a s n o t p r o v e n t o b e a n e f fe c ti v e a g e n t f o r

d e l i v e r in g I n t o tu m o r s . A n u m b e r o f I n - c h e l a t e s h a v e b e e n t e s t e d f o r

se l e c tive ly de l ive r ing In t o va r iou s t i s sue s [69 , 121] . M uc h o f the re c e n t

e m p h a s i s h a s b e e n o n c o v a le n tl y b i n d i n g I n c h e la te s t o m o n o c l o n a l a n t i b o d i e s

fo r more se l e c t i ve de l ive ry t o t umor c e l l s [122] . In t he se a pp l i c a t i ons one t a ke s

a d v a n t a g e o f t h e s lo w e r l i g a n d e x c h a n g e k i n e t ic s o f I n to h e l p p r e v e n t

d i s so c i a t i on o f the l a be l i n v ivo .

3.3

Aluminum

T h e r e i s c u r r e n t l y a g r e a t d e a l o f i n t e r e s t i n t h e b i o lo g i c a l c h e m i s t r y o f AI 3 + ,

a n d n u m e r o u s r e c e n t r e v i e w s a n d b o o k s a r e a v a i l a b l e [ 9 4 , 1 2 3 - 1 2 8 ] .A l u m i n u m w a s l o n g c o n s i d e r e d t o b e r e l a t i v e l y n o n - t o x i c , p r i m a r i l y b e c a u s e

the i n t e s t i na l ba r r i e r t o t he a b sor p t io n o f A I f ro m the gu t i s qu i t e e f fe c t ive [94,

123 , 125, 129]. H ow e ve r , A 1 tha t d oe s e n t e r t he b lo od c a n c a use ne u ro log ic a l

e f fe c t s , o s t e oma la c i a , a nd a ne mia [94 , 1 2 5 ] . A l u m i n u m t o x i c i t y h a s b e e n

d e m o n s t r a t e d m o s t d r a m a t i c al l y in t h e f o r m o f d ia ly s is d e m e n t i a , w h e r e



138 W.R. Ha rris

neuro log ica l impai rment i s observed in long te rm d ia lys i s pa t ien t s [125 , 130] .

T h e c o n d i t i o n a r i se s f r o m t h e t r a n s f e r o f t o x ic a m o u n t s o f f re e A1 f r o m t h e

d ia lysi s so lu t ion in to s e ru m due to the r e la t ive ly h igh b ind ing a f f in i ty o f s e ru m

l ig a n d s , e s p ec i a ll y t r a n s f e r r i n . A l u m i n u m n e u r o t o x i c i t y h a s a l so b e e n o b s e r v e d

i n p a t i e n t s o n t o t a l p a r e n t e r a l n u t r i t i o n [ 1 3 1 ] , w h e r e o n c e a g a i n t h e n o r m a l

p r o t e c t io n s a g a i n s t in t e s ti n a l a l u m i n u m u p t a k e a r e c i rc u m v e n t e d . A l u m i n u m -

r e l a t e d e n c e p h a l o p a t h y h a s e v e n b e e n o b s e r v e d i n n o n - d i a l y s i s p a t i e n t s ,

e s p e c i a l l y c h i l d r e n , w h o a r e t a k i n g h i g h d o s e s o f o r a l a l u m i n u m d r u g s a s

p h o s p h a t e - b i n d e r s [ 1 32 - 1 34 ] . T h e r e is e v e n s o m e c o n c e r n t h a t i n f a n ts m a y b e

expose d to h az ard ou s l eve ls o f A1 f rom cer ta in types o f in fan t fo rm ula [135] .

T h e r e h a s a l s o b e e n m u c h d i s c u s s i o n r e g a r d i n g a p o s s i b l e l i n k b e t w e e n t h e

dep os i t ion o f A1 in the b ra in and Alz he im er ' s d i sease [136-141] . Th is

a s s o c i a t i o n w a s i n i t i a l l y p r o m p t e d b y t h e f i n d i n g s t h a t t h e b r a i n s o f

A l z h e i m e r ' s p a t i e n t s h a d u n u s u a l l y h i g h A1 c o n c e n t r a t i o n s [1 42 ] a n d t h a tt h e A 1 a p p e a r e d t o b e l o c a li z e d i n t h e s p e c if ic r e g i o n s o f t h e b r a i n d a m a g e d b y

A l z h e i m e r ' s d i s e a s e [ 1 4 3 ] . T h e r e i s e v e n s o m e e v i d e n c e t h a t t r e a t m e n t o f

A l z h e i m e r ' s p a t i e n t s w i t h A 1 c h e l a t i n g d r u g s s lo w s t h e p r o g r e s s i o n o f t h e

d isease [144]. Ho wever , the pa th o log y o f A l - in duc ed ne urof ib r i l l a ry t ang les is

d i f f e ren t f rom the l es ions s een in Alzhe imer ' s d i sease [94 , 137] , and the

i m p o r t a n c e o f A1 a s a c a u s a t i v e f a c t o r i n A l z h e i m e r ' s d i s e a se h a s b e e n

ser io us ly cha l leng ed [94, 125, 137] .

T h e b i n d i n g o f A1 t o a p o T f h a s b e e n s t u d i e d b y a v a r i e t y o f s p e c t r o s c o p i c

m eth od s , inc lud ing 1H NM R [145-147] , 27A1 NM R [23, 55 , 148-150] , 13C NM R[55, 63] , c i rcular d ichroism [147, 151] , f luorescence [151] , and dif ference UV

spec t rosco py [36 , 38-40 , 65]. Bo th d i f f e rence UV and 27A1 NM R in d ica te tha t

b in d in g leve ls o ff p r io r to s a tu ra t ion o f bo th the T f b in d in g s i t es [23, 38] .

I n v e s t i g a t o r s o f t e n i g n o r e t h i s f a c t a n d a s s u m e t h a t a d d i n g t w o e q u i v a l e n t s o f

A13+ to ap oT f wi l l s a tu ra te b o th the t r a ns fe r r in b ind ing s i tes .

H a r r i s a n d S h e l d o n [ 3 8 ] h a v e c o n d u c t e d d i f fe r e n c e U V t i tr a t i o n s o f b o t h

f o r m s o f m o n o f e r r i c T f w i t h A1 a n d d e t e r m i n e d t h a t a t p H 7 .4 t h e A1 b i n d i n g i s

s t ron ger a t the C - te rmina l s it e . H igh reso lu t ion 1H and 13C NM R s tud ies ha ve

ind ica ted p re fe ren t ia l b ind ing to the N- te rmina l s i t e a t pH 8 .8 [23 , 146] . I t has

b e e n s u g g e s t e d t h a t t h e N M R d a t a m i g h t r e f l e c t a k i n e t i c p r e f e r e n c e f o r

b i n d i n g a t t h e N - t e r m i n a l s it e , a s h a s b e e n o b s e r v e d f o r th e b i n d i n g o f f e r ro u s

ion [37] . How ever , A1 equ i l ib ra tes r e la t ive ly qu ick ly w i th T f , so k ine t ic con t ro l

o f the A1 d i s t r ib u t io n i s un l ike ly . S tud ies on f e r r ic ion ind ica te th a t the r e la t ive

b i n d i n g a f fi n it ie s o f th e t w o T f b i n d i n g s it e s a re s e n s i t iv e t o b o t h p H a n d s a lt

c o n c e n t r a t i o n s [1 52 , 1 53 ]. T h e a p p a r e n t d i s c r e p a n c y b e t w e e n t h e d i f f e re n c e

U V a n d N M R d a t a o n A1 b i n d i n g m a y w e ll b e d u e t o v a r i a t i o n s i n e x p e r i m e n t a l

c o n d i t i o n s .

A 1 -T f b i n d i n g c o n s t a n t s h a v e b e e n d e t e r m i n e d b y s e v e r a l g r o u p s , a n d

log K~ gen eral ly fal ls in the ran ge 1012-1014. Based o n d if ference UV t i t ra t ion s ,Ha r r i s and She ldo n [38] h ave r ep or te d va lues o f log K~ = 13 .5 and

log K~ = 12 .5 fo r pH 7.4 and 5 mmol /1 b ica rb on a te a t 25 ~ M ar t in e t al.

[36] hav e r ep or te d som ew hat lower va lues o f log K~ ---- 12 .2 a nd

log K~ = 11 .6 . The se para t ion b e tw een log K~ an d log K~ in these s tud ies i s

in the r ange tha t has been observed fo r o ther meta l ions [21 , 22] . In con t ras t ,



Binding and T ransport of Nonferrous Meta ls 139

Co c hra n e t al. [40 ] r e po r t e d t ha t t he tw o s i t e s ha ve e s se n t i a l l y i de n t i c a l b in d in g

c on s t a n t s o f 3 x 10 ~s a t 37 ~ In t h i s p a pe r b i c a rbo na te w a s e xp l i c it l y

i n c l u d e d a s a re a c t a n t i n t h e e q u i l i b r i u m e x p r e s s i o n s . I f t h e s e c o n s t a n t s a r e

re fo rm ula t e d to c onfo rm to Eqs . (1 ) a nd (2 ), t he n log K ~ = log K ~ = 13 .7 fo r1 8 m m o l /1 b i c a r b o n a t e . T h e v a r i a t i o n b e t w e e n t h e r e s u l ts f r o m 2 5 a n d 3 7 ~

r a i s e s t h e p o s s i b i l i t y t h a t t h e s i t e s e l e c t i v i t y m a y v a r y w i t h t e m p e r a t u r e , b u t

a d d i t i o n a l s t u d i e s a r e n e e d e d t o c o n f i r m t h i s o b s e r v a t i o n .

Fa te m i e t al . [39] rep or t va lu es o f log K~ -- 12 .23 an d log K~ = 11.76 for

2 5 m m o l /1 b i c a r b o n a t e . H o w e v e r , t h e t i t ra t i o n c u r v e r e p o r t e d b y F a t e m i e t al.

i s c l e a r ly s igmoida l , i n c on t ra s t t o t he hype rbo l i c c u rve e xpe c t e d fo r t h i s

s y s t e m . F u r t h e r m o r e , t h e d a t a a r e a n a l y z e d b y s i m p l e H i l l p l o t s t h a t t a c i t l y

a s sume d i f fe re n t mola r a bso rp t iv i t i e s fo r t he tw o s i t e s a nd fa i l t o t a ke i n to

a c c o u n t i n c o m p l e t e s a t u r a t i o n o f t h e p r o t ei n .

A t t e m p t s t o c h a r a c t e r i z e t h e p r o t e i n b i n d i n g o f A1 i n s e r u m h a v e b e e ni n c o n s i s t e n t a n d c o n f u s i n g . T h e r e a r e s e v e ra l c o m p l i c a t i n g f a c t o rs . S i n ce t h e r e

is n o c o n v e n i e n t r a d i o i s o t o p e f o r A1, s e r u m f r a c t i o n a t i o n s t u d i e s h a v e b e e n

c o n d u c t e d u s i n g c o l d A1. A l u m i n u m c o n t a m i n a t i o n is s o u b i q u i t o u s t h a t it is

v e r y d i ff ic u lt t o m e a s u r e l o w A1 c o n c e n t r a t i o n s a c c u r a t e l y [ 15 4 ]. A l u m i n u m

r e c o v e r i e s f r o m s i z e e x c l u s i o n c o l u m n s a r e o f t e n h i g h l y v a r i a b l e d u e t o t h e

e x c h a n g e o f A1 b e t w e e n t h e c o l u m n a n d s a m p l e [ 1 5 5 - 1 5 9 ]. T h e a n a l y si s o f

c h r o m a t o g r a p h i c d a t a is fu r t h e r c o m p l i c a te d b y t h e a b s e n c e o f b i c a r b o n a t e i n

s o m e o f t h e e l u t i n g b u f fe r s , w h i c h w e a k e n s A 1 -T f b i n d i n g a n d e x a c e r b a t e s t h e

p r o b l e m o f d is s o c ia t io n o f t h e T f c o m p l e x o n t h e c o l u m n [1 60 ].S e v e ra l g r o u p s h a v e n o w c o n c l u d e d t h a t T f i s t h e o n l y m a j o r A l - b i n d i n g

p r o t e i n i n s e r u m [ 1 5 6 - 1 5 8 , 1 6 1 - 1 6 4 ] . T h e s t r o n g e s t d a t a o n t h i s p o i n t c o m e

f r o m i o n e x c h a n g e H P L C a n d i m m u n o a f f i n i t y c h r o m a t o g r a p h y [1 56 , 1 5 7 , 1 63 ,

1 64 ]. E a r ly r e p o r t s o f A l - a l b u m i n b i n d i n g i n s e r u m [1 60 , 1 6 5, 1 6 6 ] b a s e d o n

s i z e e x c l u s i o n c h r o m a t o g r a p h y s h o u l d b e d i s c o u n t e d , s i n c e t h e r e w a s v e r y

p o o r r e s o l u ti o n b e t w e e n T f a n d a l b u m i n f o r th e c o l u m n s t h a t w e r e u s e d [3 6,

1 25 ]. A t t e m p t s t o m e a s u r e A l - a l b u m i n b i n d i n g d i r e c tl y in v i t ro h a v e a l s o b e e n

s o m e w h a t c o n f u s i n g . F a t e m i e t a l . [ 3 9 ] r e p o r t e d t h a t a l b u m i n c o m p e t e s w i t h

t r a n s f e r r i n f o r A1 a t p h y s i o lo g i c a l c o n c e n t r a t i o n s , a n d w e a k b i n d i n g o f AI t o

a lb um in h a s be e n d e t e c t e d b y 27A 1 N M R [148] . H ow e ve r , C oc h ra n e t al. [158]

d e t e c t e d n o A l - a l b u m i n b i n d i n g b y e q u i l i b r i u m d i a l y s i s , a n d o t h e r s h a v e

p o i n t e d o u t t h a t , b a s e d o n s t a b il it y c o n s t a n t s o f a l b u m i n w i t h o t h e r h a r d m e t a l

i o n s s u c h a s C a 2§ a n d G d 3§ o n e w o u l d p r e d i c t t h a t a l b u m i n w o u l d b i n d A 13§

ve ry w e a k ly [36 , 125] . Whi l e one ma y be a b l e t o de t e c t w e a k , non-spe c i f i c A 1

b i n d i n g u n d e r c e r t a i n c o n d i t i o n s , i t i s h i g h l y u n l i k e l y t h a t a l b u m i n c a n

c o m p e t e w i t h T f , c i tr a te , a n d p h o s p h a t e i n s e r u m f o r t h e b i n d i n g o f a

s ign i f i c a n t f r a c t i on o f A1 in v ivo .

T h e i n f u s i o n i n t o s e r u m o f t h e s i d e r o p h o r e d e s f e r r io x a m i n e B ( D F O ), w h i ch

i s a l so a pow e r fu l A l -c he l a t i ng a ge n t , r e su l t s i n a l a rge i nc re a se i n t he f ra c t i onof u l t r a f i l t e ra b l e o r l ow mole c u la r w e igh t A 1 . Mos t i nve s t i ga to r s a t t r i bu t e t h i s

t o t h e f o r m a t i o n o f t h e A 13+ c o m p l e x o f d e s f e r r i o x a m i n e [1 57 , 1 61 , 1 6 3 , 1 6 7 -

1 71 ]. O t h e r s a t t r i b u t e t h i s n e w p e a k t o n e w , r e la t iv e l y s m a l l a l u m i n u m - b i n d i n g

p r o t e i n s [ 1 7 2 - 1 7 6 ] . O n e g r o u p h a s p r o p o s e d a n e w p r o t e i n , n a m e d a l b i n d i n ,

w i t h a M W o f a r o u n d 1 4,0 00 [ 1 72 , 1 74 ]. A n o t h e r g r o u p h a s p r o p o s e d a



140 W.R. Harr is

s o m e w h a t s m a l l e r ( a r o u n d 8 00 0 M W ) M - b i n d i n g p r o t e i n [1 73 , 17 5]. I t h a s

b e e n s u g g e s t e d th a t t h e s e p r o t e i n s a r e s y n t h e s i z e d a s a m e c h a n i s m t o d e t o x if y

a l u m i n u m [1 72 , 1 75 ]. H o w e v e r , n e i t h e r o f t h e s e p r o t e i n s h a s b e e n i s o l a t e d a n d

a d e q u a t e l y c h a r a c t e r i z e d , a n d i t s e e m s m o r e l i k e l y t h a t t h e i n c r e a s e i n

u l t r a f i lt e rab le A1 as soc ia ted wi th the adm in is t r a t io n o f DFO resu l t s f rom the

s i m p l e c h e l a t i o n o f A1 b y D F O . T r a n s f e r r i n r e m a i n s t h e o n l y s e r u m p r o t e i n

tha t i s c lea r ly show n to b in d A1 in v ivo .

T h e m o s t r e li ab l e d a t a o n t h e f r a c ti o n o f L M W a l u m i n u m i n s e r u m c o m e

f rom u l t r a f i l t r a t ion s tud ies [156, 167 , 168 , 177-180]. In no rm al s e rum , 15% of

t h e A1 i s u lt r a fi lt e r ab l e , a n d t h e r e m a i n i n g 8 5 % i s p r e s u m a b l y p r o t e i n - b o u n d .

The f r ac t ion o f u l t r a f i l te rab le A1 r i s es to 25% in u rem ic se rum . T he bas i s fo r

t h i s i n c r e a s e i s n o t f i r m l y e s t a b l i s h e d . A c o m p u t e r m o d e l f o r t h e e q u i l i b r i u m

s p e c i a t i o n o f A1 i n s e r u m h a s b e e n d e v e l o p e d w h i c h c a l c u l a t e s th a t t h e f r a c t i o n

o f L M W A1 i s 1 9% f o r n o r m a l s e r u m a n d 3 0 % f o r u r e m i c s e r u m , i n g o o dagre em ent w i th the u l t r a f i l t r a t ion d a ta [181 ]. In th i s mod el , the on ly d i f f e rence

b e t w e e n n o r m a l a n d u r e m i c s e r u m i s a n i n c r e a s e i n t h e c o n c e n t r a t i o n o f

inorg an ic p ho sph a te f rom 1.1 mm ol /1 fo r no rm al s e ru m to 1 .8 mm ol /1 fo r

u r e m i c s e r u m , a n d p h o s p h a t e - A 1 b i n d i n g a c c o u n t s f o r t h e d e c r e a s e i n t h efract ion of A1-Tf .

S e v e ra l o t h e r A 1 s p e c i a t i o n m o d e l s h a v e b e e n r e p o r t e d [ 1 82 - 1 84 ] . T h e

m a j o r v a r i a t i o n a m o n g t h e m o d e l s i s in t h e s e t o f A1 b i n d i n g c o n s t a n t s u s e d

f o r p h o s p h a t e a n d c i tr a te . T h e M - p h o s p h a t e b i n d i n g c o n s t a n t s a r e p a r ti c u l a rl y

d i f f i c u l t t o m e a s u r e a n d r e p r e s e n t t h e g r e a t e s t u n c e r t a i n t y i n t h e m o d e l s . I nthe Har r i s model [181] , the p r imary LMW spec ies i s [AI (PO4) (OH)] - . O ther s

hav e sug ges ted the c i t r a te com plex [Al (H~cta ) (OH)] 2 - [124 , 178 , 183] and the

b in uc lear ph os ph a te com plex [A12(PO4) (OH)2] + [182] as the m ajo r LM W

s p e c ie s . M o r e e x p e r i m e n t a l d a t a o n A1 c o m p l e x a t i o n b y p h o s p h a t e a n d c i t r a te

a re needed to r eso lve th i s i s sue .

I t ap pea r s tha t A13§ i s de l ivered to ce ll s by A1-Tf v ia the r ece p to r -m ed ia ted

i r o n u p t a k e p a t h w a y , a n d t h a t t h i s h a s a s ig n i f ic a n t i m p a c t o n i ts to x i ci ty . T h e

A 1 -T f c o m p l e x b i n d s t o t h e t r a n s f e r r i n r e c e p t o r , a l t h o u g h n o t a s s t ro n g l y a s F e -27 3+Tf [185-187] . T he A1 NM R sp ec tru m in dic ates th at A1 , l ike Fe 3+, is s ix-

c o o r d i n a t e i n t h e t r a n s f e r r i n c o m p l e x [2 3]. H o w e v e r , X - r a y s c a t te r i n g s t u d i e s

s u g g e s t t h a t t h e b i n d i n g o f A13+ c a u s e s a s m a l l e r c o n f o r m a t i o n a l c h a n g e t h a n

d o e s t h e b i n d i n g o f i r o n [1 18 ], a n d t h i s c h a n g e i n t h e p r o t e i n c o n f o r m a t i o n

may adver se ly a f fec t r ecep to r b ind ing o f A1-Tf .

D e s p i t e t h e w e a k e r b i n d i n g o f A 1-T f t o t h e T f r e c e p t o r , t h e a d d i t i o n o f A1 -T f

r a t h e r t h a n s i m p l e i n o r g a n i c A 1 s a lt s s ig n i f ic a n t ly e n h a n c e s t h e i n h i b i t i o n o f

ce ll g row th a nd d iv i s ion [188-191]. T he a dd i t io n o f A1-Tf a l so in te r fe res w i th

the ce l lu la r up t ake o f iron f rom Fe-Tf [187 , 192 , 193]. There i s ev idenc e tha t

t h e i n t e r fe r e n c e w i t h i r o n u p t a k e i n v o l v e s t h e d o w n r e g u l a t i o n o f t h e n u m b e r

of t r an s fe r r in r ecep to r s [187] .Because o f the neuro tox ic i ty o f A1 , i t i s pa r t i cu la r ly s ign i f ican t tha t the

p a s s a g e o f A1 a c r o s s t h e b l o o d - b r a i n b a r r i e r a p p e a r s t o b e m e d i a t e d b y

t ran s fe r r in r e cep to r s [185, 194-196] . T here i s r e la tive ly l i tt l e d i f f e rence in the

b i n d i n g o f F e -T f o r A 1 -T f t o t h e t r a n s f e r r i n r e c e p t o r s i n n o r m a l o r A l z h e i m e r ' s

b ra in ce ll s [185], an d the r eg iona l d i s t r ib u t ion o f A1 wi th in the b ra in m atch es



Binding and Transport of Nonferrous Metals 1 4 1

the d i s t r ib u t io n o f T f r ecep to r s [197] . There i s a lso a study , based on the

u p t a k e o f G a a s a m o d e l f o r A1, w h i c h s u g g e s t s t h a t t h is r e c e p t o r m e d i a t e d

m o v e m e n t a c r o s s t h e b l o o d b r a i n b a r r i e r i s u n i d i r e c t i o n a l , l e a d i n g t o t h e

g r a d u a l a c c u m u l a t i o n o f A1 i n t h e b r a i n a t a ra t e h i g h e n o u g h t o a c c o u n t f o rthe A1 foun d in the b ra ins o f A lz he im er ' s v ic t ims [194] .

Th ere h ave be en severa l s tud ies in w hich the e f fec ts o f A1 tox ic i ty have bee n

s u b s t a n t i a l l y r e v e r s e d b y r e l a t i v e l y s i m p l e c h e l a t i o n t h e r a p y w i t h t h e

s i d e r o p h o r e D F O [1 26 , 1 69 ]. T r e a t m e n t w i t h D F O s u b s t a n t i a l l y r e d u c e s t h e

b o d y b u r d e n o f A1 a n d i s q u i t e e f fe c ti v e f o r r e v e r s i n g b o t h t h e n e u r o t o x i t y a n d

o s t e o m a l a c i a a s s o c i a t e d w i t h l o n g t e r m d i a l y s i s p a t i e n t s . I n f a c t , A l - i n d u c e d

a n e m i a i n d ia l y si s p a t i e n t s c a n s o m e t i m e s b e r e v e r s e d s im p l y b y p r e p a r i n g t h e

d ia lysa te wi th de ion ized wate r [198] .

T h e e ff ic a cy o f D F O t r e a t m e n t i s p r e s u m a b l y r e l a te d t o i t s h i g h A1 b i n d i n g

c o n s t a n t . C o m p u t e r s i m u l a t i o n s s h o w c l e a r l y t h a t D F O s h o u l d b e a b l e t or e m o v e e s s e n t ia l l y a ll th e A 1 f r o m s e r u m t r a n s f e r r i n u n d e r i n v iv o c o n d i t i o n s

[38] . The aminocarboxy la te l igand EDTA a l so has a h igher e f fec t ive b ind ing

co ns ta n t th an T f, bu t i t is mu ch les s e f fec t ive than DFO in t r ea t ing A1 tox ic i ty

[199]. Th is r e f lec t s the d i f f e rence in the s e lec t iv ity o f these che la t ing agen ts fo r

A1 o v e r th e o t h e r m e t a l s p r e s e n t i n s e r u m . T h e c o m p u t e r s i m u l a t i o n s s h o w

t h a t A l - c h e l a t i o n i n v i v o b y D F O s h o u l d b e r e l a t i v e l y u n a f f e c t e d b y

i n t e r f e r e n c e f r o m s e r u m C a 2| an d Zn 2* [38]. In co ntra s t , th e s im ula t ion s

s h o w t h a t E D T A w o u l d b e a l m o s t c o m p l e t e l y s a t u r a t e d w i t h z i n c a n d c a l c i u m

a n d w o u l d b e u n a b l e t o b i n d s i g n if i c a n t a m o u n t s o f A1.

3 .4

P l u t o n i u m

T h e s e r u m t r a n s p o r t o f th e a c t i n i d e s h a s b e e n e x t e n s i v e ly s tu d i e d i n r e l a ti o n t o

t h e h e a l t h h a z a r d s a s s o c i a t e d w i t h v a r i o u s e l e m e n t s i n t h e n u c l e a r f u e l c y c l e .

I n g e n e r a l , t h e e a r l y a c t i n i d e s ( T h t h r o u g h P u ) a r e d i s t i n g u i s h e d b y a v e r y h i g h

b io log ica l hazard as soc ia ted wi th 0c-emi t t ing i so topes which have very long

phys ica l ha l f - l ives [200] . The g rea tes t a t t en t ion has been focused on

p l u t o n i u m , w h i c h i s p r o d u c e d i n s i g n i f i c a n t a m o u n t s d u r i n g r e a c t o r o p e r a -

t i o n s b y t h e r e a c t i o n

n , ~ / ~ - / ~ -238u ) 239 u ~ 239Np ~ 239pu (9)

Th e 239pu, an 0c-emit ter wi th a phy s ical half - l i fe of 24,400 years , i s extrem ely

t o xi c. A l t h o u g h a f ew k g o f 2 39 pu c a n b e f o u n d i n n a t u r a l u r a n i u m o r e s, t h e

a m o u n t s a r e i n c o n s e q u e n t i a l c o m p a r e d t o t h e m o r e t h a n 5 00 0 k g o f

a n t h r o p o g e n i c 2 39 pu f r o m w e a p o n s a n d o t h e r s o u r c e s t h a t h a v e a l r e a d y b e e n

r e l e a s e d i n t o t h e e n v i r o n m e n t [ 2 0 1 ] .P l u t o n i u m c a n b e f o u n d i n e v e r y o x i d a t i o n s t a t e f r o m + 3 t o + 7 [ 2 0 0 , 2 0 1 ] ,

b u t u n d e r b i o l o g i c a l c o n d i t i o n s t h e s t a b l e o x i d a t i o n s t a te i s + 4 . T h e P u 4+ i o n

h a s a c h a r g e / r a d i u s r a t i o v e r y s i m i l a r t o F e3+ , so i t i s no t su rp r i s ing tha t a

n u m b e r o f b i o lo g i c a l s t u d i e s h a v e s h o w n t h a t P u i s b o u n d i n th e b l o o d a n d

t r a n s p o r t e d b y T f [ 49 , 2 0 2 - 2 0 7] . B i c a r b o n a t e i s r e q u i r e d a s a sy n e r g i s t ic a n i o n



142 W.R. Harris

f o r f o r m a t i o n o f a s t a b le P u - T f c o m p l e x [ 20 4 ], a n d P u b i n d i n g is b l o c k e d b y

the a dd i t i on o f f e r r ic i on [203, 204]. Thus t he re i s l i tt le do ub t t ha t Pu i s

b i n d i n g a t t h e h i g h - a f f in i t y i r o n b i n d i n g s it e s o f t h e p r o t e i n .

Be c a use t he re a re no s t a b l e i so tope s o f Pu , i t i s ve ry d i f f i c u lt t o c on du c t t he

u s u a l ch e m i c a l st u d ie s o f p r o t e i n b i n d i n g t h a t r e q u i r e m a c r o s c o p i c a m o u n t s o f

t h e m e t a l i o n . N o n e t h e l e s s , t h e b i n d i n g o f P u 4+ t o a p o T f h a s b e e n f o ll o w e d b y

d i f fe r e n c e U V s p e c t r o s c o p y [ 49 , 2 0 8 ]. T h e c h a r a c t e r is t i c s p e c t r u m o f a m e t a l -

T f c o m p l e x is o b s e r v e d , w h i c h f u r t h e r c o n f i r m s t h a t P u 4+ b i n d s a t t h e i r o n

b i n d i n g s i te . A b i n d i n g c o n s t a n t o f l o g K~ = 2 1 .2 h a s b e e n r e p o r t e d f o r P u - T f

[209] . Th i s i s sl i gh t ly h ig he r t ha n the Fe -T f b in d in g c ons t a n t . S inc e it i s kn ow n

tha t Fe 3§ re a d i ly d i sp l a c e s Pu 4+ f rom t ra n s fe r r in [203 , 204] a nd tha t D TP A ,

c i t r a t e , a n d o t h e r c h e l a t o r s r a t h e r e a s i l y r e m o v e P u f r o m s e r u m t r a n s f e r r i n

[2 03 , 2 0 4, 21 0 ], th i s h i g h P u - T f b i n d i n g c o n s t a n t s h o u l d b e v i e w e d a s

p r e l i m i n a r y .T h e p e r c e n t a g e o f s e r u m P u r e p o r t e d t o b e b o u n d t o T f v a r ie s f r o m 8 0 t o

100% [49 , 204 , 206 , 207] . A k ine t i c s mode l fo r p lu ton ium de pos i t i on ind i c a t e s

t h a t t he L M W f r a ct io n o f P u c l ea r s f ro m t h e b l o o d m o r e r a p i d ly t h a n t h e P u

w h i c h i s b o u n d t o T f, so t h a t t h e f r a c t i o n o f P u - T f t e n d s t o i n c r e a s e w i t h t i m e

a n d r e p r e s e n t s > 9 0 % o f s e r u m P u a n y t i m e a f t er 2 h p o s t - i n j e c t io n [ 21 1 ].

A l t h o u g h t h e r e a r e s o m e e a r l y r e p o r t s o f P u a l so b i n d i n g t o a l b u m i n [2 05 ] a n d

t o a n 0 ~ - m a c r o g l o b u l i n [ 2 0 3 , 2 0 4 ] , t h e c h r o m a t o g r a p h y p e a k s i n t h e s e s t u d i e s

p r o b a b l y r ef le ct t h e f o r m a t i o n o f h i g h m o l e c u l a r w e i g h t P u h y d r o x i d e

p o l y m e r s , r a t h e r t h a n a d d i t i o n a l P u - p r o t e i n c o m p l e x e s . M o r e r e c e n t s t u d i e su s i n g i o n e x c h a n g e a n d a ff in i ty c h r o m a t o g r a p h y c o n s i st e n tl y s h o w t h a t T f is

t he on ly s ign i f i c a n t p ro t e in l i ga nd fo r Pu in se rum [49 , 206 , 207] .

A d d i t i o n a l i n s ig h t i n t o P u - T f b i n d i n g c a n b e g a i n e d f r o m t h e s t u d y o f th e

t e t ra v a l e n t m e ta l i ons Th 4+ a nd H f ~+. The m e ta b o l i s m o f T h 4§ i s g e ne ra l l y

s i m i l a r t o t h a t o f P u 4§ [ 21 2 ]. T h i s s i m i l a r it y e x t e n d s t o t h e c h r o m a t o g r a p h i c

p r o fi le s o f t h e se t w o m e t a l i o n s i n s e r u m , b o t h o f w h i c h s h o w o n e m a j o r b a n d

a s s o c i a t e d w i t h T f [ 21 3 ]. T h e i n v i t r o b i n d i n g o f T h 4+ t o a p o T f h a s a ls o b e e n

s t u d i e d b y d i f fe r e n c e U V s p e c t r o s c o p y [ 21 4 ]. T h e T h 4§ t it r a t i o n c u r v e w a s

l i n e a r o v e r t h e a d d i t i o n o f th e f i r s t e q u i v a l e n t o f m e t a l i o n , w i t h a s lo p e

c o r r e s p o n d i n g t o a m o l a r a b s o r p t i v it y o f a b o u t 1 6,0 00 l / m o l c m -1 p e r b o u n d

m e t a l i o n . T h e t i t r a t i o n c u r v e l e v e le d o f f a t o n l y 2 4,0 0 0 l/ m o l c m - ' , a n d t h is

w a s o r i g in a l l y i n t e r p r e t e d a s a n i n d i c a t i o n o f n o n e q u i v a l e n t b i n d i n g s it es , w i t h

t w o t y r o s i n e i n v o l v e d i n t h e s t r o n g e r s i t e a n d o n l y o n e t y r o s i n e b o u n d t o t h e

T h i n t h e w e a k e r s i te [ 2 1 4] . H o w e v e r , g iv e n t h e n u m b e r o f s u b s e q u e n t s t u d i e s

w h i c h h a v e s h o w s a t u r a t i o n o f t h e d i f fe r e n c e U V t i t r a ti o n s a t l es s t h a n t w o

me ta l i ons pe r t r a ns fe r r in mole c u le [29 , 38 , 41 , 50 , 215] , i t now a ppe a rs muc h

m o r e l i k e l y t h a t t w o t y r o s i n e s b i n d t o T h a t b o t h t r a n s f e r r i n b i n d i n g s i t e s , b u t

t h a t h y d r o l y s i s o f t h e T h l i m i t s t h e d e g r e e o f s a t u r a t i o n o f th e w e a k e r s i te t o

a b o u t 5 0 % . S i n c e P u 4+ i s e v e n m o r e p r o n e t o h y d r o l y s i s t h a n is T h 4+, t h e T hd a t a p r o b a b l y r e p r e s e n t a n u p p e r l i m i t o n t h e b i n d i n g o f P u 4+ t o a p o T f .

T h e b i o d i s t r i b u t i o n o f H f 4+ a l so r e s e m b l e s t h a t o f P u 4§ [ 2 16 ], a n d > 9 0 % o f

s e r u m H f is b o u n d i n v iv o t o t r a n s f e r r i n [ 21 6 , 2 1 7] . O n e s i g n i fi c a n t a d v a n t a g e

t o u s i n g H f 4§ a s a m o d e l f o r P u i s t h a t t h e b i n d i n g o f 1 8~ Hf4 + t o a p o T f c a n b e

s t u d i e d b y p e r t u r b e d a n g u l a r c o r r e l a t i o n s p e c t r o s c o p y ( P A C ) [ 2 0 9 , 2 1 7 , 2 1 8 ] .



Binding and Transport of Nonferrous Metals 143

I n th i s m e t h o d o n e e v a l u a te s t h e r e la t i o n s h i p b e t w e e n t h e d i r e c ti o n o f t w o

suc c e ss ive 7 ph o to ns e m i t t e d d ur i ng the de c a y o f 181H f to l a lTa [217] . A

c o m p l e x a n a l y s is o f t h e d a t a p r o v i d e s i n f o r m a t i o n r e l a te d t o t h e lo c a l

e l ec t ro n i c e n v i r o n m e n t a r o u n d t h e H f a n d t h e r o t a t io n a l m o t i o n o f t h ec o m p l e x .

U p o n a d d i t i o n o f sm a l l a m o u n t s o f c h e la t e d H f ~+ t o a p o T f , t h r e e d i s t i n c t

s i g n a ls a r e o b s e r v e d b y P A C . O n e s i g n a l, r e p r e s e n t i n g a b o u t 2 5 % o f t h e H f , is

v e r y b r o a d a n d i s a s s i g n e d t o n o n - s p e c i f i c a l l y b o u n d H f [ 2 1 7 ] . T h e o t h e r t w o

s i g n a ls w e r e i n it ia l ly a s s i g n e d t o H f b o u n d t o t h e C - a n d N - t e r m i n a l m e t a l

b in d in g s i te s o f Tf [217 , 218] . Th i s w a s c ons i s t e n t w i th a d i ffe re nc e U V

t i tr a t io n t h a t a p p e a r e d t o s h o w b i n d i n g o f a r o u n d 2 H f 4§ i o n s p e r t r a n s f er r i n

m o l e c u l e [ 2 0 8 ] . H o w e v e r , s u b s e q u e n t P A C s t u d i e s s h o w e d t h a t t h e a d d i t i o n o f

H f 4+ a b o v e a b o u t 0 .8 e q u i v a l e n t p e r a p o T f m o l e c u l e l e a d s o n l y t o n o n - s p e c i f i c

b in d in g [209] . Ba se d on the T h 4+ a nd H f 4+ da t a , i t i s un l ike ly t ha t t he b ind ingof Pu 4+ to a po T f w il l c om pe te e f fec t ive ly w i th hyd ro lys i s un l e s s t he re i s a n

e x ce ss o f ap o T f , a n d t h a t h y d r o l y s is a n d p o l y m e r i z a ti o n o f P u w o u l d b e c o m e

d o m i n a n t r e a c t i o n s n e a r s t o i c h i o m e t r i c P u : T f r a ti o s .

F u r t h e r P A C s t u d i e s o n H f b o u n d t o is o l a te d C - a n d N - t e r m i n a l l o b es o f

o v o t r a n s f e r r i n i n d i c a t e t h a t e a c h b i n d i n g s i t e e x i s t s a s a p H - d e p e n d e n t

m i x t u r e o f t w o c o n f o r m a t i o n s [ 20 9] . A s im i l a r p H - d e p e n d e n t c o n f o r m a t i o n a l

c h a n g e i n t h e v a n a d y l - t r a n s f e r r i n c o m p l e x h a s b e e n d e t e c t e d b y E S R

s p e c t r o s c o p y [ 21 9 ], b u t t h e n a t u r e o f t h is c h a n g e i s st il l u n k n o w n . I t h a s

b e e n r e p o r t e d t h a t f o r m a t i o n o f t h e lo w p H c o n f o r m a t i o n o f H f - T f r e q u i re sp h o s p h a t e [ 20 9] . T h e r e q u i r e m e n t f o r b i c a r b o n a t e h a s n o t b e e n e x p e r i m e n t a l l y

v e r i f i e d f o r H f , b u t t h e r e i s n o p r e c e d e n t a m o n g o t h e r m e t a l i o n s f o r

p h o s p h a t e a c t i n g in p l a ce o f b i c a r b o n a t e a s a s y n e r g i s t ic a n i o n . T h e

i n v o l v e m e n t o f p h o s p h a t e m a y i n d i ca t e t h e f o r m a t i o n o f a P O 4 -H f -C O 3 - T f

q u a t e r n a r y c o m p l e x , s i n c e th e P A C d a t a o n H f - T f a r e c o n s i s t e n t w i t h e ig h t -

c o o r d i n a t e H f [ 2 1 7 ] .

The tw o c r i t i c a l o rga ns fo r Pu tox i c i t y a re l i ve r a nd bone [220] . In ge ne ra l ,

m o n o m e r i c P u c o m p l e x e s w ill d e p o s i t a b o u t 7 0 % o f t h e P u i n t h e s k e l et o n a n d

a b o u t 3 0 % i n t h e l i v er [ 22 1 ]. T h e r e a p p e a r s t o b e n o s i g n i f ic a n t u p t a k e o f P u f r o m

Pu -T f i n to l i ve r [211]. In he pa to c y te c el l c u l tu re s , so lub le l ow -m ole c u la r -w e igh t

c o m p l e x e s , su c h a s P u - c i tr a t e , a re t a k e n u p m o r e r e a d i l y th a n P u - T f [ 2 2 2 -2 2 4 ] .

I n f ac t, th e a d d i t i o n o f a p o T f a l m o s t t o t a l ly i n h i b i t s P u u p t a k e i n t o h e p a t o c y t e s

[222], w h i l e t he a dd i t i on o f d i fe r r i c Tf h a s a lm os t no e f fe c t [222, 225] .

E v e n t h o u g h a p o T f i n h i b i t s c ell u p t a k e o f P u , s o m e P u d o e s b i n d t o th e c ell

m e m b r a n e s f o l lo w i n g e x p o s u r e t o P u - T f [ 22 3, 2 26 , 2 2 7 ]. O n e p o s s i b i l it y is t h a t

P u - T f b i n d s a t t h e n o r m a l F e - T f r e c e p to r , b u t c a n n o t b e i n t e r n al i ze d a n d / o r

re l e a se d f ro m T f fo r i nc o rp ora t ion in to t he c e ll [223 , 227] . H ow e ve r , t he

a d d i t i o n o f d e te r g e n t t o so l u b il iz e m e m b r a n e p r o t e i n s f r o m l y m p h o b l a s t s

s h o w s t h a t t h e P u i s n o t p r e s e n t e i t h e r a s P u - T f o r as t h e P u - T f - r e c e p t o rc o m p l e x [ 22 6 ]. I n s t e a d , t h e P u a p p e a r s t o d i ss o c i a t e f r o m T f a n d b i n d t o s o m e

o t h e r m e m b r a n e p r o t e i n .

T a y l o r a n d c o - w o r k e r s [ 2 2 5 ] s t u d i e d P u u p t a k e i n l i v e r c e l l s g r o w n a s

m u l t i c e l l u l a r s p h e r o i d s r a t h e r t h a n a s a m o n o l a y e r a n d f o u n d m u c h h i g h e r

u p t a k e o f P u f r o m P u - T f . H o w e v e r , t h e r e w a s st il l n o c o m p e t i t i o n f r o m F e -T f ,



144 W.R. Harris

s o i t a p p e a r s t h a t e v e n u n d e r c o n d i t i o n s l e a d i n g t o h i g h e r c e l l u l a r u p t a k e o f

Pu , Pu -Tf does n o t d e l ive r Pu to the ce ll v ia the usua l Fe -Tf r ecep to r s .

S m a l l a n g le X - r a y s c a t t e r i n g s t u d i e s h a v e s h o w n t h a t t h e b i n d i n g o f H f ~+ t o

a p o T f d o e s n o t t r i g g e r t h e u s u a l p r o t e i n c o n f o r m a t i o n c h a n g e t h a t o n eobse rves fo l lowing the b i nd in g of Fe ~+, Ga 3+, or In 3+ [118] . S ince H f 4+ an d

In 3§ hav e es sen t i a l ly the s a m e s ize , it has b een sugge s ted th a t th e f a i lu re o f

H f 4+ t o p r o m o t e t h e n o r m a l c l o su r e o f t h e a p o T f c o n f o r m a t i o n m a y b e d u e t o

t h e p r e f e re n c e o f th i s t e t r a v a l e n t c a t i o n f o r a c o o r d i n a t i o n n u m b e r o f e ig h t

[118] . Tay lo r has sugges ted tha t the t r ans fe r r in r ecep to r f a i l s to r ecogn ize Pu-

T f b e c a u s e P u 4 +, l ike Hf 4+, f a il s to p rom ote the c onf o rm at io n chan ge to the

" c l o s e d " c o n f o r m a t i o n c h a r a c t e r i s t i c o f f e rr i c t r a n s f e r r i n [2 09 ].

I t is w el l e s t a b l i s h e d t h a t s o l ub l e , m o n o m e r i c P u i s p r e f e r e n ti a l l y ta k e n u p

b y b o n e , a n d t h a t t h e d e g r e e o f b o n e d e p o s i t i o n i s d i r e c t ly r e l a te d t o b o n e

growth [220] and b lood f low [228 , 229] . The Pu does no t appear to subs t i tu tef o r c a l c i u m w i t h i n t h e m i n e r a l c o m p o n e n t o f t h e b o n e , n o r i s t h e r e a n y

i n d i c a t i o n t h a t t h e p r o c e s s i n v o l v e s t r a n s f e r r i n r e c e p t o r s . I n s t e a d , i t a p p e a r s

t h a t t h e r e i s a l i g a n d e x c h a n g e r e a c t i o n b e t w e e n t r a n s f e r r i n a n d e i t h e r

p h o s p h o l i p i d s o r p r o t e i n s a t th e m i n e r a l i z i n g s u r fa c e s o f t h e b o n e [2 30 ].

S e v e ra l b o n e p r o t e i n s a r e r e p o r t e d t o h a v e P u - b i n d i n g a f f in i ti e s g r e a t e r t h a n

tha t o f ap oT f [231] . Soon a f t e r the me ta l i s dep os i t ed on the bon e su r face , i t i s

c o v e r e d o v e r b y n e w l y d e p o s i t e d b o n e m a t r i x a n d b e c o m e s p h y s i c a l l y

inacces s ib le to s e rum che la t ing agen t s [201] .

T h e r o l e o f T f i n P u m e t a b o l i s m a p p e a r s t o b e r a t h e r i n d i r e c t i n t h a t t h ep ro te in does no t ac t ive ly "de l ive r " Pu to spec i f i c s i t e s . In s tead , the p ro te in

f u n c t i o n s i n l a r g e p a r t b y p r e v e n t i n g e x c r e t i o n a n d f o r m i n g a l o n g - l i v e d P u

b u f f e r i n b l o o d [2 11 ]. T h e c i r c u l a t i n g p r o t e i n c o m p l e x i s i n e q u i l i b r i u m w i t h a

s m a l l p o o l o f r e a c t i v e l o w - m o l e c u l a r - w e i g h t P u c o m p l e x e s , w h i c h a r e m o r e

a v i d l y t a k e n u p b y c e l l s a n d r e a c t w i t h m i n e r a l i z i n g b o n e s u r f a c e s .

T h e c h e m i c a l c o m p o s i t i o n o f t h i s l o w m o l e c u l a r w e i g h t P u p o o l i n s e r u m i s

s ti ll n o t c l e ar . A t t e m p t s t o s i m u l a t e t h e s p e c i a t i o n o f th i s p o o l w i t h e q u i l i b r i u m

c o m p u t e r m o d e l s a r e s e v e r e l y h a m p e r e d b y t h e l a c k o f r e l i a b l e s t a b i l i t y

c o n s t a n t s f o r P u w i t h i m p o r t a n t s e r u m c o m p o n e n t s s u c h a s c i t r a t e ( c t a ) a n d

phospha te . Duf f i e ld e t a l . [ 232] ca lcu la ted tha t th i s LMW Pu shou ld be p resen t

a s a m i x t u r e o f 7 1 % [ P u ( c t a ) ( O H ) 2 ] - a n d 2 9 % [ P u ( c t a ) ( O H ) ] . A s u b s e q u e n t

r e a n a l y s is s u g g e s t e d t h a t t h e P u s h o u l d b e p r e s e n t a l m o s t e x c l u si v e ly a s

[Pu(c ta )2 (OH)2] 4 - [233]. H owev er , these au tho r s p o in t o u t th a t the p red om -

i n a n c e o f a c h a r g e d s p e c i e s d o e s n o t a c c o u n t f o r t h e e f f i c a c y o f c i t r a t e i n

m e d i a t i n g t h e t r a n s p o r t o f P u a c r o s s c el l m e m b r a n e s . T h u s t h e c o m p u t e r

m o d e l i n g s t i l l n e e d s m o r e r e l i a b l e d a t a o n P u - c i t r a t e c o m p l e x a t i o n .

3.5

Other Actinides

There a re b io log ica l da ta ava i l ab le fo r U , Am, Cm, and to a l e s se r ex ten t Np

a n d P a . T h e i r s e r u m c h e m i s t r y is s tr o n g l y i n f lu e n c e d b y t h e o x i d a t i o n s t a te o f



Binding and Transport of No nferrous Meta ls 145

t h e m e t a l i o n s . I n t h e c a s e o f u r a n i u m , t h e u r a n y l i o n , U O 2 +, is m u c h m o r e

s t ab l e t h a n U 4§ u n d e r p h y s i o l o g i c al c o n d i t i o n s [ 20 0 ]. T h e s e r u m c h e m i s t r y o f

u r a n i u m is a c c o r d i n g l y q u i t e d i s t i n c t f r o m T h 4 . o r P u 4 . . T h e b i n d i n g o f t h e

U O 2+ i o n t o p u r i f i e d s e r u m t r a n s f e r r i n h a s b e e n d e m o n s t r a t e d b y d i f f e re n c eU V s p e c t r o s c o p y [ 23 4 ], b u t t h e T f c o m p l e x o f t h i s d io x o c a t i o n is m u c h

w e a k e r t h a n t h e c o m p l e x e s o f t h e t e t r a v a l e n t a c t in i d e s . A s a re s u lt , a b o u t 6 0 %

o f s e r u m u r a n i u m e x is ts a s a u r a n y l - c a r b o n a t e c o m p l e x , w h il e o n ly a b o u t 4 0 %

i s b o u n d t o T f [ 2 3 4 ] . T h i s w e a k e r p r o t e i n b i n d i n g l e a d s t o m u c h m o r e r a p i d

c l ea r a n ce o f u r a n i u m v i a t h e k id n e y s [ 23 5] , a n d e x p o s u r e t o h ig h u r a n i u m

le ve l s l e a ds t o r e na l t ox i c i t y . The re i s r e l a t i ve ly l i t t l e bone de pos i t i on

c o m p a r e d w i t h P u .

S e r u m f r a c t i o n a t i o n s t u d i e s h a v e s h o w n t h a t e s s e n t i a l l y a l l N p a n d P a i s

c a r r i e d b y t r a n s f e r r i n [207 , 236] . Ta y lo r e t a l . [207] ha ve sugge s t e d tha t t he

a p p a r e n t a v i d i ty w i th w h i c h T f b i n d s t h e s e t w o m e t a l i o n s i n v i v o i n d i c a te stha t t he p ro t e in i s a b l e t o s t a b i l i z e t he t e t r a va l e n t s t a t e a nd thus fo rm s t a b l e

M 4 + - T f c o m p l e x e s , w h i c h a r e e x p e c t e d t o m i m i c t h e c h e m i s t r y o f T h 4 + - T f a n d

pu4+ -Tf . The N p 4+ ion i s r e l a ti ve ly s t a b l e w i th re spe c t t o o x id a t ion to N pO 2+

[ 20 0 ], a n d o n e w o u l d e x p e c t t h a t T f b i n d i n g w o u l d f u r t h e r s t ab i li z e th e

N p 4+ i o n . H o w e v e r , t h e t i g h t p r o t e i n b i n d i n g o f P a is s o m e w h a t s u r p r i s i n g ,

s i n c e o x i d a t i o n o f P a 4+ t o P aO 2 is e v e n m o r e f a v o r a b l e t h a n t h e o x i d a t i o n o f

U 4+ to UO~+ [ 2 0 0 ] , a n d o n e w o u l d h a v e e x p e c t e d t o o b s e r v e t h e w e a k e r

p r o t e i n b i n d i n g c h a r a c t e r i st i c o f o t h e r d i o x o c a t i o n s .

A s o n e p r o g r e s s e s a c r o s s t h e p e r i o d i c t a b l e p a s t P u , th e m e t a ls m o r e c l os e lyr e s e m b l e t h e l a n t h a n i d e s , a n d t h e t r i v a l e n t o x i d a t i o n s t a t e b e c o m e s m o r e

s t a b l e [74 ] . Thus one f i nds on ly A m 3+ a n d C m 3+ u n d e r p h y s i o l o g i c a l

c o n d i t i o n s . A l t h o u g h t h i s i s t h e s a m e c h a r g e a s t h e f e r r i c i o n , t h e m u c h

la rge r i on i c r a d i i o f A m 3+ a n d C m 3+ l ea d s t o m u c h w e a k e r c o m p l e x a ti o n , a n d

t h e b i n d i n g o f A m 3+ a n d C m 3+ t o m o s t p r o t e i n s ( i n c l u d i n g t r a n s f e r r i n ) i s

w e a k e r t h a n t h e b i n d i n g o f P u 4+ [2 31 , 2 37 ] . A l t h o u g h T f b i n d i n g c o n s t a n t s

h a v e n o t b e e n m e a s u r e d d i re c tl y fo r A m 3 + o r C m 3+ , H a r r i s [ 5 0 ] h a s e s t i m a t e d

a K~ ' va lue of only about 1 0 6 .5 f o r b o t h m e t a l i o n s b a s e d o n l i n e a r f r e e e n e r g y

r e l a t i o n s h i p s w i t h t h e l a n t h a n i d e s N d 3+ a n d S m 3+ .

E a r ly s tu d i e s s u g g e s te d t h a t A m a n d C m w e r e b o u n d i n s e r u m t o T f a n d / o r

a l b u m i n , b u t t h e i n t e r a c t i o n a p p e a r e d t o b e w e a k , p o s s i b l y r e f l e c t i n g n o n -

s p e ci fi c b i n d i n g [ 23 7 ]. T h e i s s u e w a s c o m p l i c a t e d b y t h e t e n d e n c y o f t h e T f

c o m p l e x e s t o d i s s o c i a t e d u r i n g g e l f i l t r a t i o n c h r o m a t o g r a p h y . A m o r e r e c e n t

s t u d y o n t h e f r a c ti o n a t io n o f r a d io l a b e le d s e r u m u s e d a f fi ni ty c h r o m a t o g r a p h y

t o s e p a r a t e t h e a l b u m i n a n d T f f r a c ti o n s , a n d s h o w e d t h a t a s ig n i f i ca n t f r a c ti o n

o f b o t h A m a n d C m is r e c o v e r e d i n t h e T f f r a c t io n [ 23 8 ]. T h i s s t u d y a p p e a r s t o

r u le o u t b i n d i n g o f A m o r C m t o a l b u m i n b u t , b e c a u s e o f lo w r e C ov er ie s o f t h e

me ta l i ons f rom the c o lumns , i t i s s t i l l d i f f i c u l t t o a s se s s qua n t i t a t i ve ly t he

d i s t r i b u t i o n o f t h e s e m e t a l i o n s b e t w e e n T f a n d L M W c o m p l e x e s . T a y l o r e t al.[4 9] h a v e e s t i m a t e d t h a t o n l y a b o u t 2 0 % o f s e r u m A m 3+ a n d C m 3+ is b o u n d t o

T f. T h e l a rg e p o o l o f L M W c o m p l e x e s i n v i v o le a d s t o t h e r a p i d c l e a r a n c e o f3 + 3 +

A m a n d C m f r o m t h e b l o o d [ 23 9, 2 40 ].



146 W .R. Harris

3 . 6

Lanthanides

B e c a u se o f t h e i r f a v o r a b le l u m i n e s c e n t p r o p e r t i e s , l a n t h a n i d e - T f c o m p l e x e s

h a v e b e e n i n v o l v e d i n a l a rg e n u m b e r o f s p e c t r o s c o p i c s t u d i e s [ 1 - 3 ] . P r i o r t o

t h e s u c c e s s f u l c r y s t a l l o g r a p h i c w o r k o n F e - T f , l u m i n e s c e n t e n e r g y t r a n s f e r

s t u d i e s h a d d e t e r m i n e d t h a t t h e t w o m e t a l b i n d i n g s i t e s w e r e a b o u t 3 6 A a p a r t

[241] a nd w e re bur i e d a bo u t 15 ~ , un de r t he ou t e r su r fa c e o f t he p ro t e in [242] .

T h e s e n u m b e r s a g r e e r e a s o n a b l y w e l l w i t h t h e c r y s t a l l o g r a p h i c v a l u e s o f

a ro un d 42 a nd 10 A [13].

T h e b i n d i n g o f th e l a n t h a n i d e s t o T f c a n e a s il y b e d e t e c t e d b y d i f f e re n c e U V

s p e c t r o s c o p y [ 24 3 ]. H a r r i s [5 0] m e a s u r e d t h e b i n d i n g c o n s t a n t s o f N d 3§ a n d

Sm 3§ a s m ode l s fo r A m 3§ a n d Cm 3+, a nd ob ta ine d log K~ va lue s o f 6 .1 fo r

N d 3+ a nd 5 .4 fo r Sm 3§ Th e re i s a de l i c a te b a l a nc e re g a rd ing the e f fe c t o f theb i c a r b o n a t e c o n c e n t r a t i o n o n l a n t h a n i d e - T f b i n d i n g . S o m e b i c a r b o n a t e m u s t

b e p r e s e n t t o e n a b l e th e f o r m a t i o n o f t h e u s u a l M - C O 3 - T f t e r n a r y c o m p l e x .

H o w e v e r , t h e l a n t h a n i d e c a r b o n a t e s ( M 2 ( C O 3 ) 3 ) a r e ve ry inso lub le , w i th K sp

v a l u e s in t h e r a n g e o f 1 0 - 3 ~ T h u s t h e a d d i t i o n o f a f ew m m o l / 1 b i c a r b o n a t e , a s

is ty p i c a ll y d o n e i n m o s t m e t a l - T f b i n d i n g s t u d i es , a c t u a l ly r e d u c e s t h e e x t e n t

o f l a n t h a n i d e b i n d i n g r e la ti v e t o t h a t o b s e r v e d a t a m b i e n t b i c a r b o n a t e ( a r o u n d

2 0 0 p m o l / 1 ) . E v e n a t a m b i e n t b i c a r b o n a t e c o n c e n t r a t i o n s , t h e f i n a l s a t u r a t i o n

o f T f w i t h 2 e q u i v a l e n t s o f th e m e t a l is p r e c l u d e d b y t h e f o r m a t i o n o f t h e

m e t a l - c a r b o n a t e c o m p l e x e s [ 4 7 , 5 0 ] .L u m i n e s c e n c e s t u d i e s h a v e p r o v i d e d a d d i t i o n a l i n f o r m a t i o n a b o u t t h e

b i n d i n g o f l a n t h a n i d e s t o T f. L u m i n e s c e n c e l i f e ti m e s t u d i e s i n d i c a t e t h a t T f-

b o u n d T b 3+ r e t a i n s o n e w a t e r m o l e c u l e [ 24 3 ], p r e s u m a b l y a r e f le c t io n o f t h e

t e n d e n c y o f t h e l a n t h a n i d e s t o w a r d h i g h e r c o o r d i n a t i o n n u m b e r s . T h e r e h a s

e v e n b e e n s p e c u l a ti o n t h a t a n e x p a n d e d c o o r d i n a t i o n s h el l o f t h e l a n th a n i d e s

m i g h t i n c l u d e a t h i r d t y r o s i n e l i g a n d i n t h e t r a n s f e r r i n c o m p l e x [ 4 8 ] . T h e

c i r c u la r l y p o l a r i z e d l u m i n e s c e n c e s p e c t r a h a v e b e e n r e p o r t e d f o r t h e T b 3+

c o m p l e x e s o f tr a n s f e r r i n , l a c t o f e r ri n , a n d o v o t r a n s f e r r i n [ 24 4 ]. T h e r e s u l ts

i n d i c a t e t h a t t h e l a n t h a n i d e c o o r d i n a t i o n g e o m e t r i e s a r e v e r y s i m i l a r i n

t ra ns fe r r in a nd l a c to fe r r in , bu t t ha t t he re i s a s ign i f i c a n t d i f fe re nc e be tw e e n

t h e s e t w o p r o t e i n s a n d o v o t r a n s f e r r i n .

Re c e n t ly t he re ha s b e e n g re a t i n t e re s t i n G d3+ -Tf, l a rge ly be c a use o f the

g r o w i n g i n t e r e s t i n G d c o m p l e x e s a s c o n t r a s t a g e n t s i n m a g n e t i c r e s o n a n c e

i m a g i n g . T h e G d - T f b i n d i n g c o n s t a n t f o r th e C - t e r m i n a l s it e h a s b e e n r e p o r t e d6 8 8 0

a s 10 " [48 ] a nd 10 " [47] . Th e re a pp e a rs t o be a n unu sua l ly w ide ga p be tw e e n

t h e G d b i n d i n g c o n s t a n t s o f t h e t w o T f s it es . O n e s t u d y [4 8] r e p o r t e d n o

d e t e c t a b l e b i n d i n g a t t h e N - t e r m i n a l s i t e , w h i l e a n o t h e r s t u d y [ 4 7 ] r e p o r t e d a

b i n d i n g c o n s t a n t o f o n l y 10 5 .9 . B i n d i n g t o t h e w e a k e r N - t e r m i n a l s i t e i s m o r e

d i ff ic u lt t o c h a r a c t e r i z e b e c a u s e o f t h e e x p e r i m e n t a l d i ff i cu l ty o f c o p i n g w i t ht h e G d - c a r b o n a t e s o l u b i l i t y p r o b l e m .

T h e d a t a b a s e o f th e K s v'S f o r la n t h a n i d e - c a r b o n a t e s is i n c o m p l e t e [ 10 9 ],

b u t t h e a v a i l a b l e d a t a w o u l d s u g g e s t t h a t t h e s e m e t a l i o n s s h o u l d f o r m

i n s o l u b l e c o m p l e x e s w i t h c a r b o n a t e a n d / o r p h o s p h a t e i n v i v o . N e v e r t h e l e s s ,

l o w co n c e n t r a t i o n s o f l a n t h a n i d e s i n s e r u m a p p e a r t o b e r ea s o n a b l y s t a b le a n d




t o b i n d t o s e r u m p r o t e i n s , p r i m a r i l y a l b u m i n [ 2 4 5 ] . S i n c e t h e b i n d i n g c o n s t a n t

o f G d t o b o v i n e s e r u m a l b u m i n is o n l y a b o u t 104 [ 2 45 ] , i ts r o l e in G d b i n d i n g

i n v iv o p r e s u m a b l y r e fl ec ts i t s re l a ti v e ly h i g h c o n c e n t r a t i o n i n s e r u m .

T h e r e i s s o m e d i s a g r e e m e n t a s t o t h e l i k e l i h o o d t h a t t r a n s f e r r i n w i l l b i n d

l a n t h a n i d e s i n v iv o . T h e b i o d i s t r i b u t i o n o f T m 3§ is u n a f f e c t e d b y t h e a d d i t i o n

o f e i t h e r F e o r G a , w h i c h i n d i c a t es t h a t T m is n o t b o u n d t o T f i n s e r u m [ 24 6 ].

S o m e a u t h o r s h a v e c o n c l u d e d t h a t G d - T f b i n d i n g is to o w e a k t o b e o f

s i g n if i ca n c e in v iv o b e c a u s e o f t h e h i g h b i c a r b o n a t e c o n c e n t r a t i o n i n s e r u m

[4 7, 4 8] . H o w e v e r , a c o m p u t e r s i m u l a t i o n o f t h e d i s t r i b u t i o n o f G d 3+ i n s e r u m

i n d i c a t e d t h a t f o r l o w c o n c e n t r a t i o n s o f G d ( 10 - s m o l/1 ), t h e m e t a l i o n w o u l d

b e a b o u t e q u a l l y d i s t r i b u t e d b e t w e e n T f a n d c i t ra t e [ 93 ]. A s i m i l a r m o d e l f o r

S m 3+ i n d i c a te d t h a t 9 5 % o f t h e S m i n s e r u m w o u l d b e b o u n d t o t r a n s f e r r in a t

e q u i l ib r i u m , a l t h o u g h t h e a u t h o r s a ls o n o t e d t h a t t h e f o r m a t i o n o f S m - T f w a s

s l o w a n d s e v e r a l h o u r s m i g h t b e r e q u i r e d t o r e a c h t h i s e q u i l i b r i u m [ 2 4 7 ] . I na d d i t i o n , T a y l o r e t a l. h a v e r e p o r t e d t h a t T f b i n d s a b o u t 2 0 % o f E u a n d Y b

i n v iv o [ 49 ]. T h u s t h e r e c o u l d b e a r o l e fo r T f i n t h e t r a n s p o r t o f l a n t h a n i d e s a t

l o w c o n c e n t r a ti o n s w h e r e t h e f o r m a t i o n o f c o ll o id a l c a r b o n a t e a n d / o r

p h o s p h a t e p r e c i p i t a t e s i s s u p p r e s s e d .

T r a n s f e r r i n b i n d i n g i s st il l u n l i k e l y t o b e i m p o r t a n t w i t h r e s p e c t to t h e u s e o f

G d a s a n M R I c o n t r a s t a g e n t. T o r e d u c e i ts t o x i c i ty t h e G d 3+ m u s t b e i n t r o d u c e d

a s a s t a b l e c he l a t e , e .g ., G d (D T PA ) 2 - . I t is v i r t ua l ly c e r t a in t ha t t he c he l a t i ng

a g e n t u s e d t o b i n d t h e G d w il l b e a m u c h s t r o n g e r l i g a n d t h a n T f , s o o n e w o u l d

e x p e c t v e r y li tt le d o n a t i o n o f G d f r o m t h e l o w m o l e c u l a r w e i g h t l i g a n d t o T f.

3.7

Vanadium

V a n a d i u m i s u n i q u e i n t h a t c o m p l e x e s w i t h b o t h t r a n s f e r r i n [ 2 4 8 ] a n d

la c to fe r r in [249] ha ve be e n re por t e d fo r t h re e d i f fe re n t ox ida t ion s t a t e s : V ( I I I ) ,

V ( I V ) , a n d V ( V ) . T h e b e s t c h a r a c t e r i z e d s y s t e m i n v o l v e s V ( I V ) i n t h e f o r m o f

t h e v a n a d y l c o m p l e x . B e c a u se t h e V O a+ i o n h a s o n l y o n e d - e l e c t r o n a n d is

e a si ly s t u d i e d b y E S R s p e c t r o s c o p y , i t h a s s e r v e d a s a v e r y u s e f u l p r o b e o f th e

t r a n s f e r r i n m e t a l b i n d i n g s i t e s [ 1 , 2 ] . E S R s t u d i e s h a v e ( a ) s h o w n t h e s t r o n g

b i n d i n g o f t w o v a n a d y l i o n s p e r t r a n s f e r r i n m o l e c u le i n t o t w o s p e c t r o s c o p -

i ca ll y d i s t i n c t s it es [ 2 50 ] , ( b ) d o c u m e n t e d p H d e p e n d e n t c o n f o r m a t i o n a l

c h a n g e s i n t h e t r a n s f e r r i n m e t a l b i n d i n g s i t e s [ 2 1 9 , 2 5 0 ] , a n d ( c ) p r o v i d e d

e a r l y e v i d e n c e t h a t t h e s y n e r g i s t i c a n i o n i n t h e m e t a l - a n i o n - T f t e r n a r y

c o m p l e x w a s d i r e c t l y c o o r d i n a t e d t o t h e m e t a l i o n [ 2 5 1 ] .

T h e v a n a d y l i o n a p p e a r s t o b i n d r a t h e r t i g h t l y t o a p o T f , a l t h o u g h n o

e q u i l i b r i u m c o n s t a n t s f o r s e r u m T f h a v e b e e n r e p o r t ed . A b i n d i n g c o n s t a n t o f

a r o u n d 10 9 h a s b e e n r e p o r t e d f o r t h e v a n a d y l c o m p l e x w i t h o v o t r a n s f e r r i n

[ 67 ]. H o w e v e r , t h e r e l ev a n c e o f t h is c o n s t a n t t o s e r u m t r a n s f e r r i n is d e b a t a b l ed u e t o t h e f a c t t h a t t h e V O Z + - o v o t r a n s f e r r i n c o m p l e x f o r m s i n t h e a b s e n c e o f

b i c a r b o n a t e [ 6 7 ] , w h e r e a s a s y n e r g i s t i c b i c a r b o n a t e a n i o n i s r e q u i r e d f o r

f o r m a t i o n o f t h e v o z + - T f c o m p l e x [5 2, 25 0 ].

T h e v a n a d y l - T f c o m p l e x i s r a t h e r e a s i l y o x i d i z e d t o V ( V ) b y m o l e c u l a r

oxyg e n [52 , 252] , w i th a tl z 2 o f on ly 8 m in in a i r - s a tu r a t e d he p e s buf fe r a t



148 W.R. Harris

37 ~ Th is V(V ) -Tf spec ies can a l so be fo rmed d i rec t ly and rev er s ib ly by the

a d d i t i o n o f v a n a d a t e ( V O 3 - ) t o a p o T f a t p h y s i o l o g i ca l p H [5 2]. O t h e r

t e t r a h e d r a l a n i o n s s u c h a s a r s e n a t e a n d p h o s p h a t e a l s o b i n d t o a p o T f [ 5 8 ] , s o

i t i s p l a u s i b le t h a t v a n a d a t e c o u l d b i n d t o a p o T f a s a n a n i o n . H o w e v e r , t h e

d i ff e r en c e U V s p e c t r u m p r o d u c e d b y a n i o n - b i n d i n g t o a p o T f is q u i t e d i s t i n c t

f r o m t h a t p r o d u c e d b y m e t a l - b i n d i n g [ 5 8 , 5 9 ] . T h e d i f f e r e n c e U V s p e c t r u m o f

V ( V ) - T f v s a p o T f is c h a r a c t e r i s t i c o f m e t a l b i n d i n g , w h i c h i n d i c a t e s t h a t t h e

v a n a d i u m i s d i r e c t ly c o o r d i n a t e d t o t h e t w o t y r o s in e s o f t h e m e t a l b i n d i n g s it e

[52].

V ( V ) m o s t o f t e n r e a c t s w i t h m u l t i d e n t a t e l i g a n d s to f o r m c o m p l e x e s o f th e

VO + cat ion , bu t com plexe s of V 5+, VO 3+, an d VO(OH) 2+ are a l so kno w n [248] .

T h e a b s e n c e o f s t r o n g m e t a l - l i g a n d c h a r g e t r a n s f e r b a n d s i n t h e s p e c t r u m o f

the V(V ) -Tf com plex ru les o u t b in d in g o f e i ther V 5+ o r V O 3+ [253]. Th ere a re

p H - d e p e n d e n t c h a n g e s i n t h e d i f fe r en c e U V s p e c t r u m o f V ( V ) - T f w h i c hi n d i c a t e t h a t t h e r e i s a p r o t o n a t i o n e q u i l i b r i u m w i t h a pK a o f a p p r o x i m a t e l y 8

[5 2]. I t h a s b e e n s u g g e s t e d t h a t V ( V ) r e a c t s w i t h T f t o f o r m a V O + - T f c o m p l e x

a t h i g h p H , w h i c h i s t h e n p r o t o n a t e d t o f o rm VO(OH)/+-Tf at neu t ra l pH [52] .

T h i s t y p e o f p r o t o n a t i o n e q u i l i b r i u m h a s b e e n o b s e r v e d f o r t h e V O + c o m p l e x

w i t h t h e t r a n s f e r r i n m o d e l c o m p o u n d e t h y l e n e b i s [ ( o - h y d r o x y b e n z y l ) g l y c i n e ]

[253].

T h e d i a m a g n e t i c V ( V ) - T f s y s t e m h a s a l s o b e e n i n v e s t i g a t e d b y 5 * V N M R

[2 48 ]. T h e a d d i t i o n o f tw o e q u i v a l e n t s o f a m m o n i u m v a n a d a t e t o a p o T f r e s u l ts

i n t w o p a r t i a ll y r e so l v e d p e a k s i n t h e 51V N M R s p e c t r u m w h i c h a r e a s s i g n e d t ov a n a d i u m b o u n d a t t h e C - a n d N - t e r m i n a l b i n d i n g s i te s . T h e c h e m i c a l s h if ts o f

t h e s e t w o p e a k s a r e c o n s i s t e n t w i th d i r e c t c o o r d i n a t i o n o f t h e t w o t y r o s i n e

res idues [254] .

T h e 5 1V N M R s p e c t r u m i n d i c a t e s t h a t t h e V ( V ) i o n i s in a t r i g o n a l

b i p y r a m i d a l e n v i r o n m e n t [2 48 ]. I t h a s b e e n p r o p o s e d t h a t t h e V O + c a t i o n

b i n d s t o t h e t w o t y r o s in e s a n d t h e a s p a r t i c a c i d o f t h e t r a n s f e r r i n b i n d i n g s it e ,

w i t h h y d r o g e n b o n d s b e t w e e n t h e t w o o x o g r o u p s a n d t h e b i n d i n g s i t e

h i s t i d i n e a n d a r g i n i n e r e s i d u e s [2 48 ]. R e t e n t i o n o f t h e c o o r d i n a t e d o x y g e n

l ig a n d s i n V O + - T f a n d V O ( O H ) + - T f p r e s u m a b l y a c c o u n t s f o r th e f o r m a t i o n o f

these V(V) complexes in the absence o f a synerg i s t i c ca rbona te an ion [52 , 62] .

A va lue o f log K1 = 7 .45 fo r the b ind ing o f the f i r s t equ iva len t o f van ada te

t o a p o T f h a s b e e n d e t e r m i n e d b y c o m p e t i t io n b e t w e e n v a n a d a t e a n d

p h o s p h a t e [ 52 ]. T h e s e c o n d e q u i v a l e n t o f v a n a d a t e b i n d s m o r e w e a k l y , s o

t h a t o n e c a n d e t e r m i n e a b i n d i n g c o n s t a n t o f K2 ~ 106.5 e i th er f ro m the s im ple

t i t r a t i o n o f a p o T f w i t h v a n a d a t e [5 2, 25 5] o r b y c o m p e t i t i o n w i t h e i t h e r

p h o s p h a t e o r s u l f a t e [ 5 9 ] .

A V ( I I I ) c o m p l e x w i t h T f h a s b e e n c h a r a c t e r i z e d b y U V - v i s a n d c i r c u l a r

d ich ro i sm spec t rosco py [256, 257]. I t i s m os t r ead i ly d i s t ing u ish ed f rom VO 2+-

T f b y c h a n g e s i n t h e d - d b a n d s i n t h e v i s ib l e +e g i o n a n d f r o m V O 2 - T f b y t h ep r e s e n c e o f i n t e n s e c h a r g e - t r a n s f e r b a n d s i n t h e U V s p e c t r u m o f t h e V ~ +-T f

c o m p l e x . T h e m o s t r e m a r k a b l e a t t r i b u t e o f t h i s c o m p l e x i s i t s p u r p o r t e d

s t a b i l i t y t o w a r d o x i d a t i o n . T h e V3+-Tf c o m p l e x , a f t e r p r e p a r a t i o n u n d e r a n

iner t a tm osp he re to avo id ox id a t ion o f fr ee V 3+, was r ep or te d to be s tab le to

o x i d a t i o n b y a t e n - f o l d e x ce s s o f h y d r o g e n p e r o x i d e [2 56 ]. W e w e r e i n i t ia l l y




qu i te skep t ica l o f th is r e po r t [52] becau se VO 2+-Tf i s eas i ly a i r -ox id ize d to

VO2+-Tf. M os t low molec u la r w eigh t phe no la te l igands such as e thy lene b is [ (o -

h y d r o x y p h e n y l ) g l y c i n e ] a l s o fo r m V ( I II ) c o m p l e x e s w h i c h a r e u n s t a b l e t o w a r d

a i r ox ida t ion [253] . Howev er , the re i s a r ecen t r epo r t o f an a i r - s tab le V( I I I )

c o m p l e x w i t h t h e h e x a d e n t a t e l i g a n d N,N'-bis(o-hydroxybenzyl)-N,N'-bis(2-p y r i d y l m e t h y l ) - e t h y l e n e d i a m i n e [ 2 5 8 ] .

Very r ecen t ly Smi th e t a l . [249] p repared the V( I I I ) , V( IV) , and V(V)

c o m p l e x e s o f l a c t o fe r r in . T h e s e w o r k e r s r e l ie d p r i m a r i l y o n c h a n g e s i n t h e E S R

s p e c t r u m t o f o l l o w o x i d a t i o n r e a c t i o n s , s i n c e o n l y t h e V ( I V ) c o m p l e x i s E S R

d e t e c t a b l e a t r o o m t e m p e r a t u r e . A t n e u t r a l p H , t h e V ( I I I ) - l a c t o f e r r i n c o m p l e x

i s a i r - o x i d i z e d t o v o E + - l a c t o f e r r i n i n a b o u t 5 r a i n . T h i s v a n a d y l c o m p l e x a i r -

o x i d iz e s t o a v a n a d a t e - l a c t o f e r r i n c o m p l e x i n a b o u t 1 h .

V a n a d i u m i s a n e s s e n t i a l e l e m e n t a n d i s f o u n d i n n o r m a l b l o o d a t a

co nc en t ra t io n o f abou t 0 .5 I .tmol/1 [259] . The d i s t r ib u t io n o f th i s van ad iumb e t w e e n p l a s m a a n d r e d b lo o d c ells d e p e n d s o n a n i n t r ig u i n g c o m b i n a t i o n o f

p r o t e i n b i n d i n g a n d v a n a d i u m r e d o x c h e m i s t ry . I t w a s n o t e d e a r l y o n t h a t t h e

b i o d i s t r i b u t i o n o f v a n a d i u m w a s e s s e n t ia l l y t h e s a m e f o ll o w i ng t h e i n j e c t io n o f

e i ther V( I I I ) , V( IV) , o r V(V) [260] . I t appear s tha t vanad ium cyc les be tween

V(IV) and V(V) wi th in b lood . Fo l lowing in jec t ion o f VOW- or V O 2+, the re i s a

v e r y ra p i d e l i m i n a ti o n p h a s e , i n w h i c h a b o u t 4 0 % o f v a n a d a t e a n d 7 0% o f

vanady l l eaves the b lood [261] . Af te r th i s in i t i a l phase , the r emain ing

v a n a d i u m c le a rs f ro m b l o o d w i t h a t l / 2 of 24 h r eg ard les s o f w het her the in i t ia l

f o r m o f t h e i n j e c te d v a n a d i u m w a s v a n a d y l o r v a n a d a t e [2 61 ]. I n a d d i t i o n , t h ep e r c e n t a g e o f t o t a l v a n a d i u m f o u n d i n t h e b l o o d c e ll s r e m a i n s c o n s t a n t d u r i n g

t h e s e c o n d p h a s e o f v a n a d i u m c l ea r an c e . T h u s t h e v a n a d i u m m u s t b e m o v i n g

b e t w e e n t h e p la s m a a n d c e l l u l a r c o m p o n e n t s o f b l o o d a t a r a t e c o m p a r a b l e t o

t h e r a t e o f b l o o d c l e a ra n c e .

I t h a s b e e n e s t a b l i s h e d t h a t e r y t h r o c y t e s t a k e u p v a n a d a t e i n a b i p h a s i c

proces s [262] . The in i t i a l phase , which has a t l / 2 o f o n ly 4 m i n , i s a t t r i b u t e d t o

t h e r a p i d m o v e m e n t o f v a n a d a t e a c r o s s t h e ce ll m e m b r a n e t h r o u g h t h e

p h o s p h a t e t r a n s p o r t s y s t e m a n d p r o d u c e s e q u a l i n t r a c e l l u l a r a n d e x t r a c e l l u l a r

v a n a d i u m c o n c e n t r a t i o n s [2 63 ]. T h e s e c o n d , s lo w e r p h a s e o f v a n a d i u m u p t a k e

o c c u r s a t t h e s a m e r a t e a s th e r e d u c t i o n o f i n t r a c e l l u l a r v a n a d a t e t o v a n a d y l ,

p r e s u m a b l y b y g l u t a t h i o n e [ 2 6 2 ] .

Chas teen e t a l . [252] have shown tha t vanada te i s a l so r educed wi th a t l / 2 o f

o n l y 5 m i n i n f r e s h , a n a e r o b i c s e r u m . F o r s e r u m t r e a t e d t o r e m o v e r e d u c t a n t s

and the n a era ted , the ha l f -l i fe fo r a i r ox id a t ion o f VO 2+ bac k to v ana da t e was

15 min . Essen t ia l ly the s am e ox ida t ion ha l f- l if e was obse rved fo r ox ida t ion o f

v a n a d y l i n h e p e s b u f f e r i n t h e p r e s e n c e o f e x c e s s T f . T h e r a t e o f o x i d a t i o n

i n c r e a s e d w i t h d e c r e a s i n g a p o T f c o n c e n t r a t i o n , s o t h a t i t a p p e a r s t h a t t h e

ox id a t ion r eac t ion a c tua l ly p roce eds v ia a smal l poo l o f fr ee vana dy l ion [252] .

B e c a u s e o f t h e s e c o m p e t i n g o x i d a t i o n a n d r e d u c t i o n r e a c t i o n s , i t h a s b e e np r o p o s e d t h a t t h e v a n a d i u m i n s e r u m w i l l c o n s i s t o f a d y n a m i c m i x t u r e o f

v a n a d y l a n d v a n a d a t e [ 2 5 2 ] .

W h e n v a n a d a t e u p t a k e is c a r ri e d o u t w i t h e r y t h r o c y te s s u s p e n d e d i n bu f fe r ,

9 0 % o f t h e v a n a d i u m a c c u m u l a t e s w i t h i n t h e c e ll s a s v a n a d y l i o n [2 62 ]. T h e

m e t a l a p p e a r s t o b e b o u n d i n t r a c e l l u la r l y t o h e m o g l o b i n [ 26 2 ], a l t h o u g h



150 W.R. Harris

n e i t h e r v a n a d y l n o r v a n a d a t e b i n d s t o h e m o g l o b i n i n v i t r o [ 2 6 4 ] . I n c o n t r a s t ,

o n l y a b o u t 4 0 % o f v a n a d i u m i n b l o o d a c c u m u l a t e s i n c el ls fo l lo w i n g

i n t r a v e n o u s i n j e c ti o n o f v a n a d a t e o r v a n a d y l [ 26 1 ], a n d o n l y 1 0% o f b l o o d

v a n a d i u m is f o u n d i n b l o o d c ells w h e n t h e v a n a d i u m is a b s o r b e d f r o m t h e g u t[2 55 ]. T h u s i t a p p e a r s t h a t t h e c o m b i n a t i o n o f r e d u c t a n t s a n d v a n a d i u m

l ig a n d s i n p l a s m a h a v e a s t r o n g e ff ec t o n t h e d i s t r ib u t i o n o f v a n a d i u m b e t w e e n

t h e p l a s m a a n d r e d b l o o d c e l l s .

I t h a s b e e n d i ff ic u lt t o o b t a i n r e l ia b l e e s t i m a t e s o f t h e e x t e n t o f v a n a d i u m

bin d in g to Tf i n v ivo . In i t ia l l y , Sa b b ion i a n d M a ra fa n t e [265] f ra c t i o na t e d

l a b e l e d s e r u m b y g e l c h r o m a t o g r a p h y a n d r e p o r t e d t h a t t h e p e r c e n t a g e o f

p l a s m a v a n a d i u m b o u n d t o T f s t a r te d a t a b o u t 3 % 3 0 m i n a f t er i n je c ti o n a n d

rose s low ly to a bou t 90% a f t e r 4 da ys . H a r r i s e t a l . [261] a na lyz e d the p ro t e in

b i n d i n g o f v a n a d i u m i n s e r u m b y a g a ro s e g el e l e c tr o p h o r e s is a n d f o u n d t h a t

a b o u t 2 5 % o f t h e v a n a d y l - T f a n d e s s e n t i a l l y a l l o f t h e v a n a d a t e - T f d i s s o c i a t e dd u r i n g t h e 2 - h e l e c t r o p h o r e s i s e x p e r i m e n t . A f t e r c o r r e c t i n g f o r t h i s d i s s o c i -

a t io n , t h e y c o n c l u d e d t h a t o n l y a b o u t 5 % o f t h e v a n a d i u m w a s in i ti a ll y b o u n d

( a s v a n a d y l ) t o T f r e g a r d l e s s o f w h e t h e r v a n a d y l o r v a n a d a t e w a s i n je c te d . T h e

p e r c e n t o f v a n a d i u m b i n d i n g t o tr a n s f e r r i n r o s e to 7 0 % a f t er 2 d a y s. T h e

p e r c e n t a g e w a s e s s e n t ia l ly i d e n t i c a l f o ll o w i n g i n j e c ti o n o f e it h e r v a n a d y l o r

v a n a d a t e . S i n ce o n l y v a n a d y l - T f s u r v i v e d t h e e l e c t r o p h o r e s i s , t h e r e w a s n o w a y

t o d e t e r m i n e t o w h a t e x t e n t v a n a d a t e b i n d s t o T f i n v i v o .

Ch a s t e e n e t a l. [252] ha ve use d ESR to me a su re t he d i s t r i b u t io n o f V O 2+

b e t w e e n T f a n d a l b u m i n , b o t h i n s e r u m a n d i n h e p e s b u f fe r . T h e y e s t i m a t e t h a tt h e v a n a d y l - T f b i n d i n g c o n s t a n t i s a b o u t s i x t i m e s l a r g e r t h a n t h e v a n a d y l -

a l b u m i n c o n s t a n t . H o w e v e r, s in c e th e c o n c e n t r a t i o n o f a l b u m i n i n s e r u m is

t e n t im e s l a r g e r t h a n t h e T f c o n c e n t r a t i o n , t h e y s u g g e s t t h a t a l b u m i n m a y a l so

c a r r y a s i g n i fi c a n t fr a c t io n o f t h e v a n a d y l i o n i n s e r u m . T h e v a n a d y l - a l b u m i n

c o m p l e x i s r a p i d l y a i r - o x i d i z e d a n d w o u l d h a v e li k e ly e s c a p e d d e t e c t i o n i n t h e

e l e c t rop ho re s i s s t ud i e s o f H a r r i s e t al. [261].

T h e d i f f e r e n c e s b e t w e e n t h e E S R a n d e l e c t r o p h o r e s i s d a t a o n t h e b i n d i n g o f

v a n a d a t e t o t r a n s f e r r i n i n s e r u m m a y r e f l e c t i n p a r t a d i f f e r e n c e i n v a n a d i u m

c o n c e n t r a ti o n s . T h e E S R s t u d ie s u s e d v a n a d i u m c o n c e n t r a t i o n s t h a t w e r e

c o m p a r a b l e t o t h e s e r u m t r a n s f e r r i n c o n c e n t r a t i o n . B i n d i n g t o a l b u m i n m a y

b e c o m e a m o r e s i g n i f i c a n t f a c t o r a s t h e s t r o n g e r t r a n s f e r r i n b i n d i n g s i t e h a s

b e e n s a t u r a t e d a n d v a n a d a t e m u s t b i n d t o t h e w e a k e r s it e. C h a s t e e n e t al. [ 25 2]

n o t e t h a t t h e b i n d i n g t o a l b u m i n i s h i g h e r a t h i g h e r t o t a l v a n a d i u m

c o n c e n t r a t i o n s . I n c o n t r a s t , t h e s e r u m s a m p l e s a n a l y z e d b y H a r r i s e t a l .

[261] ne ve r e xc e e d 3 ~ tmol/1 , w h ic h i s on ly a b ou t 10% of t he se r um t ra n s fe r r in

c o n c e n t r a t i o n .

T h e o v e r al l m o d e l t h a t h a s e m e r g e d f o r t h e b e h a v i o r o f v a n a d i u m i n s e r u m

is a c o m b i n a t i o n o f e x t ra c e ll u la r p r o t e i n b i n d i n g a n d o x i d a t i o n c o u p l e d w i t h

i n t r a c e l l u l a r r e d u c t i o n . E x t r a c e l l u l a r v a n a d i u m c o n s i s t s o f voZ +-T f, VO 2+-a l b u m i n , a n d a m i x t u r e o f f re e v a n a d a t e a n d V O ( O H ) Z + -T f . T h e v o Z + - p r o t e i n

c o m p l e x e s i n s e r u m a r e o x i d i z e d , p r e s u m a b l y b y f r e e o x y g e n . T h e r e s u l t i n g

v a n a d a t e a n i o n r a p i d l y e q u i l i b r a t e s w i t h a n i n t r a c e l l u l a r v a n a d a t e p o o l i n t h e

e ry th roc y te s . Th i s i n t ra c e l lu l a r poo l i s s l ow ly re duc e d to va na dy l , w h ic h l e a ve s

t h e ce ll a n d b i n d s t o T f a n d a l b u m i n i n t h e s e r u m c o m p a r t m e n t .



Binding and Transport of Nonferrous Meta ls 151

3.8

Manganese

T h e b i n d i n g o f m a n g a n e s e t o T f b e a r s s o m e r e s e m b l a n c e t o th e i r o n -

t r a n s f e r r i n s y s te m . T h e h i g h e r o x i d a t i o n s t at e m a n g a n i c i o n ( M n 3§ is b o u n d

v e r y t ig h t ly , w h il e th e l o w e r o x i d a t i o n s t at e m a n g a n o u s i o n ( M n 2§ is b o u n d

v e r y w e ak l y . A s w i th i r o n , t h e M n 3 + - H C O 3 - T f t e r n a r y c o m p l e x c a n b e p r e p a r e d

b y a i r - o x i d a t i o n o f M n 2§ i n t h e p r e s e n c e o f a p o T f a n d b i c a r b o n a t e [ 26 6 ]. B u t

w h i le t h e o x i d a t i o n o f f e r r o u s i o n t o f o r m f e rr i c t r a n s f e r r i n i s v e r y r a p i d , t h e

ox ida t io n o f M n z+ i s ve ry s low . Ind e e d , t he syn the s i s o f M n3+ - l a c to fe r r in

r e q u i r e s p e r o x i d e a s t h e o x i d a n t [ 2 6 7 ] .

T h e v is ib l e a b s o r p t i o n s p e c t r u m o f th e M n 3 + - T f c o m p l e x is d o m i n a t e d b y

i n t e n s e c h a r g e t r a n s f e r b a n d s a t 4 3 0 a n d 3 3 0 n m w i t h t ;'s o f a b o u t

5000 1/mol cm -1 [ 2 6 6 ] . S t u d i e s o n m o d e l c o m p o u n d s h a v e c o n f i r m e d t h a tt h e s e ar e a s s o c i a te d w i t h th e c o o r d i n a t e d p h e n o l i c g r o u p s o f t h e tw o b i n d i n g

s i t e t y r o s i n e r e s i d u e s [ 2 6 8 ] . T y r o s i n e c o o r d i n a t i o n h a s b e e n c o n f i r m e d b y t h e

e n h a n c e m e n t o f a r o m a t i c ri n g v i b r a t io n a l m o d e s i n th e r e s o n a n c e R a m a n

s p e c t r u m o f M n 3 + - T f [2 6 9] . T h e t r i v a le n t o x i d a t i o n s t at e o f th e m e t a l h a s b e e n

c o n f i r m e d b y m a g n e t i c s u s c e p t i b i l i t y m e a s u r e m e n t s a n d E S R spectroscopy

[266].

N o b i n d i n g c o n s t a n t f o r M n 3 + - T f h a s b e e n r e p o r t e d , a n d , g i v e n t h e p o o r l y

b e h a v e d a q u e o u s c h e m i s t r y o f th e f re e M n 3+ i o n , n o n e i s l ik e l y t o b e m e a s u r e d

i n t h e f o r e se e a b l e f u t u re . T h e f r ee M n ( I I I ) a q u o i o n i s n o t v e r y s t ab l e i n n e u t r a la q u e o u s s o l u t i o n a n d i s s u b j e c t t o d i s p r o p o r t i o n a t i o n t o M n ( I I ) a n d M n ( I V )

(MnO 2) [270] . In a c id i c me d ia , t he re d uc t ion po te n t i a l fo r t he re a c t ion M n 3+ +M n 2+e - ---) i s + 1 . 5 V vs N H E, m a k ing M n 3§ a po t e n t ox id i z ing a ge n t . Th us

m a n y M n ( I I I ) c o m p l e x e s a r e s u b j e c t t o i n t e r n a l r e d o x r e a c t i o n s i n w h i c h t h e

l i g a n d i s o x i d i z e d a n d t h e m e t a l i s r e d u c e d t o M n ( I I ) . E v e n c o m p l e x a t i o n b y3 + 3 +

E D T A o n l y r e d u c e s th e M n r e d u c t i o n p o t e n t i a l t o + 0 .8 V , a n d th e M n -

E D T A c o m p l e x s l o w ly d e c o m p o s e s b y o x i d a t i o n o f t h e l i g a n d [ 27 0 ].

G i v e n t h e h i g h r e d u c t i o n p o t e n t i a l o f M n 3+, i t m i g h t s e e m s u r p r i s i n g t h a t

o n e c a n f o r m a s t a b le M n 3 + - T f c o m p l e x b y s i m p l e a i r o x i d a t i o n . T h i s u n u s u a l

s t a b il it y o f t h e M n 3 + - T f c o m p l e x c a n b e a t t r i b u t e d t o t h e v e r y h i g h s e le c ti v it y3 + 3 +

o f t h e c o o r d i n a t e d p h e n o l i c g r o u p s f o r M n . F o r e x a m p l e , t h e M n c o m p l e x

o f t h e d i p h e n o l i c l i g a n d E H P G h a s a r e d u c t i o n p o t e n t i a l o f 0.0 V v s N H E a n d

i s s t a b l e to i n t e rn a l r e d ox r e a c t ions [268]. In t he a b se nc e o f re l i a b le

e x p e r i m e n t a l d a t a o n t h e s t a b i li t y o f M n 3 +- T f, o n e c a n u s e a n L F E R b e t w e e n

M n 3+ a n d F e 3+ t o e s t i m a t e a b i n d i n g c o n s t a n t f o r t h is c o m p l e x . A n L F E R b a s e d

p r i m a r i l y o n a m i n o c a r b o x y l a t e l i g a n d s s h o w s a g o o d l i n e a r r e l a t i o n s h i p

( r = 0 .987) be tw e e n the M n ~+ a nd Fe 3+ b in d in g c on s t a n t s w h ic h i s de sc r ibe d

b y t h e e q u a t i o n

log KMn = 1.09(log KFe) + 1.09 (10)

Ba se d on the K M va lue fo r Fe -Tf [22 ], th i s e q ua t io n p re d i c t s a l og KM

va lue o f 23 .4 fo r M n3+-Tf . Th i s i s 2 l og un i t s l a rge r t ha n the Fe 3+ b in d in g

c o n s t a n t . W i t h s u c h a l a r g e b i n d i n g c o n s t a n t , o n e w o u l d e x p e c t t h a t e s s e n t i a l l y



152 W.R. Harris

a ll M n 3+ i n s e r u m w i ll b e b o u n d t o T f , a n d t h a t t h e c o m p l e x w i ll b e s t a b le

e n o u g h t o s u rv i v e m o s t m e t h o d s w h i c h m i g h t b e u s e d t o s e p a ra t e t h e p r o t e i n s

i n s e r u m .

I n s h a r p c o n t r a s t t o M n 3+, t h e M n 2+ io n b i n d s v e r y w e a k l y t o a p o T f[33] . S inc e t he M n 2+ doe s no t hy dro ly z e a t ne u t ra l p H , t h i s i s one o f the ra re

m e t a l - T f s y s te m s f o r w h i c h o n e c a n m e a s u r e t h e s i m p l e e q u i l ib r i u m b e t w e e n

t h e p r o t e i n a n d t h e f r e e m e t a l i o n w i t h o u t t h e n e e d f o r c o m p e t i t i v e c h e l a t i n g

a g e n t s . A n a d d i t i o n a l e x p e r i m e n t a l a d v a n t a g e i s t h a t o n e c a n u s e E S R

s p e c t r o s c o p y t o m e a s u r e d i r e c t ly th e c o n c e n t r a t i o n o f f re e , h e x a a q u o M n ( I I ) i n

e qu i l i b r ium w i th t he p ro t e in . The f re e i on g ive s a s imple , i so t rop ic s ix - l i ne ESR

s p e c t r u m w h i c h c a n b e d e t e c t e d a t m i c r o m o l a r c o n c e n t r a t i o n s , w h i l e t h e s l o w

r o t a t i o n a l m o t i o n o f t h e p r o t e i n b r o a d e n s t h e M n Z + - p r o t ei n s ig n a l t o th e p o i n t

t h a t i t i s n o t o b s e r v e d a t r o o m t e m p e r a t u r e [ 2 7 1 , 2 7 2 ] .

A va lue o f l og K ~ = 3 . 98 fo r M nZ+ -T fw a s m e a s ur e d by t i t r a t i on o f 50 ~ tmol/1

f re e Mn 2+ in p H 7 .4 H e pe s buf fe r w i th a po Tf [33]. S im i l a r t i tr a t i on s w i th

d i f e r r i c t r a n s f e r r i n w e r e u s e d t o c o r r e c t f o r n o n - s p e c i f i c b i n d i n g . T h e

m a c r o s c o p i c b i n d i n g c o n s t a n t f o r t h e b i n d i n g o f th e s e c o n d M n 2+ i o n t o

a p o T f w a s d e t e r m i n e d b y d if f e re n c e U V s p e c t r o s c o p y t o b e l o g K~ - - 2.9 6 . T h e

b i n d i n g o f M n 2+ t o t h e v a c a n t m e t a l b i n d i n g s it es o f a p o T f a n d b o t h C - a n d N -

t e r m i n a l m o n o f e r r i c t r a n s f e r r i n s w a s a l s o m e a s u r e d b y d i f f e r e n c e U V

t i t r a t i on s o f t he p ro t e ins w i th a r e l a t ive ly l a rge e xc e ss o f M n 2+ [33] . S it e -

spe c if i c b ind ing c on s t a n t s o f log k c = 3 .74 a n d log kN = 3 .05 w e re de t e r -

m i n e d , w h e r e t h e s u b s c r i p t d e n o t e s t h e s it e a t w h i c h t h e M n 2+ is b i n d i n g . T h ed i f fe r e n c e o f 1.0 l o g u n i t b e t w e e n t h e m a c r o s c o p i c b i n d i n g c o n s t a n t s c a n b e

a c c o u n t e d f o r b y a c o m b i n a t i o n o f a st a ti s ti c a l s e p a r a t i o n f a c t o r fo r tw o

b i n d i n g s it e s a n d t h e d i f f e r e n c e o f 0 .7 l o g u n i t s b e t w e e n t h e i n t r i n s i c b i n d i n g

c o n s t a n t s f o r t h e t w o s i t e s .

T h e b i n d i n g c o n s t a n t s f o r M n 3 + - T f a n d M n 2 + - T f d if fe r b y a p p r o x i m a t e l y

1 9 l o g u n i t s, w h i c h w o u l d s h i ft t h e M n 3 + - T f r e d u c t i o n p o t e n t i a l 1.1 V n e g a t i v e

o f t h e E ~ f o r f r e e M n 3+. T h i s c o n t r i b u t e s t o t h e u n u s u a l s t a b il i ty o f t h e M n 3 + - T f

t o w a r d r e d u c t i o n t o M n 2+. T h e r e i s a s i m i l a r g a p o f a b o u t 1 7 lo g u n i t s b e t w e e n

t h e b i n d i n g c o n s t a n t s f o r F e3 + -T f a n d F e2 +- Tf . S i n ce t h e s t a n d a r d r e d u c t i o n

p o t e n t i a l f o r F e 3+ i s m u c h l o w e r t h a n t h a t o f M n 3+, c o m p l e x a t i o n s h if ts t h e

F e 3+ -T f r e d u c t i o n p o t e n t i a l t o v a l u e s s u f f ic i en t ly n e g a t i v e t o a c c o u n t f o r t h e

fa c il e a i r -o x id a t ion o f Fe2+ -Tf.

T h e r e h a s b e e n c o n s i d e r ab l e c o n f u s i o n r e g a r d i n g t h e s p e c i at io n o f M n i n

s e r u m . T h e r e h a v e b e e n r e p o r t s t h a t M n i n s e r u m is b o u n d t o t r a n s f e r r in [ 2 7 3 -

2 7 9 ], s e r u m a l b u m i n [2 80 , 2 8 1 ], a p r o t e i n i n it ia l ly i d e n t if i e d a s t r a n s m a n g a n i n

[ 2 8 2 ] , a n d a m u c h l a r g e r p r o t e i n w h i c h m i g h t b e a 2 - m a c r o g l o b u l i n [ 2 7 7 - 2 7 9 ] .

I t h a s a ls o b e e n p r o p o s e d t h a t a s ig n i fi c an t p o r t i o n o f s e r u m m a n g a n e s e is

p re se n t a s i on i c M n 2+ o r l ow m ole c u la r w e igh t c he l a t e s [273, 277 , 283] .

P a r t o f t h e c o n f u s i o n s t e m s f r o m t h e fa c t t h a t t h e e x t e n t o f t r a n s f e r r i nb i n d i n g i n s e r u m d e p e n d s o n t h e i n c u b a t i o n t im e o f M n 2+ w i t h a p o T f in e i t h e r

se rum or bu f fe r [153 , 274 , 278 , 279 , 283] . The de l a y in b ind ing to a poTf i s

a s s o c i a t e d w i t h t h e n e c e s s i ty o f o x i d i z in g t h e M n 1+, w h i c h i s v e r y w e a k l y

b o u n d , t o M n 3+, w h i c h is v e r y s t ro n g l y b o u n d . W h e n s e r u m w a s l a b e le d

i n v i t r o w i t h M n 2+, i n c u b a t e d f o r 1 m i n , a n d t h e n f r a c t i o n a t e d b y g e l



Binding and Transpor t o f Nonfer rous Meta ls 153

p e r m e a t i o n c h r o m a t o g r a p h y , t h e m a j o r i t y o f t ile M n w a s a s so c i a te d w i t h l ow

m o l e c u l a r w e i g h t s p e c i e s , a b o u t 1 5 % w a s a s s o c i a t e d w i t h T f , a n d a s i m i l a r

p e r c e n t a g e a p p e a r e d a t t h e v o i d v o l u m e o f t h e c o l u m n [ 27 3 ]. A f t e r i n c u b a t i o n

f o r 1 h , t h e T f p e a k h a d g r o w n t o 4 0 % o f t h e t o t a l M n . A l ar g e f r a c t io n o f io n i cMn w a s a l so obse rve d fo l low ing in v i t ro l a be l ing by H a nc oc k e t a l . [277] , bu t

t h e i n c u b a t i o n t i m e w a s n o t g i v en . S c h e u h a m m e r a n d C h e r i a n [2 79 ] r e p o r t e d

t h a t i t t a k e s a b o u t 6 h a t 3 7 ~ f o r T f b i n d i n g t o m a x i m i z e i n s e r u m l a b e le d i n

v i t ro w i th M n z+ .

M a n g a n e s e o x i d a t i o n i n s e r u m a p p e a r s t o b e fa s te r t h a n o x i d a t io n i n b u f fe r ,

a n d i n v i t r o s t u d i e s h a v e s h o w n t h a t t h e o x i d a t i o n a n d b i n d i n g o f M n z+ t o

a p o T f c a n b e c a t al y z e d b y c e r u l o p l a s m i n [2 78 , 2 8 3 ]. W h e t h e r c e r u l o p l a s m i n

c a r r i e s o u t t h i s f u n c t i o n i n v i v o h a s n o t b e e n e s t a b l i s h e d . T h e o b s e r v a t i o n o f

n o r m a l T f l o a d i n g w i t h M n i n t h e p l a s m a o f c o p p e r - d e f i c i e n t r a ts w i t h l o w

c e r u l o p l a s m i n l e v el s c a s ts s o m e d o u b t o n t h e r o le o f t h is p r o t e i n a s t h e p l a s m a

ox ida se fo r Mn [273] .

W h e n M n 2+ i s in j e c te d , t h e r e i s a v e r y r a p i d c l e a r an c e o f th e m e t a l f r o m t h e

b l o o d , a n d o n l y a b o u t 1 % o f t h e i n j ec t e d d o s e r e m a i n s i n th e c i r c u l a t io n a f te r

10 ra in [278 , 284] . A l a rge f ra c t i on o f t he do se o f f r e e Mn 2§ is t a ke n up by the

l iv e r [ 2 8 4 ]. F o l lo w i n g t h i s r a p i d c l e a r a n c e o f m o s t o f t h e M n d o s e , o n e

o b s e r v e s a s m a l l b u t p e r s i s t e n t M n f r a c t i o n w h i c h i s p r i m a r i l y a s s o c i a t e d w i t hT f [278]. 3+i b b o n s e t al. [2 78 ] i n je c t e d t h e p r e - f o r m e d M n - T f c o m p l e x a n d

f o u n d t h a t t h e c o m p l e x h a s a p l a s m a c l e a r a n c e h al f- li fe o f a b o u t 3 h , w h i c h is

s im i l a r to t ha t o f Fe3+ -Tf [278] . The se w ork e rs p ro po se d tha t i t i s M n3+ -Tfw h i c h i s t h e s p e c i e s r e s p o n s i b l e f o r t h e l o n g e r - t e r m t r a n s p o r t a n d c o n t r o l l e d

d e l i v e ry o f M n t o e x t r a h e p a t i c t is s u e s. T h e p r o t e i n o r i g i n a ll y i d e n t if i e d a s

t r a n s m a n g a n i n [ 2 8 2 ] i s n o w p r e s u m e d t o h a v e b e e n t r a n s f e r r i n .

T h e r e h a v e b e e n s e v e r al r e p o r t s o f a v e r y h i g h m o l e c u l a r w e i g h t M n

c omple x in se rum, w hic h i s o f t e n i de n t i f i e d a s a c omple x w i th 0~ 2-ma c rog lob-

u l in [273, 277 -279 , 285] . G ibb ons e t al. [278] ha v e sh ow n tha t M n 1+ w i ll b in d

t o p u r i f i e d ~ 2 - m a c r o g l o b u l i n . H o w e v e r , w h e n t h e M n 2+ c o m p l e x o f ~ 2-

m a c r o g l o b u l i n w a s i n je c t ed , t h e M n 2+ w a s c l e a r e d f r o m t h e b l o o d a l m o s t a s

ra p id ly a s f r e e M n z+ . Th us i t doe s n o t a pp e a r t ha t t h i s c o m ple x a t ion by 0~2-m a c r o g l o b u l i n p l a y s a m a j o r r o l e i n M n 2§ t r a n s p o r t .

T h e r e h a v e b e e n c o n f l ic t in g r e p o r t s o n t h e b i n d i n g o f M n t o a lb u m i n . T h e

M n z+ i o n b i n d s t o p u r i f i e d a l b u m i n a t a s in g l e b i n d i n g s i te w i t h a b i n d i n g

c on s t a n t o f l og Kz = 4 .3 [27 1 , 286 ] . T he re m a y be a dd i t i ona l l ow a ff in it y s i te s ,

b u t b e c a u s e o f t h e h ig h c o n c e n t r a t i o n o f a l b u m i n i n s e r u m , t h e s e s h o u l d n o t

b e i m p o r t a n t w i t h r e s p e c t t o b i n d i n g M n i n v i v o . T w o e a r l y r e p o r t s i d e n t i f i e d

a l b u m i n b y g e l p e r m e a t i o n c h r o m a t o g r a p h y a s t he m a j o r M n b i n d i n g a g e n t in

s e r u m [2 80 , 2 81 ] . M o r e r e c e n tl y , t h e p r o t e i n b i n d i n g o f M n 2+ i n s e r u m h a s

b e e n s t u d i e d u n d e r c o n d i t i o n s d e s i g n e d t o l i m i t o x i d a t i o n t o M n 3§ [2 87 ]

U l t ra f il tr a ti o n d a t a s h o w e d t h a t 7 0 % o f th e M n 2§ i n s e r u m w a s p r o t e i n - b o u n d[ 28 7 ]. A t t e m p t s t o i d e n t i f y t h e s p ec if ic p r o t e i n ( s ) w h i c h b i n d M n z+ b y g e l

f il tr a ti o n c h r o m a t o g r a p h y w e r e t h w a r t e d b y t h e d i s so c i a ti o n o f th e w e a k

M n - p r o t e i n c o m p l e x e s o n t h e c o l u m n .

A c o m p u t e r m o d e l f o r t h e s p e c i a t io n o f M n z+ i n s e r u m h a s a ls o b e e n

r e p o r t e d [ 33 ]. W h e n b o t h a l b u m i n a n d T f a r e i n c lu d e d i n th e m o d e l , t h e



154 W .R. Harr is

c a l c u l a ti o n s p r e d i c t t h a t o v e r 8 0 % o f M n 2+ w o u l d b e b o u n d t o a l b u m i n . A b o u t

1 2 % o f t h e M n w o u l d b e e v e n l y s p l it b e t w e e n M n (H 2 0)6 2+ a n d M n ( H C O 3 ) + ,

a n d o n l y 1 % w o u l d b e b o u n d t o Tf. T h i s is i n g o o d a g r e e m e n t w i th t h e

u l t r a f i l tr a t i o n d a t a a n d c l e a r l y i n d i c a t e s t h a t a l b u m i n , r a t h e r t h a n T f, i s t h e

p r i m a r y t r a n s p o r t p r o t e i n f o r M n 2+.

T h e b u l k o f t h e d a t a o n M n b i n d i n g i s c o n s i s t e n t w i t h t h e ba s ic s e r u m

t r a n s p o r t m o d e l p r o p o s e d b y G i b b o n s e t a l. [2 78 ]. U p o n e n t e r i n g t h e b l o o d ,

M n 2+ i s l o o se l y b o u n d t o a l b u m i n a n d p o s s i b l y a z - m a c r o g l o b u l i n . T h i s f re e o r

l o o s e l y b o u n d M n 2+ i s r e m o v e d v e r y e f f ic i e n tl y ( e s s e n t i a l l y i n o n e p a s s ) b y t h e

l iv e r d u e t o t h e p r e s e n c e o f a h i g h a f fi n it y , h i g h c a p a c i t y M n 2+ t r a n s p o r t

s y s t e m i n h e p a t o c y t e s [ 28 8 ]. M o s t o f th i s M n 2+ i s p a s s e d q u i c k l y i n t o b i l e f o r

e x c r e t i o n [ 28 8 ]. H o w e v e r , a s m a l l f r a c t i o n o f th e M n 2+ i s o x i d i z e d t o M n 3+ a n d

r e a p p e a r s i n th e s e r u m a s M n 3 + -T f . T h i s o x i d a t i o n m a y b e c a t a ly z e d b y9 9 3 +

c e r u l o p l a s m m . T h i s M n - T f h a s a l o n g e r h a l f- li fe in p l a s m a , a n d i s l i k e ly t or e p r e s e n t t h e t r a n s p o r t s p e ci e s f o r t h e c o n t r o l l e d d e l i v e r y o f M n t o

e x t r a h e p a t i c t i s s u e s [ 2 7 9 ] .

F r e e M n ( I I ) i n s e r u m i s r a p i d l y a s s i m i l a t e d i n t o o t h e r t i ss u e s i n a d d i t i o n t o

l iv e r. F re e M n ( I I ) c r o s s e s th e b l o o d - b r a i n - b a r r i e r a n d is i n c o r p o r a t e d i n t o

b r a i n i n a v e r y r a p i d , s a t u r a b l e p r o c e s s [ 28 7 , 2 8 9 ]. It h a s b e e n p r o p o s e d t h a t

t h e r e i s a f a m i l y o f m e t a l t r a n s p o r t s y s t e m s w i t h d i f f e ri n g s e l e c t iv i t ie s f o r a

s e r ie s o f d i v a l e n t m e t a l i o n s , i n c l u d i n g M n 2+ [ 29 0 ]. O t h e r s t u d i e s h a v e s h o w n

t h a t t h e re i s r e c e p t o r m e d i a t e d c e l lu l a r u p t a k e o f M n f r o m t h e M n 3 + - T f

c o m p l e x [ 28 3 , 2 8 9, 2 91 ] . O n e h y p o t h e s i s i s t h a t t h e u p t a k e o f M n 2+ is t o o r a p i da n d d i ff ic u l t to c o n t r o l . T o a l l o w f o r a m o r e c o n t r o l l e d d e l i v e r y o f M n t o

t is s u e s w h i c h n e e d it , t h e l iv e r r a p i d l y r e m o v e s M n 2+ f r o m t h e s e r u m a n d

r e l e a se s l o w c o n c e n t r a t i o n s o f M n 3 + - t r a n s f e r r i n . U p t a k e o f t h i s sp e c i e s is t h e n

r e g u l a t e d b y t h e u s u a l t r a n s f e r r i n r e c e p t o r m e d i a t e d e n d o c y t o s is .

4

Conclusions

A s d e m o n s t r a t e d b y t h e d a t a i n T a b le 1, s e r u m t r a n s f e r r i n i s c a p a b l e o f

b i n d i n g a v e r y w i d e v a r i e t y o f m e t a l i o n s i n v it r o . T h e s t a b i l i t y o f t h e s e

c o m p l e x e s v a r i e s w i d e l y f r o m a r o u n d 104 f o r F e 2§ a n d M n 2+ t o a r o u n d 1 02 2 f o r

F e 3§ [ 2 1, 2 2 ] . T r a n s f e r r i n c a n a l s o b i n d o x o c a t i o n s , s u c h a s V O 2+ a n d U O 22+,

b u t t h i s b i n d i n g i s re l a t iv e l y w e a k . T h e M 3+ a n d M 4§ c a t io n s w h i c h t e n d t o

f o r m s t r o n g t r a n s f e r r i n c o m p l e x e s a l s o h a v e a s t r o n g t e n d e n c y t o h y d r o l y z e ,

a n d t h e r e is o f te n a cl o se b a l a n c e b e t w e e n h y d r o l y s i s a n d p r o t e i n b i n d i n g . F o r

s e v e r a l m e t a l i o n s , t h e s t r o n g e r C - t e r m i n a l b i n d i n g s i t e c a n c o m p e t e e f f e c t i v e l y

w i t h h y d r o l y s i s , b u t t h e w e a k e r N - t e r m i n a l s i t e c a n n o t . A s a r e s u l t , f o r m a n y

m e t a l i o n s i t i s d i f f i c u l t t o s a t u r a t e t h e N - t e r m i n a l s i t e .

T h e e x t e n t o f t r a n s f e r r i n b i n d i n g i n v iv o v a ri e s s i g n if i ca n t ly f r o m o n e m e t a li o n t o a n o t h e r . T r a n s f e r r i n a p p e a r s t o b i n d a l m o s t a l l o f s e r u m M n 3§ [2 7 3, 2 7 4,

276 ] , Ga 3+ [71 , 85 , 87 , 8 8 ] , In 3+ [88 -9 0 , 112 ] , H f 4+ [216 , 2 17 ] , Pa 4§ [236] , a n d

N p 4§ [ 20 7 , 2 9 2 ]. I t a l s o a p p e a r s t o b i n d a r o u n d 9 0 % o f s e r u m P u 4§ [ 74 , 2 0 4,

2 0 6 , 2 0 7 ], a r o u n d 8 0 % o f s e r u m A 13 § [ 16 7 , 1 68 , 17 7, 1 7 9] , a n d a r o u n d 2 0 % o f

t h e t r i v a l e n t a c t i n i d e s A m 3§ a n d C m 3+ [ 49 ]. T r a n s f e r r i n b i n d s a b o u t 4 0 % o f



Binding and Transport of Nonferrous Metals 15 5

UO~ + [234] a nd a s igni f ican t f ra c t ion of se ru m VO 2+ [252, 261] , bu t the r e i s

l i t t l e e v i d e n c e t h a t t r a n s f e r r i n b i n d s a n y o t h e r d i v a l e n t c a t i o n s i n s e r u m .

T h e r e a r e c o n f li c ti n g vi ew s o n t h e e x t e n t t o w h i c h T f b i n d s l a n t h a n i d e i o n s.

T a y l o r e t a l. h a v e r e p o r t e d t h a t T f b i n d s a b o u t 2 0 % o fE u 3+

a n dY b 3+

[49].E q u i l i b r i u m m o d e l s f o r s e r u m s p e c i a t io n o f t h e l a n t h a n i d e s t e n d s t o g iv e

m u c h h i g h e r e s t i m a t e s f o r th e d e g r e e o f T f b i n d i n g [9 3, 2 4 7 ], b u t o t h e r s h a v e

p o i n t e d o u t t h a t T f b i n d i n g i s l ik e l y t o b e s e r i o u s l y r e s t r ic t e d b y t h e s m a l l

s o l u b i l i t y p r o d u c t s f o r t h e l a n t h a n i d e - c a r b o n a t e c o m p l e x e s [ 4 7 , 4 8 ] .

T h e i m p a c t o f t r a n s f e r ri n b i n d i n g o n t h e i n vi v o b e h a v i o r o f a m e ta l i o n

d e p e n d s o n b o t h t h e f r a ct io n o f t h e m e t a l b o u n d t o T f i n s e r u m a n d t h e

i n t e r a c t i o n s o f t h e m e t a l - T f c o m p l e x e s w i t h t h e t r a n s f e r r i n r e c e p t o r . S i n c e

L M W m e t a l c o m p l e x e s t e n d t o b e c l e a r e d f r o m b l o o d r a t h e r q u i c k l y , b l o o d

c l e a r a n c e r a te s t e n d t o b e i n v e r s e ly r e l a t e d t o t h e e x t e n t o f t r a n s f e r r i n b i n d i n g .

E v e n a r el a ti v e ly s m a l l p o o l o f l ab i le L M W c o m p l e x e s m a y p r o v i d e as i g n i f i c a n t s o u r c e f o r t r a n s f e r r i n - i n d e p e n d e n t m e t a l u p t a k e .

T h e r e is a ls o a s ig n i f ic a n t v a r i a t i o n i n t h e e x t e n t t o w h i c h m e t a l - t r a n s f e r r i n

c o m p l e x e s c a n u t i l i z e t h e t r a n s f e r r i n r e c e p t o r t o e n h a n c e c e l l u l a r u p t a k e .

C e l l u l a r u p t a k e v i a r e c e p t o r m e d i a t e d e n d o c y t o s i s h a s b e e n d e m o n s t r a t e d f o r

G a 3+ [ 82 , 9 9 ] , M n 3+ [ 2 8 3, 2 8 9 , 2 9 1 ] a n d A 1 3 + [ 1 8 5 , 1 8 6 , 1 8 9 , 1 9 1 , 1 9 2 ] a l t h o u g h

t r a n s f e r r i n - i n d e p e n d e n t p a t h w a y s m a y a l s o b e i m p o r t a n t [ 9 8 ] . T h e I n - T f

c o m p l e x c a n b i n d t o t h e t r a n s f e r r i n r e c e p t o r , b u t t h e I n 3+ i s n o t t r a n s p o r t e d

a c r o s s th e m e m b r a n e [9 4, 11 6]. T h e a b s e n c e o f r e c e p t o r - m e d i a t e d u p t a k e o r a

s i g n i fi c a n t L M W p o o l o f I n le a d s t o a m u c h l o n g e r r e s i d e n c e t i m e i n s e r u m f o rt h e I n - T f c o m p l e x . T h e P u - T f co m p l e x d o e s n o t a p p e a r t o b i n d t o t h e

t r a n s f e r r i n r e c e p t o r [ 22 5, 2 2 6] . F o r t h is m e t a l i o n , t h e i m p a c t o f t r a n s f e r r i n

b ind ing i s t o r e t a rd b lood c l e a ra nc e , w h ic h e xpose s t i s sue s t o a r e l a t i ve ly l ong-

l iv e d , p o o l o f m o r e r e a c t iv e L M W P u c o m p l e x e s .

T h e v a r i a t i o n i n th e r e s p o n s e o f M - T f c o m p l e x e s t o th e t r a n s f e r r i n r e c e p t o r

m a y b e d u e t o s e v e ra l f a c to r s . C o n f o r m a t i o n a l d i f fe r e n c e s b e tw e e n M - T f a n d

Fe3+-Tf , due e i t he r t o t h e i on i c r a d ius o f t he m e ta l i on [86 , 118, 119 ] o r t o a

h i g h e r c o o r d i n a t i o n n u m b e r f o r t h e m e t a l [ 1 1 8 ] , m a y r e d u c e t h e a v i d i t y w i t h

w h i c h t h e M - T f c o m p l e x b i n d s t o t h e t r a n s f e r r i n r e c e p t o r . I n a d d i t i o n , t h e

ina b i l i t y t o r e du c e m e ta l s l i ke G a 3+ a nd In 3+ m a y h in de r t he re l e a se o f t he

m e t a l f r o m t h e c o m p l e x b e t w e e n M - T f a n d t h e tr a n s f e r r i n r e c e p t o r [9 8, 1 96 ].

5

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