MATERIALS AND METHODS

COLUMN MATERIALS

AH - Sepharose-4B, Sephadex G-100, Sephadex G-200,'

Blue dextran, Low molecular weight markers f~r gel

filtration, BSA (67,000), Ovalbumin (43,000), chymotryp­

sinogen A (25,000), Ribonuclease A (13,700), were pro­

ducts of Pharmacia Fine Chemicals,Sweden. All columns

used in the purific~tion were made of glass or Bio-Rad

columns.

Electrophoresis Materials

Acrylamide, N-N'-methylene bis-acrylamide and

brqrqophenol blue were purchased from Bio-Rad L~boratories,

USA. Commassie brilliant blue R-250, sodium dodecyl

sulfate, 2-mercaptoethanol, ammonium persulfate, TEMED,

were obtained from Sigma Chemical Co., St. Louis.

Molecular weight marker kit for SDS-PAGE, e. g'., Bovine

sert;tm albumin (67,000), ovalbumin (43,000), carbonic

anhydrase (30,000) ,Trypsin inhibitor (20,000), Lactal­

bumin' (14,400) was purchased fromPharmacia Fine Chemi­

cals, Sweden.

Radioactive Chemicals

r 125 (specific activity 100 mCi) were obtained

from the Radiochemical Centre, Amersham, Bucks, UK.

Ch~micals

Hyaluronic acid type-r (from human umbilical. cord) ,

gl utaraldehyde, glllcuronic acid, N-acetyl-gl ucosamine,

,D-glucuronolactone, N-acetyl-neuraminic acid, chondroitin-

4-sulphate, chondroi tin-6-sulphate, Protease type XIV

- 32 -

(Pronase E) ,Tris, Conunassie brilliant blue R-25,O,

p-dimethylaminobenzaldehyde, EDTA, glycine, hyaluroni­

dase; chondroitinase ABC, heparin, blue dextran, sialic

aCid,'thiobarbituric acid, cyanogen bromide, dye amido

black, cyclohexanone, Toluidine Blue, toluidine were

obtained from Sigma Chemical Co., St. Louis. Polyethy­

lene glycol-20~000, was from Fluka, BuCha Switzerland,

Protein reagent was purchased from Bio-Rad Laboratorie~.

Iodogen was from Pierce Chemical Col, Rockford, USA.

Iodogenis a trademark for chemical 1,3,4,6-Tetrachloro-

30(.., 6 oc;..-Diphenyl-glycouril. Mono-sodium citrate,

methyl-cellosolve, Thiodiglycol, Brij-35, sodium-hydroxide,

sodium chloride, sodium acetate hydrazine-sulphate,

Ninhydrin were .from E. Merck., England .. Some of the other

chemicals were purchased from E.Merck and BDH (AR grade) .

All other solvents and acids used, were obtained from

conunercial sources in highest purity grade available.

Millipore immercible CX-10 single use - ultrafiltration

unit were purchased from Millipore, USA~

Processing of Dialysis Tubing

Dialysis tubing obtained from Medicell International

Ltd., Liverpool, London, (England) ,was boiled for fi~e

min in 10 mM EDTA solution and then washed with distilled

wa ter.

Maintenance of Rats

Wistar rats of different age groups were purchased • from Haryana Agricultural University, Hissar and All

In~ia Institute of Medical Sciences, New D~lh{. The

rats ~ere maintained with proper facility and bred f6r

- 3.3 -

newborn (2-3 days), 3-4 week (young ones) of age rats in

our animal house. Animals were fed ad libitum -the diet

obtairied from Hindustan Lever Ltd.

Activation of AH-Sepharose-4B with Glutaraldehyde

Cambiaso's(142) method was used for the activation

of AH-Sepharose-4B beads with ,glutaraldehyde. The glu­

taraldehyde acts as a homobifunctional reactant. To

couple, the desired ligand, -Le., hyaluronic acid on one

hand and the commercially availableSepharose-4B beads

already substituted with 6-amino-hexyl side chains (AH­

Sepharose-4B, from Pharmacia) on the other hand, made·

the task to conjugate HA to amino hexyl sepharose easier,

by means or two step reaction with glutaraldehyde and

later 'the conjugation ,with HA. The aminated gel, was

first washed with ten volumes of PBS (0. aIM Phosphate

buffer saline; pH 7.2,0.80% NaCl) on a sintered glass

filter. 7.0 ml PBS b~ffer containing one ml of 25%

glutaraldehyde was added to 3.0 ml of packed gel with

constant stirring so that the final concentration of

glutaraldehyde was 2.5% •. This represents a lOa-fold

molar excess of glutaraldehyde over the amino groups .of'

the aminated gels (8 p-mole NH 2/ml packed gel). A

yellow colour soon developed after the addition of gluta-

'raldehyde. The reaction was allowed to proceed for

twenty minutes at 40 C after which the tinreacted glutaral­

dehyde' was eliminated by washing five times with 20 ml

PBS ..

Coupling of Hyaluronic Acid to the Glutarladehyde Acti­

vated Amino-Hexyl Sepharose

As mentioned above, the aminated gel, which is now

- 34 -

glutaraldehyde activated gel, we added 1 ml of hyaluronic

acid solution (Grade I) (10 mg/gm of gel) in PBS to 3 ml

of glutaraldehyde activated gel,under continuous mild

stirring. The reaction was allowed to proceed at 40

C ·for

20 min. After'the incubation period, the gel was washed

five times with 20 ml of PBS to allow the non-conjugated

hyaluronic acid to diffuse out of the gel beads. A

further washing with 0.2 M'glycine;buffer, pH 8.5 was

carried out to remove non-covalently bound hyaluronic

, acid~

The conjugation of hyaluronic acid to glutaral­

dehyde activated gel was checked quantitatively. As the

quantity used to couple'HA to glutaraldehyde activated

gel,was known, and the total content of'HA in the wash

was determined according to Bitter and Muir(145) and

deducted from the absolute amqunt used for conjugation.

Thus, it was seen'that 2.2 mg of HA was bound perml of

packed Sepharose gel. The HA-coupled sepharose, AH­

Sepharose-4B was scanned in Shimadzu UV240 from 200 to

300 nrnwavelength.Gel was suspended. in 42% sucrose

solution and scanned (Fig. 3). The gel was further in­

cubated for sixteen hours at 40 C with 0.2 M glycine­

buffer, pH 8.5, to block unreacted aldehyde groups, and

to completely prevent freshly activated gel from cova­

lently binding hyaluronic acid.

prepara'tion of Chondroi tin-4-Sulphate-sepharose-4B Affinity

Gel

The method used to couple-chondroitin'"-4-sulphate­

to AH-Sepharose-4B via glutaraldehyde was same as men­

tioned in hyaluronate-Sepharose affinity gels. Accord.-

•

>-t---Vl Z UJ 0

---1 0.1 « LJ

t--0... 0

I I I I' I , , \ \ \ \ \ \ \ \ \ \ \ \ \

\ \ ,

\ \ \

'-..-.-....

'\ , " " " " , '" '"

\ .'

\

"

\ \ \ \ \

\

" " " ----'0.0 0 '---______ -----JL....-____ -_-_--=-:.==-~...oL

Fig. 3

200 300 WAVELENGTH nm

Absorbance spectrum of HA, HA-Sepharose, and AH-sepharose-4B. AH-Sepharose-4B and HA-sepharose Were suspended in 40% sucrose and their absorption spectra at 200-400 nm. __ ' ___ Hyaluronic acid -------- AH-Sepharose-4B -.-.-.-.-. HA-sepharose

400

- 35 -

. , ing to Cambiaso et al. (142) to 3 ml of glutaralciehyde

activated gel, j ml of chondroitin-4~sulphate (Type-C)

(10 mg/gm of gel in PBS) was added, under constant mild

stirring. The reaction was allowed to proceed at 40

C

for twenty minutes after th'? incubation period.' The gel

was washed with 20 ml of buffer for five times to allow

the non-conjugated chondroitin-4-sulphate to diffuse out

of the gel beads. A further washing with 0.2 M glycine

buffer, pH 8.5, Vlas carried out to remove non-covalently (

bound chondroitin-4.;..sulphate.

In order to check the coupling of chondroitin -4~

sulpha te to the gl utaraldehyde-acti va ted gel, the gel

was scanned in ShimadzuUV-240 . from 200 to 300 nm wave

length. The gel was suspended in 40% sucrose solution and

scanned (Fig. 4). The amount coupled was determined by

chondroitin-4.;..sulphate estimation of coupling solution

before and after coupling. Approximately 1.7mg of chondroitin-

4-sulphate was bound per ml ·of packed gel.

The gel was incubated for sixteen hours at 40 C with

0.2 M glycine buffer, pH 8.5, to block unreacted aldehyde

gr:o,ups, arid to completely prevent freshly activated gel

from covalently binding to chondroitin-4-sulphate. When

the gel was nOt in use, they were stored in 0.02% sodium

'azide in buffer to prevent microbial growth.

Activation of Sepharose .with Cyanogen Bromide:

Porath's (143) procedure was used for activation

of Sepharose 6B beads with cyanogen bromide. 10 gms of

Sepharose 6B was washed with 40 ml of 2M potassium phosphate

buffer pH 12.1 on a sintered glass funnel (G2 ). 'I'hen the

gel was suspended in 15 ml of 5M potassium phosphate

>-~ -C/)

z w 0

~

<i u -~ Q... 0

O. 2 .-r-~;----.---~--------~---4

0.1

I' I" I '\

l \ I I \ \ \ \ \ \ \

\ \ \

\ \

"

\

\ . \

" " ;\

" '- ....... \ .........

........ ....... \

" O.OO~--------------~--------~~~--~~

. 200

Fig. 4

300 WAVELENGTH nm

400

Absorption spectrum af chondraitin-4-sulphate chondroitin-4-sulphate­sepharose, and Jl.H-sepharos-48. AH-sepharose-48 and chondroitin-5-sulphate-sepharose, were suspended in 40% sucrose and their absorp­tion spectra at 200-400 nm.

-.-.-.-.-.

Chondroi tiri-4-sulphate AH-sepharose:.48 Chondra i t i n-4 -sulpha te -sepharose

- 36 -

buffer pH 12.1. 6 ml of cyanogen bromide solution(lOOmg/

ml) was added in portions during a period of 2 min and

the reaction was allowed to continue for 8 more minutes

with gentle stirring. The temperature of S°";;lOoC was

maintained during the reaction time. The product was

washed with cold distilled water to neutral pH. Immediately;

after the washing the activated gel was tran~ferred ~o

heparin i.e., ligand solution, activation was done in a

well ven'tila ted place or under· furnehood. Precautions was

taken not to touch or inhale CNBr. vapours.

Activation of gel was checked qualitatively according

to the procedure described by Kohn and Wilchek (144). '1'0 a

few drops_of activated material one or two ml of qualitat­

ive reagent was added. After 30 seconds a red purple

colour developed which,became maximum after 10 min. The

qualitative reagent was prepared by mixing 12 ml of pyridine ~

slowly with 2.5 ml of concentrated hydrochloric acid. 0.5 g

of barbituric acid was added ,to this and volume was made , .

upto 20 ml with distilled water. A clear. colourless solution

was obtained after stirring for 10 min.

Preparation of Heparin Sepharose:

20g of activated Sepharose 6B was suspended in 100 ml.

of heparin (1 mg/ml) solution made in 0.1 !vI N,aHC0 3 contain­

ing O.SM Kcl. The suspension was kept at 300 C 'for overnight

with gentle stirring. The gel was filtered on a sintered

glass funnel (containing O.lM NaHC03 and O.SM Kcl) and O.SM

Kcl solution. Heparin concentration in coupling solution

was determined before and after coupling reaction by using

to~uidene blue. To 20 pl of sample, 0.5 mI. of O.lSM Nacl

- 37 -

solution was added. To thisO.Sml buffer containing O.lM

Boric acid, O.ISM NaCl, pH 8.5 and 0.2Sml. of Toluid:tne

Blue (0.015%) were added absorption at SDQ nm was

recorded (Fig. S);

All these column chroma~ographic analysis were

conducted in Pharmacia Frac-100 Fraction Collector with

uV-I monitor, peristaltic pump and Recorder I with the

efficiency of monitoring as low as S fl9 of protein.

'Assay of Hyaluronic acid:

I, Hyaluronic acid was estimated according' to the

method of Bitter and Muir (14S) S ml. of sulphuric

acid reagent (0.02SM sodium tetraborate, 10H2 0 in

sulphuric acid sp. gr. 1.84) was cooled at 4o C, 1 mI.

of the sample is lay~redon to the acid. The tubes were

closed with glass stoppers. The tubes were shaken at

first gently and then vigorously with conStant cooling;

Th,e tubes were then heated for 10 min. in a boiling

water bath and cooled at room temperature. 0~2 ml of

carbazole reagent (0.12S% carbazole in absolute ethanol)

was then added, the tubes were shaken again and heated

in a boiling water bath for further lS mi~. It was then

cooled to room temperature. The optical density was read

at S30"rnu. Glucuronolactone standards 'of 20-120 umoles/ml

were used as shown in Fig 6 by dilution with deionized

water saturated wi,ttl benzoic acid from a stock standard

in water saturated with benzoic acid.

'Fig. 5

2

.-. I \. .

w I \ u . z I \ <i "'-, (I) 1 I

I , /. , \ a:: \

0 i: .' \ Vl , .,

m II , " \. <i

., .~'

,. ~- ---...........

.........

OL..------....I.------....J 190 240 .290

WAVELENGTH (nm)

Absorbance spectrum of heparin, CNBr-Sepharose and sepharose CN3r-sepharose and heparin-sepharose were in 40% sucrose and their absorption spectrum were taken.

- ..... -.-.-.

heparin CNBr-sepharose Heparin-sepharose

heparin­suspended

1.2

·1.0

E c 0.8

(:)

rn LI"I

.t-:- 0.6 4:

>-t-:- . -l/) 0.4 ·z UJ Cl

. ....J 4: 0.2 u -t-o... 0

0.02 0.04 0.06 0.08 0.10 0.12 ..

( 0 NeE NT RAT ION 0 F G L U CUR 0 NO LAC TON E (f mol e·s )

~6.. Standard Curve for hyaluronic acid.

- 38 -

Assay of Hyaluronidase

Hyaluronidase activity was measured by the method of

polansky and Toole (88). Hyaluronidase activity was

determined by the measurement of the·terminal N-accetyl­

glucosarnine in oligosaccharides released during incubation

of hyaluronate with hyaluronidase.

Hyaluronidase'activity was measured simultaneously

for standard hyaluronidase and for purified hyaluronic

acid binding protein.

For the standard hyaluronidase, the incubation·

mixture consisted of 0.8 ml of HA (0.8 mg/ml, in 0.1 M \

sodium formate buffer,· pH 3.7, containing 0.15 M Nacl)

and O. '4 mI. of standard hyaluronidase (Sigma, from umbi­

lical cord) (200ug ml of 0.1 M sodium formate buffer,

pH 3.7) whereas, the incubation mixture for purified

hyaluronic aciq binding protein consisted of 0.8 ml of

HA, (0.8 mg/ml, in 0.1 M'sodium formate buffer, pH 3.7),

containing 0;15 M Nacl) and 0.4 ml of purified HB~

(200 ug in PBS). The standard hyaluronidase as well

purified, HBPwith the 'above incubation mixture was

, incubated for six hours in a water bath at 37o C. pre­

c~ution,was taken to. check microbial growth during the

i'ncuba tion by adding 15 rnM Saccharolactone to the

incubation mix~ures. The blank corisisted 0.4 ml of

purified HBP and'0.8 ml of 0.1 M sodium formate buffer,

PH 3.7 containing 0.15 M NaCl. Enzymatic reaction was

terminated by boiling the tube at 100~C for 3 minutes:.

The enzym'atic activity of blank was' terminated at zero

minut,e. Release of terminal reducing N-Acetyl hexosamine

as a result of hyaluronidase action was measured accord-,

, ing to Reissig ~t al (146).

- 39 -

N- Acetylglucosarnine was estimated according to

Reissig et al (1461 .. To 0.5 ml of samples was added O.lml

of sodium tetraborate, pH 9.2. The tubes were kept in a

boiling water~bath for exactly 3 minutes and cooled in

tap water. 3 m'l. of DMAB reagent (10 .,9 of p.-DIV'lAB·was

dissolved in 100 ml of, glaci,al acetic acid which

contained 12.5% wjV ION Hcl. Before using it was diluted

with 9 volumes of glacial acetic acid) was then added

and immediately after mixing the tubes were then placed

in a water bath at 36-380 C. After precisely 20 minutes the

tubes are cooled in tap water ,and the optical density was

read at 578 mu. Standard curve for N-acetylglucosamine

is presented in Fig. 7.

Assay of Sialic Abid:

. The Sialic Acid content was measured according to

Warren etal (147). To a sample of 0.2 ml (approximately . -o .o? ~ole of N -acetylneuramn.ic acid) 0.1 ml,of periodate

in 9 M phosphoric acid} was added. The tubes were shaken

and a~lowed to stand at room temperature for 20 minutes.

Iml of arsenite solutism (10% sodium arsenite arid O.OSM

sodium sulfate in 0.1 M sulphuric acid) was added. The

tubes were shaken until a' yellow brown colour disappears.

Thiobarbituric acid solution (0.6% in O. 5M sodium, sulfate)

,was added, tubes were shaken capped with glass stopper

and heated in a boiling water bath for 15 min. The tubes

are then placed in cold water for five miriutes. This

,~ixture4.3 ml is tran;ferred to a tube containing 4.3 mI.

of cyclbhexanone. The tubes are shaken twice qnd then

centrifuged for 3' minutes in a centrifuge. The clear

upper cyclohexanone phase was red and the colour is more

0.6

-. E 0.5

-00 " 0.4 L()

t-e:(

>- 0.3 t- '

Ul Z w 0.2 c ...J

~ 0.1

t-o... o

.02 .04 .06 .08 ·10 '·1 ,1

CONCENTRATION OF N-Ac-Gm (fJ mol ) ,

~I Standard curve for N .... AcetyJ-gJucosamine.

- .40 -

intense than in wat~r. Optical density of the organic

phase are -determined ,at 549 nm. Blank contained 0.2 mI.

of distilled water. N-acetylneuraminic acid was used

for the standard curve and presented in Fig. 8.

Protein Determination:

The protein content of the column effluent was

monitored at 280 nm using an ISCO UA-5 absorption

monitor with type 6 optical unit.

Protein is assayed by the method of Bradford

(148) using Biorad Bradford reagent and BSA as standard.

This method is based on the observation that cOmniassie

brilliant blue G-250 exists in two different colour

forms~ red and blue, the red form is converted to the

blue upon binding of the dye to the protein. The protein

dye has a high extinction coefficient thus leading to

great sensitivity in measuring protein. Th~ binding of

the dye to protein is a very rapid process (approx. 2 min)

and the protein-dye complex remains dispersed in solution

for relatively longtime (approx. 1 hr), thus making the

procedure very rapid and yet not requiring critical

timing for assay.,

preparation ot Bradford Reagent:

Commassie brilliant blue G-250 (100 mg) was

dissolved in 50 ml. of 95% ethanol. To this 100 ml.of

85% (V/V) phosphoric acid and 50 ml. distilled water

was added. Final concentration in the reagent were 0.05%

(W/vt Commassie brilliant blue G-250, 23.5% (W/V)

ethanol, and 42.5% (W/V) phosphoric acid.

0.6 E c

C3' 0.5· ~

LI1

r-« 0.4 >-r-(/) 0.3 z w 0 --l 0.2 « LJ

r- 0.1 a.. 0

0.2 0.4 0·6 '0 .. 8 1.0 1.2 1.4 (0 nc.o f N - Ace t y 1- N e u r Q min i C Q C i d ()1m 0 Ie s 1

Fig. 8 Standard Curve for N-acetyl-neurarnic acid.

- 41 -

Assay Procedure:

. Protein standard containing 1 to 20 JIg in a volume

of 0.1 ml. then diluted the sample to 0.8 ml. with

distilled water and 0.2"ml. in Bradford reagent was

added to it and mixed several times by gentle inversion

of the test 'tube. ,The absorbance of 595 nin was measured

after 5 miri. to one hou~ against a reagent bl~nk prepared

from 0.8 ml. of the appropriate buffer water anq 0.2 ml.

of Bradford reagent~

ELECTROPHORESIS

a) Polyacrylamide gel Electrophoresis (PAGE),:

The purity of the enzyme was checked by disc gel

~lectrophoresis at pH 8.9 by the method of Davis (1491.

Elettrophoresis was carried out at 4o C. The separation

gel was made of 7.5% aorylamide and 0.27% N, N-methyl

bi~-acrylamide coritained 0.375M Tris-Hcl buffer, pH

8.9. 'The stacking gel. of 3% acrylamide and 0.08% N,- N­

methyl bis-acrylamide contained 0.067 M,. Tris-Hol pH 6.1. '"'

Then;the solution was polymerised b"y adding 0.050%

ammonium persulfate and 0.0"50% TEMED. The gels, were

poured in glass tube ( 6 x 100 mm). The sample protein

(10~40 ng) with 10% glycerol was loaded. The running

buffer containedO.05M Tris-Hcl and G.3 M glycine at .

pH 8.3 and electrophoresis was commenced by applying 1

rnA current per gel. After 30 min. the current was , .

raised to 4 rnA per gel, till" the marker dye reached about

0.5 cm from the bottom of the gel tube.

- 42 -

After electrophoresis, the gels were sta~ned for 4

hours for protein with 0.2% corrunassie brilliant blue

R-250 in methano~, glacial-acetic acid and water (25 : 10

65) and then destained in methanol, acetic acid and water

(20: 10: 70).

b)SDS - Polyacrylamide gel'Electrophoresis(SDS-PAGE):

"',',;

SDS-PAGE was done according to the method of Laemmli

(150). The ~eparating gel was made of 10% acrylamide 0.27%

N~N' methylene bis - acrylamide, 0.1% SDS and 0.375 M tris­

Hcl buffer (pH 8.8). 'l'he various_ components were mixed

deaerated-and polymerization was initi'!l-ted by adding \

0.025% ammunlum persulfate. The, solution was quickly

poured into 6 x 130mm gel tubes, and water layered on

top. After polymerization was over,stacking gel contain­

ing 3% acrylamide, 0.08% N-N' ..:. methylene - bisacrylamide

O~l% SDS and 0.125 M Tris-Hcl (pH 6.8) was poured and

allowed to polymerize. The separation gel of 100 rom were

prepared. The running buffer was composed of Tris(0.025M)

glyCine (0~192 M), buffer, pH 8.3 ~nd 0.1% SDS~

The sample buffer for SDS-PAGE had the following

composition:

, Tris-Hcl

SDS

'Glycerol

2- Mercaptoethanol

Bromophenol blue

0.0645 M(pH 6.8)

2 %

10%

5%

0.001%

The samples were irrunersed for 2 min. in boiling

water bath cooled and loaded onto the gels. Standard

proteins were also run each time alongwith the sample.

- 43 -

After loading the samples, stacking was carried out at

1 rnA per gel in constant current mode and electrophoresis was

donee; by ·using a current of 4mA per gel at 30oe, till the

marker dye reached about I em from the bottom of the gel

tube. The gels were stained overnight after the run in a

staining solution and detained as mentioned in neutral

gel electrophoresis.

Molecular, Weight Determination:

Molecular weight determination of Hyaluronic acid

binding protein was determined by gel filteration on a

Sephadex G-100 column ( 1 x 57cms) according to the

method of Determan and Michel (151). The column was first

equilibrated with PBS (0.01 M Phosphate buffer pH 7.2 Nacl

8 gm/L) by passing at least five column volumes of the -

buffer through the column at a constant pressure and

flow rate. After equilibration, the void volume of the

column was determined by passing 2 rnl (2 mg/ml) Blue

Dextran. The position of the maximum of the peak in ;the

elution volume was taken as the elution volume of blue

dextran as well as the void volume (Vo) of the column

as in Fig. 9. Fo:!;, calibration of the column four prot'eins

of known mole6ular weight (ribonuclease, 13,700);

Chymotrypsinogen, (25 i 000); ovalbumin (43,00 0) and bovine

serum alb}lmin 6 mg/2ml (67,000) were run and from the

elution volume of each proteinKav and from the elution

volume was determined with the help of the following

relationship:

0.30

0'.25

0.20 0 co 0.l5 ("oJ

<!

0.10

0.05

0, 5 10 15 20 25 30 35 40 F R A[ T ION N U M B E R

Elution rofileof Blue-Dextran on se hadex G-100 column: 2 ml Blue dextran (2 mg mJ) in 100 mM Sodium phosphate buffer containing 0.8% NaCI, pH 7.2, was applied to a Sephadex G-100 column (1x54cm) equilibrated previously with the 'same buffer. Elution was done with' the same buffer at a constant flow rate of 16 ml/hr.

K " av

K av

- 44 -

=

=

=

=

=

v - V e 0

Vt - Vo

elution volume of standard proteins.

Void volume.

Total volume of the gel bed.

Partition coefficient betwee

the liquld phase and the gel

phase.

By plotting K values against logarithm of mole~ av cular weights (in a semi-logarithmic plot) a standard

curve was obtained. Purified HBP and low mol.wt.protein

markers were run through the column and from their

elution volumes (V ) molecular weight was determined , e using the calibration curve.

AMINO ACID ANALYSIS

Amino acid analysis was done according to Moo~e

et ai. (152) ~n the Technicon Sequential multisample(TSM)

amino acid analyzer. It is an automated instrument

designed to separate, detect and quantitate amino acids.

Principle:

The rate of travel of amino acids on a column of

a sulfonated polystyrene resin is a function of both the

charges possessed by an am~no acid and the riature of its

side chain, it is a result of the affinity of the resin

for both the ionic and'non-ionic portions of the molecule.

- 45 -

Thus" an amino acid with a branched side chain will be

~luted earlier than a straight sid~ chain, likewise,

an amino acid, with the presence of an acidic group on

side chain will be eluted earlier than the basic group

on side.chain.

Reagents Used:

1. Buffer No.1 - 100 mM Citrate Buffer, pH 3.25

To 3 litre of 100 mM citrate buffer in IN NaOH

was added,

a) 240 ml of methyl cellosolve

b) 1.0 ml thiodiglycol

c) adjusted pH 3.25 with 6N HCl

d) added 40 ml 6f Brij-35.

Volume made to 4 litres with distilled water.

2. Buffer No.2 - 100 roM Citrate Buffer, pH 4.25

To 3 litres of 100 mM citrate buffer in IN

NaOH was added.

a) 1. Q ml of thiodiglycol

b) pH adjusted to 4.'25

c) added 4'0 ml of Brij-35.

Volume made upto 4 litres.

3. Buffer No.3 - 100 mM Citrate Buffer, pH 5.25

q'o·3 litres of 100 mM citrate buffer in IN

NaOH added

a.) 1.0 ml of thiodiglycol

b) pH adjusted to 5.25

c) added 40 ml of Brij-35

d) added 40.0 gm of NaCl.

Volume made upto 4 litres with distilled water.

? - 46 -

4. Ninhydrin Reagent

1% Ninhydrin reagent is used in AN sodium

,acetate buffer and methyl cellosolve in 4 litres of

total buffer.

5. Hydrazine Solution

2 mM solution of hydrazine sulfate was made in

.distilledwater and 30% of Brij-3S, making the volume

to 4 litres.

6. Methyl Cellosolve

50% of methyl cellosolve was made in distilled

water.

PROCESSING OF THE SAMPLE

a) Hydrolysis

Hydrolysis of the sample. was 'done according to

Moore et al.(lS2) and as modified by Chatterjee 'and

Abrol (NRL, IARI., New D'elhi, unpublished) .• (153) .

One mg of HBP (purified) was taken in hydrolysing

tubes (18 x 150· mm) and 12.0 ml of 6 N HCl was added.

The sample in the tubes were chilled and evacuated for

30 min. The hydrolyzing tubes were sealed under vacuum

and kept at 1100 + lCC for 24 hrs. After the hydrolysis,

the tubes are brought to room temperature (If the

analysis is not to be performed immediately, the hydro­

lysis tubes can be stored in the deep freeze) .

- 47 -

b) Concentration by Flash Evaporation

After hydrolysis, the seal was broken and the

contents filtered through a sintere4 glass funnel. The

hydrolysing tube and the funnel were washed several

times with double distilled water to ensure qualitative

recovery of the amino acids. The filtrate was quanti­

tatively transferred to a round bottomed flask of a

rotary flash evaporator. The sample was evaporated to

dryness at 50-550 C under 'reduced pressure. The residue

was washed 5-6 times with small volumes of double

distilled water to get rid of the excess HCl. Finally,

the residue was dissolved in one ml of sample buffer

(0.1 M sodium citrate" pH 2.0). This was the stock

supply of amino 'acids.

c) Loading of Sample for Analysis on TSM

An aliquot, of stock' (100 ul) was loaded on 'to two

sample cartridges, care, was taken not to leave sample

on the side walls of the, cartridges.

While keeping it vertical, a vacUum was applied

to the bottom of the cartridge. As soon as the sample

was drawn from the top filter, vacuum was stopped and

a Teflon plug was inserted. Norleucine was used as

an internal standard.

d) Chromatographic Ana!'ysi~

The automatic recording equipment is used in

conjunction with 2 ion-exchange columns of sulfonated

polystyrene resins. The long column is for the separa­

tion of neutral and acidic amino acids while;shorter

- 48 -.

for basic amino acids. Buffers'are delivered to the

columns-through the sampler, from two high pressure

pumps. The sequence of these buffers from the storage

reservoirs is controlled bi a peristalic valve. This'

valv~sequence~ the buffer, as well as th~ flow of

effluent to the analytical system or waste. It is

electromechanically controlled.

The column effluent that flows into the analytical

system are mixed with a segmented stream of reagents

into the' system through the proportioning pump. The

combined streams are then passed through a heating bath

equipped on its output side, with a finned cooler. The

separated amino acids react to form a coloured product

in the reagent stream, whose absorbance is monitored

continuously in a colorimeter and recorded by a two-pen

recorder as a linear trace of each amino acid.

To calculate the amount of each amino acid

present in an·unknown sample the following formula is

used

A, )l moles = ~p x NE x )lMsp.

).m sp

where, A = Area unknown peak )lP

Aun = Norleucine area from the same chromatogram

NE = Norleucine equivalent to the corresponding sp peak from a standard chromatogram

)lMsp = )lffioles in the standard sample (0.025 value

was used for calculations).

- 49 -

IODINATION OF PROTEIN

Protein Iodination by Iodo-Gen

a) Principle

Iodo-Gen was first described by Fraker and J •.

Speck'as a reagent for the iodinatio? of proteins and

cell membranes. 1,3,4, 6-Tetrachloro-30\,,6 G(.-diphenyl­

glycoluril (Iodo-gen) is extremely stable and insoluble

,in water allowing rapid iodination in the solid phase

with aqueous solutions of I and proteins. The incidence

of side chain reactions with this method is negligible.

Iodo-gen dissolved in chloroforms are plated on walls of

the test-tubes. Iodinations are terminated by decanting

the r~action solution from the solid Iodo-Gen. There­

fore, no reducing agent is needed. This reagent can

iodinate micrograms of protein.

I

b) Iodination of hyaluronic acid binding protein

The Iodination of hyaluronic acid binding protein

was performed according to. Fraker et ai. (154) by using

a sparingly soluble chloramide, 1,3,4,6, tetrachloro-3oc.,

6 OC-dipenylg lyco11.lril, . called as Iodo-gen. A thin

film of Iodo"':gen was made by taking 10 ).1g of Iodo-gen

in 20 »1 of.chloroform in a test tube (10 x 75 mm).

The chloroform waS evaporated by rotating th~ tube in

a 370 C bath so that a thin film of Iodo-gen is formed

at the bottom. This is done, because of the fact that

Iodo-gen reacts rapidly in solid phase with aqueous

mixtures of iodine and proteins to yield iodinated pro­

teins.

- 50 -

To 20 pI of chloroform containing 10 pg of Iodo­

gen (23 x 10-25 mole) was placed in a 10 x 75 nun test

tube, the chloroform was evaporated by rotating the tube

in a 370 bath so that a thin film of IOdo-gen is formed

at the bottom. To this Iodo-gen ~oated tube 20 pI (6 pg)

of HBP in 0.01 M phosphate buf~er, pH 7.2, was added.

20 ul of Iodination buffer (0.1 M phosphate buffer, pH

7.2, with 0.1% BSA and 0.1 sodium azide) :5 )..lCi of (NaI*)

of 125 t and 20 ul of KI (50 ug). The reaction was

allowed to proceed for 5-10 min at 42 0 C with gentle stir­

ring; the reaction was terminated by loading this iodi­

nated,mixture on G-25 column which has been previously

packed on disposable 10 ml pipette and equilibrated with

0.1 M phos'phate buffer, pH 7.2, containing 0.1% BSA and

0.1% sodium azide. Eluates of 500)..11 were collected,

counted, iodinated proteins were seperated from ·free

1 125 and were pooled and then used for bi~ding assays.