immunochemical studies on blood groups: liii. a study of various conditions of alkaline borohydride...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 148, 394-314 (1972)

Immunochemical Studies on Blood Groups

LIII. A Study of Various Conditions of Alkaline Borohydride Degradation on

Human and Hog Blood Group Substances and on Known Oligosaccharides’

BYRON ANDERSON,2 LUCIANA ROVISa AND ELVIN A. RABAT

Departments of Microbiology, Neurology and Human Genetics and Development, College of Physicians and Surgeons, Columbia University and the Neurological Institute, Presbyterian Hospital,

New York, New York 10032

Received September 21, 1971; accepted October 21, 1971

Blood group A, B, H, Lea, Leb, and I substances, their products of periodate oxidation and Smith degradation, and disaccharides containing S-O-substituted reducing N-acetylhexosamines were treated with base-borohydride under three defined sets of conditions. Procedures for the assay and quantitation of the possible reduced base- degradation products, including hexenetetrol(s), 3-deoxygalactitol, galactitol, reduced chromogens, N-acetylglucosaminitol, and N-acetylgalactosaminitol are described. Extensive degradation occurred by two methods. 1 M NaBHl in 0.05 N NaOH at 50’ cleaves the glycosidic linkage of the oligosaccharide chains from serine and threonine with reduction of the terminal-reducing N-acetylgalactosamine with minimal base degradation. The method is useful for isolation of complete reduced oligosaccharides from blood group substances; the structural implications of the free and oligosaccharide-bound N-acetylgalactosaminitol released are discussed.

The A, B, Lea, Leb, and I blood group active substances isolated from mutinous secretions are glycoproteins with oligosaccharide chains glycosidically linked through N-acetylgalactosamine to serine or threonine of the protein portion of the molecule. This linkage is characterized by its alkali lability (1, 2). According to the proposed composite structure of the megalosaccharide (3), the main chain of the oligosaccharide is made up of alternating galactosyl and N-acetylglucos- aminyl residues predominantly in /3(1 -+ 3) linkage. Such S-O-substituted reducing

1 Aided by Grants GB-8341 and GB-25686 from the National Science Foundation and a General Research Support Grant from the United States Public Health Service.

2 Fellow of the Helen Hay Whitney and the National Cystic Fibrosis Research Foundations, 1968-1971. Present address: Department of Bio- chemistry, Northwestern University Medical School, Chicago, Ill. 60611.

8 Fulbright Senior Scholar.

sugars are particularly susceptible to base degradation so that the reducing oligosaccharide chains released from the protein are subject to further degradation by a base- catalyzed peeling reaction (4, 5). However, if the aldehydic group released initially is reduced with sodium borohydride, the degradation is stopped. In the presence of both base and borohydride, the base- catalyzed peeling and the borohydride reduction will compete (1).

Several studies have made use of the base- borohydride reaction on the blood group active glycoproteins producing both de- terminant and nondeterminant-containing oligosaccharides of a size amenable to structural studies (1, 6-9). The conditions used, 1% NaBH4 in 0.2 N NaOH or 1% NaBD4 in 0.2 N NaOD, result in considerable degradation as shown by the isolation of a galactitol and of hexenetetrol(s), unsaturated alditol residues formed by the double &elimination

304 Copyright @ 1972 by Academic Pm& Inc.

STUDIES ON BLOOD GROUPS LIII 305

mechanism on a 3,4-disubstituted galactose (7), and by the isolation of oligosaccharides terminated by galactitol, hexanepentols, and hexenetetrol(s) (6-9). The extent of the resulting degradation was advantageous since the oligosaccharides produced were of a size amenable to fractionation and since the unsaturated alditols provided information about the nature of the galactosyl branch points. The base-borohydride degradation of oligosaccharides has also been studied and the differential lability of the various linkages determined (10-12).

Various other baseborohydride propor- tions have been used to produce reduced oligosaccharides from ovine and bovine submaxillary mucins (13) and from M-active sialoglycopeptides (14). Weber and Winzler using still other conditions with several glycoproteins showed varying extents of reduction to N-acetylgalactosaminitol from N-acetylgalactosamine (15). Carlson treated porcine submaxillary mucin with 1 M NaBH4 in 0.05 N KOH at 45” and isolated N-acetylgalactosaminitol and five oligosaccharides terminated by N-acetylgalactosaminitol (16, 17) indicating that these reaction conditions liberated oligosaccharide side chains with reduction of the GalNAc4 before further degradation could occur. Iyer and Carlson (18) using similar conditions, 1 M NaBH4 in 0.05 N NaOH at 50”, showed minimal degradation of oligosaccharide side chains of human blood group H substance; N-acetylchondrosine, ,L?DG~cUA (1 -+ 3) DGalNAc, was completely reduced under these conditions with no evidence of p- elimination from carbon 3 (19).

The following studies are an application of the Iyer and Carlson technique (18). Gas- liquid chromatographic identification and quantitation of the possible degradation products formed by base-borohydride on several blood group active glycoproteins and on various disaccharides are presented. Several reduced chromogens can be formed from 3-O-substituted reducing N-acetylhexosamines and the amounts of each vary

4 Abbreviations: Gal = galactose, GalNAc = N-acetylgalactosamine, GlcNAc = N-acetyl- glucosamine, ManNAc = N-acetylmannosamine, GlcUA = glucuronic acid.

depending on the conditions of degradation. The methods presented can be used to establish the extent of or the absence of degradation of released oligosaccharide chains. The amount of free and oligosaccharide-bound N-acetylgalactosaminitol indicates the amount of unsubstituted and substituted GalNAc bound to the protein portion of the glycoproteins.

MATERIALS AND METHODS

Monosaccharides were obtained from Mann Research Laboratories or Nutritional Biochemi- cals Co. @Gal (1 + 3) DG~cNAc and @DGal (1 -+ 6) DG~cNAc were kindly supplied by Dr. A. Gauhe (20) and talosamine from the late Prof. R. Kuhn. PDGal (1 -+ 3) DGalNA@ was isolated from a partial acid hydrolyzate of hog gastric mucin (11); N-acetylchondrosine and a sample of reduced chromogen (21) were provided by Dr. Karl Meyer; truns-3-hexene-erythro-1,2,5,6-tetrol was obtained from Dr. E. F. L. J. Anet and the D-three

isomer from Dr. R. S. Tipson. The following human blood group active sub-

stances were used: Lea, N-l-l (lo%, 2X) (7); Beach (B) phenol-insoluble (22), MSS (A) (lo%‘,, 2X) (l), OG200j, 2~ (8) hog mucin (A + H) (1) and some products of periodate oxidation and Smith degradation (23); JS (H) phenol-insoluble (1) and its first, second and fourth stages of se- quential periodate oxidation and Smith degradation (3).

Nitrogen, methylpentose (fucose), hexosamine, N-acetylhexosamine, and hexose (galactose) were determined by calorimetric methods (6, 24). The direct N-acetylhexosamine reaction was done according to the method of Reissig et al. (25). Gas-liquid chromatography (glc) was carried out using an F and M (Hewlett Packard) 810 gas chromatograph. Separations were made on a glass column (200 X 0.3 cm) with ECNSS-M on Gas Chrom Q (26) using a temperature gradient as follows: hold at 150’ for 5 min, then rising at 2”/ min to 210’ and holding at this temperature until all peaks were eluted. Peak areas were measured and product formation was quantitated from the molar response factors of known standards.

Three reaction conditions were used to degrade the disaccharides containing 3-O-substituted hexosamines or the blood group substances: (1) 0.05 N NaOH plus 1 M NaBHG for 16 hr at 50” (18); (2) 0.2 N NaOH plus 1% NaBHa (0.27 M) for 24 hr at 24’ (1, lo), or for 1 week for the blood group sub-

5 This disaccharide was separated from the A-active disaccharide reported in Ref 11. Data on this oligosaccharide were not reported.

306 ANDERSON, ROVIS, AND KABAT

stances; and (3) 0.5 N NaOH plus 0.5 M NaBH4 for 20 min at 24’ (21).

Known amounts of the disaccharides (ca. 40@- 900 rg) were treated under the above conditions in a reaction volume of 0.1-0.7 ml. An exact amount of erythritol (ca. 40 pg) was added as an internal standard, the reactions stopped by adding an excess of Dowex 50-H+ (20-50 mesh), the mixture allowed to stand with excess Dowex for 15 min, the supernatant removed, and the resin washed. The combined solutions were dried in vacua over Pz05, and borate was then removed by the repeated addition and evaporation of absolute methanol. The sample was dissolved in water and divided into four parts. One part was dried in vacua and 0-acetylated by adding 40 pl each of dry pyridine and acetic anhydride and heating for 20 min in an oven at 100”. After cooling 1 ml of CHCl, was added and the solvents removed in a stream of Nz. A portion of the sample was injected into the glc apparatus for the quantitation of galactitol and reduced chromogen.

One part of the sample was hydrolyzed in 100 ~1 of 2 N HCl at 100’ for 2 hr, the HCl removed in vacua, and the sample 0-acetylated. Glc was run for the quantitation of bound N-acetylhexosaminitols.

Two of the parts were each treated with 0.25% Brz in water (300 pl) for 30 min at 24’ and the samples dried in vacua. One part was 0-acetylated as above and the other was hydrolyzed in 100 ~1 of 2 N HCl for 2 hr at 100”. The HCl was removed in vacua over P205 and NaOH and the samples 0-acetylated. Glc was then run for the identification of unsaturated reduced chromogens.

The blood group substances (2-10 mg) were degraded with base-borohydride at concentrations of 5-10 mg/ml, a known amount of erythritol added, and the products worked up in two ways. The reactions were stopped by neutralizing with HCI, the solution passed through a 1 X lo-cm column of mixed bed resin (Amberlite MB-3) and the resin washed with two column volumes of water. The eluates were evaporated to dryness, and any borate present was removed by repeated addition and evaporation of absolute methanol. It was found that all the salt occasionally was not removed by the mixed-bed resin and it was re- placed by the following alternate procedure. After adding erythritol, the base-borohydride degradations were stopped by the addition of an excess of Dowex 50-H+, the supernatant removed, and the resin washed. The combined solutions were dried in uacuo, borate removed with methanol and one half of each of the samples 0-acetylated as above. Glc was then run for the quantitation of free hex- entetrol(s), galactitol, reduced chromogens, N-

acetylglucosaminitol and N-acetylgalactosaminitol. The other half was methanolyzed in 0.5 ml of 3.3% methanolic-HCl in a sealed tube for 16 hr at 65’. The methanolic-HCl was evaporated with Ns, 1 ml methanol added and evaporated, then 1 ml CHCl, added and evaporated with Nz. The samples were then 0-acetylated and glc run for the quantitation of bound plus free galactitol, 3- deoxygalactitol, N-acet,ylglucosaminitol, and N- acetylgalactosaminitol. Because bound hexenetetrol(s) are destroyed in the methanolysis step, a portion of the base-borohydride-degraded blood group substance was treat,ed with an excess of Brz in water, hydrolyzed, and 0-acetylated as above. The brominated hexenetetrol(8) were identified by glc.

RESULTS

Several blood group glycoproteins and their stages of periodate oxidation and Smith degradation were treated with 1 M NaBH4 in 0.05 N NaOH at 50” for 16 hr. The products of the degradations were quantitated by glc as their per-O-acetylated derivatives and are listed in Table I. The total amounts of hexenetetrol(s), 3-deoxygalactitol and galactitol formed are small as compared with that of N-acetylgalactosaminitol. In only one instance was a very small peak attributable to N-acetylglucosaminitol seen. For comparison, three of the glycoproteins were also treated under the conditions of 1% NaBH4 in 0.2 N NaOH at 24” for 1 week and showed extensive degradation. The signifi- cance of the quantities measured and the calculated ratios of N-acetylgalactosaminitol/GalNAc and free N-acetylgalactosaminitol/total N-acetylgalactosaminitol are discussed below.

Figure 1 is a chromatogram showing the elution times of the products; the reduced chromogens are not included but are discussed below. Using a temperature gradient with the glc, the retention times are some- what variable, and therefore, the samples were also mixed with known alditol standards to establish the exact position of any peak. Several minor peaks were found on the glc which were not from impurities in the solvent and which did not co-chromatograph with the known alditol standards. These may be due to 0-acetylated amino acids or their products formed in the base-borohydride


TABLE I

QUANTITATION BY GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF THE I)EGRADATION PRODUCTS FORMED

FROM BASE-B• ROHYDRIDE TREATMENT OF BLOOD GROUP GLYCOPROTEINS

0.05 N NaOH + 1 M Na BHab JS phenol-insoluble (H) JS 1st 104 JS 2nd 104 JS 4th 104 Beach phenol-insoluble (B) Beach 1st 104 MSS (10%2x) (A) MSS 1st 104 N-l (10%2X ) (Lea) OG (20%2x) Hog mucin l(A + H) Hog mucin (1st 104)

0.2 N NaOH -I- 1% NaBHbc JS phenol-insoluble (H) JS 1st 104 N-l (10%2X) (Lea)

- I

E

-

Alditols formed (mg/g blood group substance)

Free Y-acetyl. :alactosa minitol

-

0.04 0.30 2.6 0.21 0.25 17.3 0.49 0.66 12.6 0.32 0.82 15.9 0.48 - 20.9 0.15 0.16 11.4 0.29 - 19.1 0.06 0.06 6.7 0.05 0.26 5.8 0.13 - 17.9 1.9 - 5.2 0.57 0.88 39.7

3.2 23.8 6.7 8.7 17.8 42.2 3.3 26.8 8.9

Total (free and i oligosaccharide-bound) Total

GalNAc -

1 i

1.20 0.7: 1.53 0.3: 1.44 1.3E 0.83 0.8; 0.47 - 1.11 0.5:

12.3 - 1.06 0.31 1.04 0.38 4.86 - 1.54 - - 2.7

10.3 35.6 9.4 46.7

18.9 41.8

-

:ylgal- rctosa- ninitol

-

-- -.

79.0 103 77 3.3 98.0 182 54 17.6 71.0 135 53 17.8 56.0 93 60 28.4

125. 131 95 16.7 64.0 138 46 17.8

111. 246 46 17.2 52.7 105 50 12.8 74.2 110 67 7.8

114. 127 90 15.7 30.0 80 38 17.3

122. 161 76 32.5

57.2 146. 53.7

Total v-acetyl.

galac- tosa-

minitol Total/

GalNAc (%)

Free / total N- acetyl- galac- tosa-

minitol (%I

a GalN was measured calorimetrically after hydrolysis of the glycoproteins for 2 hr at 100’ in 2 N HCl.

b Base-borohydride degradations were done at 50” for 16 hr and products assayed as described in the text. A’-Acetylglucosaminitol was not found in the products of any of the degradations except for a trace with hog mucin 1st IO+

c One week at 24’.

10 20 30 LO 50 60

Timetmin)

FIG. 1. Gas-liquid chromatogram of a standard mixture of alditols as their per-O-acetylated derivatives. The dotted peaks show the elution of the brominated hexenetetrols.

procedure. Many of the amino acids O- acetylated as described above as well as an unrelated protein, pepsin, treated under the base-borohydride conditions and O-acetylated, gave several minor peaks on glc, which eluted in the same general regions as the unidentified peaks.

There were one to three major peaks, which chromatographed with retention times between those of erythritol and of hexenetetrol(s), and which did not correspond to any known alditol or methyl glycoside. These products were found only in the glycoproteins treated with 1 M NaBH4 in 0.05 N

NaOH at 50” for 16 hr and not in those treated in 0.2 N NaOH plus 1% NaBH4 at

308 ANDERSON, ROVIS, AND KAEiAT

room temperature, suggesting that the higher temperature may result in some peptide hydrolysis. One of these peaks was found in the products of the base-borohydride degradations of the first, second, and fourth 104 stages of JS which also may imply its origin from the peptide chain.

Oligosaccharide-bound hexenetetrol( s) could be assayed by treating the base-borohydride product with aqueous bromine solution followed by hydrolysis. An equal mixture of the D-three and D-erythro isomers of hexenetetrol was treated in this manner and glc of the 0-acetylated products gave one major and two minor peaks as shown in Fig. 1. Portions of two of the glycoprotein products from the 1 M borohydride in 0.05 NaOH treatment of JS and JS first 104 were assayed in this manner, and on glc no peak was found with a retention time of the major peak for the brominated hexenetetrol derivative. An amount of this derivative equal to the free hexenetetrol formed, could have been detected.

Of the possible degradation products derived from a 3-O-substituted reducing N- acetylhexosamine, reduced chromogen 1 (see below) is predominantly formed under two of the sets of conditions used to degrade the blood group active glycoproteins (Table I). Reduced chromogen 1 has the same retention time and is not separable from hexenetetrol(s) and brominated reduced chromogen 1, likewise is not separable from the main peak of brominated hexenetetrol(s) on glc. The values calculated in Table I for hexenetetrol(s) formation also include any reduced chromogen 1.

As a control, hexenetetrol(s), galactose, galactitol, and N-acetylgalactosamine were treated with 1 M NaBH4 in 0.05 N NaOH at 50” for 16 hr. Recoveries of hexenetetrol(s) and galactitol and of monosaccharides as their reduced alditols, were nearly quantita- tive, indicating stability of the products formed.

Base-borohyclride degradation of S-O-substituted hexosamines and, glc assay of reduced chromogens. Reduced chromogens formed from the action of base-borohydride on 3-0- substituted GlcNAc or GalNAc are possible degradation products of the blood group

glycoproteins, and methods were developed for their assay. The reduced chromogens could not be assayed calorimetrically by the usual methods. Chromogens were generated by heating GlcNAc with KzB40, and measured by the method of Reissig et al. (25). If 1 M NaBH4 in 0.05 N NaOH is added to the KzBdO,-treated N-acetylhexosamines followed immediately by the Ehrlich’s reagent and concentrated HCl, the molar color yields are the same as for untreated samples. However, if the base-borohydride is added and the reaction mixture left for 5 min, addition of the Ehrlich’s reagent gives no color. Apparently, 5 mm in borohydride is sufficient to reduce the chromogens. In addition, chromogen formation from a blood group substance treated with base alone could be detected calorimetrically while the same substance gave no color when treated with base-borohydride followed by the Ehrlich’s reagent.

Reduced chromogens could be assayed by glc. Three disaccharides were treated with base-borohydride under three different sets of conditions : Iyer and Carlson (IS), Lloyd and Kabat (lo), and Bray et al. (21). Table II lists the percentage of products formed from the 3-O-substituted GlcNAc and GalNAc. Of the three procedures studied, the extent of &elimination by 1 M NaBHa in 0.05 N NaOH is least. Moreover, @Gal (1 -+ 3) nGalNAc is degraded to a lesser extent (6 %) than is @Gal (1 + 3) DG~cNAc (28%). The percentage of /3-elimination with the latter disaccharide was the same at the two concentrations tested, 0.75 and 3 mg/ml. With increasing base concentration, an additional compound was obtained from &Gal (1 ---) 3) DGIcNAc which co-chromatographed with N-acetylmannosaminitol. For the 3-O-substituted GalNAc no other peak was detected; the 2-epimer, N-acetyltalos- aminitol was formed to the extent of only 3 % on treatment of GalNAc with 0.5 N NaOH plus 0.5 M NaBH4; it elutes just before N- acetylglucosaminitol.

Six reduced chromogen peaks can be detected on glc of the 0-acetylated products and their relative elution times are shown in Fig. 2. The amounts of these reduced chromogens varied depending on the conditions

TAB

LE

iI @

ELIM

INAT

ION

O

F 3-

O-S

UBS

TITU

TED

G

lcN

Ac

OR

Gal

NA

c U

ND

ER

VA

RIO

US

C

ON

DIT

ION

S O

F B

AS

E-B

• KO

HYD

RID

E TR

EATM

ENT

Com

pmd

BD

Gal

(1

--t

3) D

GIc

NA

c

finG

al

(1 +

6)

DG

~cN

Ac

0.5

N

@&a

l (1

4 3)

DG

alN

Ac

0.05

N

0.2

N

0.5

N

@D

G~c

UA

(1 +

3)

-nG

alN

Ac

0.05

N

0.2

N

0.5

N

Con

cent

ratio

ns o

f

NaO

H

0.05

N

0.

2 N

0.

5 N

- ph

- -I-

+ + + f + + z + - - s - -

-

NaB

Hd

1M

28

1%

57

0.5

M

45

1M

CP

0.5

M

w

1M

1%

0.5

M

1M

1M

1%

0.5

M

6 44

37 w

-

Com

poun

ds id

entif

ied

(%)

-

1

-

V-Ac

etyl

gl

uco-

‘V

-Ace

tyl-

mam

la-

;am

inito

l sa

min

ftol

71

32

17

56

0 10

28

10

V-A&

y1

g&c&

sa

min

ito

87

35

49

30

14

5

I -

- Fr

ee re

duce

d chr

omog

ens fo

rmed

(am

ount

and

%)

1 I

z I

3 I

* I

5 I

6 Fr

ee re

duce

d ch

rom

ogen

s from

J-O

-sub

stitu

ted

Glc

NAc

z

0.58

” (lQ

)“O

.O=

0.28

(8

) 0.

0 1.

17 (

35)

0.0

0.15

(5

) 0.

46

(14)

8::4

(1

):::8

8

(2)

# 0.

13

(6)

0.23

(ll

)O.O

0.

42

(20)

0.0

5 (2

)O.ll

(5

) m

%

1 I

I

Free

redu

ced c

hrom

ogen

s from

3.O

-sub

stitu

ted

Gal

NAc

~___

____

0.

05

0.0

0.0

0.0

0.0

0.0

2

0.64

(1

6)

0.0

0.27

(6

)0.4

1 (1

0)0.

34

(8)0

.21

(5)

2 0.

04

(1)

0.19

(6

) 0.

22

(6)0

.36

(10)

0.33

(9

)0.1

7 (5

) z F

0.0

0.0

0.0

0.0

0.0

0.0

z 0.

94

0.06

0.

30

0.42

0.

37

0.21

0.

08

0.32

0.

30

0.44

0.

41

0.20

a Ar

ea o

f th

e re

duce

d ch

rom

ogen

pe

ak o

ver

the

area

of

the

eryt

hrito

l pe

ak.

b Per

cent

age

of r

educ

ed

chro

mog

en

form

ed

calc

ulat

ed

from

th

e pe

rcen

tage

of

p-e

limin

atio

n.

c 0.0

indi

cate

s th

at

no d

etec

tabl

e pe

ak

was

fou

nd.

d N

o fre

e re

duce

d ch

rom

ogen

de

tect

ed.

310 ANDERSON, ROWS, AND KABAT

O~GCII(I-~QG~CNAC 7

0.2 N NoOH.I’/.N&H~ 8 A

I

,o , s 6 A

,\; I ‘:I , ::’ \.

10 20

A&IA, 30 40 50 60

Time(min)

RDGaI(l4)LJGalNAc

0.2 N NaOH t 1 ‘I. NaBH,,

0

10 20 30 LO 50 60 Timctmin)

FIG. 2. Gas-liquid chromatograms of the per- 0-acetylated products formed from base-borohydride treatment of 3-O-substituted GlcNAc(A) and GalNAc (B).

used (Table II). The first number in the table under each reduced chromogen refers to the ratio of its area to that of erythritol. This ratio permits a comparison of the relative amounts of each reduced chromogen for a given disaccharide under the three conditions. Thus for pnGa1 (1 --+ 3) DGIcNAc twice as much reduced chromogen 1 is formed in 0.2 N NaOH plus 1% NaBH4 than in 0.05 N NaOH plus 1 M NaBH4. With 0.2 N NaOH plus 1% NaBH4 and with 0.5 M NaOH plus 0.5 M NaBHa, the amounts of reduced chromogen 4 formed are about the same; this was also true for reduced chromogens 5 and 6 from 3-O-substituted DG~cNAc and with reduced chromogens 3, 4, 5, and 6 from S-O-substituted GalNAc. The numbers in parentheses are

the percentages of reduced chromogen calculated as the ratio of its area to the total area of reduced chromogens multiplied by the percent’age of p-elimination, as calculated from the galactitol found. Thus for @Gal (1 + 3) DG~cNAc treated with 0.05 N NaOH plus 1 M NaBH4, 28% of reduced chromogen was found, 19 % of which was reduced chromogen 1 and 8% reduced chromogen 3. For N-acetylchondrosine, the N-acetylgalactosaminitol values are low perhaps because of the difficulty of hydrolyz- ing the reduced disaccharide, and the percentage of reduced chromogens cannot be calculated. This calculation implies that all of the products of the p-elimination of the 3-O-substituted N-acetylhexosamines can be detected. This is not strictly t#rue because for ,&Gal (1 -+ 3) DGalKAc in 0.05 N NaOH plus 1 M NaBH4, only a trace of reduced chromogen 1 is formed while there was 6% &elimination, as measured by galactitol. However, the sum of the areas of the reduced chromogens, when formed in considerable amounts, and assuming them to have about the same molar response factor as N -acetylhexosaminitols, very nearly equals the amount of N-acetylhexosamine degraded. For example, PDGal (1 -+ 3) DG~cNAc, treated with 0.2 N NaOH plus 1% NaBH4, the area of the reduced chromogen peaks over the sum of the areas of the reduced chromogens plus N-acetylhexosaminitols was 5970, as compared with 57 % galactitol formed.

From PDGal (1 + 3) DG~cNAc, reduced chromogen 2 is formed only with 0.5 N

NaOH plus 0.5 M NaBH4, and reduced chromogen 3 appears only under the other two sets of conditions. Only a trace of reduced chromogen 1 was formed from @Gal (1 -+ 3) DGalNAc in 0.05 N NaOH plus 1 M NaRH4 and no reduced chromogens were detected under these conditions with N-acetylchondrosine.

Reduced chromogens 1 and 2, from 3-0- substituted GlcNAc and GalNAc, disappear after acid hydrolysis. Since reduced chromogen 3 from 3-O-substituted GlcNAc co- chromatographs with one of the peaks of per-O-acetylated galactose, it could not be ascertained whether this too was lost on hydrolysis. The position of elution of reduced


chromogen 1 after treatment with bromine in water is shown in Fig. 2, and this new peak is partially stable to hydrolysis. La- bility of reduced chromogen 1 to hydrolysis and the formation of a new peak on treatment with bromine resembles that seen with hexenetetrol(s) and suggests that this compound may be unsaturated. It was not determined whether reduced chromogen 2 behaved in a similar manner, nor could it be determined whether reduced chromogen 3 from 3-O-substituted GlcNAc was unsaturated.

Treatment of either @Gal (1 + 3) DGIcNAc or BoGal (1 -+ 3) DGalNAc with 1 M NaBHI for 16 hr at 50” resulted in complete reduction of the N-acetylhexosamines and no ,&elimination since no detectable reduced chromogen or galactitol were formed. Treatment of PnGal (1 -+ 6) DG~cNAc with 0.5 M NaBHZr in 0.5 N NaOH also gave no galactitol or reduced chromogen, as expected; moreover no reduced chromogen was found after acid hydrolysis and the amounts of N-acetylglucosaminitol and N- acetylmannosaminitol found after hydrolysis were 56 and lo%, respectively (ratio 85 to 15).

DISCUSSION

To establish the overall structure of biologically active glycoproteins, such as the blood group active substances, it is desira- ble to isolate entire oligosaccharide chains. In those glycoproteins for which the carbohydrate-protein linkage involves serine or threonine the oligosaccharide is split off by an alkaline-ca,talyzed elimination and with the baseborohydride conditions of Iyer and Carlson (18) no further degradation of the eliminated oligosaccharide chains occurred. The findings in this paper permit the assay and quantitation of the possible degradation products.

Such degradation products were found in very small amounts compared to the mono- saccharide from which they were derived. Thus with JS first IO4 only 0.1% of galactitol was formed, and the quantity of free and bound 3-deoxygalactitol plus galactitol was but 1.1% of the total galactose content. If only 50 % of the oligosaccharide chains were cleaved from the protein, this value would be

but 2.2%. Methanolysis followed by O- acetylation was used to assay the bound reduced alditols. This treatment converts the monosaccharides to methyl glycosides leaving the alditol unchanged. With a sequence such as R + GlcNAc + galactitol, for example, methanolysis of the acetamido bond before glycosidic cleavage may reduce the extent of splitting of the GlcNAc + galactitol bond. Even if only 50% of this bond were methanolysed, the degradation of released galactose would be only 4.4 %.

The maximum extent of chain degradation in 0.05 N NaOH plus 1 M NaBH4 may be calculated by adding the moles of hexenetetrol(s), 3-deoxygalactitol, and galactitol and comparing this value as a ratio to the moles of total N-acetylgalactosaminitol formed. For JS, the maximum number of chains degraded is 3.3 % and for MSS first IO+ 3.6 %. The other values range from 3 to 4 %, except for MSS 10 ‘/b 2 X for which a value of 14 % was found.

By contrast, for the three blood group substances treated with 0.2 N NaOH plus 1% NaBH4, these ratios were 1.08,0.57, and 1.53 for JS phenol-insoluble, JS first 104, and N-l 10 % 2X, indicating substantial degradation of the liberated oligosaccharidc chains.

Not all oligosaccharide chains were eliminated from the protein by 0.05 N NaOH plus 1 M NaBH4 because the ratio of N- acetylgalactosaminitol formed to the total N-acetylgalactosamine varied between 38 % for hog mucin A + H and 95 % with Beach phenol-insoluble. When larger amounts of JS phenol-insoluble and JS first 104 were degraded and passed through a mixed bed resin, 59 and 60% of galactose and methylpentose, respectively, were recovered with JS phenol-insoluble; 56 % of galactose was recovered with JS first 104 which contains no methylpentose. Thus about 40% of the oligosaccharide chains were retained by the mixed-bed resin. Either a longer reaction time is needed for further base elimination or the oligosaccharide chains are linked to N-terminal serine or threonine residues and cannot be eliminated. It is also possible that the base-borohydride conditions produce some hydrolysis of peptide bonds leaving some of the oligosaccharides linked to N-

312 ANDERSON. ROVIS, AND KABAT

terminal serines or threonines. Some of the carbohydrate could also be attached to the protein through the relatively alkali-stable glycosylamine to asparagine linkage.

The amount of free to total N-acetylgalactosaminitol varied from 3.3 % for JS phenol-insoluble to 32.5% for hog mucin first 104. Assuming that the extent of the base-catalyzed elimination was the same for unsubstituted and substituted N-acetylgalactosamine, only 3% of the N-acetylgalactosamine in the original JS phenol- insoluble is unsubstituted. The larger amount of free N-acetylgalactosaminitol formed from JS first 104 indicates that about 17 % of the oligosaccharide chains in JS phenol-insoluble are small and that these are further reduced to GalNAc in the periodate oxidation and Smith degradation, or that larger chains are attached to GalNAc in a manner leaving it susceptible to periodate oxidation. The latter explanation would require that either a 4- or 6-substituted galactosyl, or a 6-substituted N-acetyl- glucosaminyl residue be linked to the GalNAc; no evidence for such linkages has been found and in the first stage of periodate oxidation and Smith degradation substantial amounts of GlcNAc should be lost. However, the recoveries of GlcNAc from the first 104 products of JS phenol-insoluble, MSS 10 % 2X and Beach phenol-insoluble were 87, 89, and 92%, respectively, without taking into account losses due to the procedure, making it unlikely that such linkages occur to a significant extent. Thus, it is more probable that smaller oligosaccharide chains are being degraded to terminal GalNAc residues in the first 104 product. In JS phenol-insoluble, for example, a chain such as /3nGal (1 + 3) crnGalNAc ---) protein would be oxidized to anGalNAc-protein. Since both the JS second and fourth IO4 stages each had considerable amounts of unsubstituted GalNAc linked to protein, the original JS phenol-insoluble was heterogeneous with respect to the lengths of sugar units.

An assay was developed for the detection of reduced chromogens using model disaccharides. The results are of interest because there is an apparent differential reactivity to the base-borohydride conditions depending on the nature of the reduc-

ing N-acetylhexosamine. 3-O-substituted GlcNAc is more labile than the correspond- ing GalNAc derivative, and on the basis of the results with the disaccharide @Gal (l---f 3) DGalNAc, the maximum percentage of chain degradation expected from a blood group glycoprotein would be 6 %. This percentage assumes that all the GalNAc linked to serine or threonine is 3-O-substituted. However, the proposed composite oligosaccharide sequence of the ovarian cyst blood group glycoprotein (3) shows GalNAc to be both 3- and 6-O-substituted and oligosaccharides have been isolated containing this disubstituted N-acetylgalactosaminitol (7, 8). Base-catalyzed elimination of the megalosaccharide and of the galactosyl group at carbon 3 of GalNAc and subsequent reduction would yield bound galactitol plus a g-o-substituted reduced chromogen. The predominant reduced chromogen formed from the Iyer and Carlson conditions be- haves as an unsaturated compound. This reduced chromogen and its brominated derivative are not separable on glc from hexenetetrol(s) and its brominated derivatives. No evidence for the latter compounds could be detected in the degraded blood group substances after bromination and hydrolysis. Furthermore, the small amounts of galactitol detected are further evidence that this type of degradation did not occur to any significant extent.

No reduced chromogen could be detected from the 0.5 N NaOH plus 0.5 M NaBH4 degradation of finGal (1 + 6) DG~cNAc, and only the reduced N-acetylhexosaminitols were found after hydrolysis. Therefore, if, in the blood group glycoproteins some of the GalNAc were only 6-O-substituted, complete reduction without degradation would be expected of the base-eliminated oligosaccharides.

From results of the base-borohydride treatments of model disaccharides, three competing reactions occur after the oligosaccharide chains are eliminated from the glycoprotein: (1) a base-catalyzed elimination of the substituent at C-3 of the reducing N-acetylhexosamine with subsequent chromogen formation and reduction; (2) an alkaline-catalyzed epimerization followed either by reduction of the N-acetylhexos-


amine or by base-elimination of the 3-0- substituent and reduction of the chromogens formed; and (3) reduction of the C-l alde- hyde before base-catalyzed elimination. From these findings it is clear that the last reaction is predominant in 0.05 N NaOH plus 1 M NaBH4. The glc results show that at least six reduced chromogens can be formed by base-borohydride action on S-O-substituted GlcNAc or GalNAc. Base-catalyzed elimination of S-O-substituted N-acetylhexosamines produced a 2-acetamido-2,3- anhydroglycofuranose (Chromogen I) and a 5-dihydroxy-3-acetamidofuran (Chromo- gen III) (27). These two chromogens have been isolated as their 6-0-sialyl derivatives from ovine submaxillary mucin after treatment with dilute base (28). Borohydride can reduce the Cl of chromogen I to an alcohol and can also reduce the carbon-carbon double bond conjugated to an amide (21,29). The result of the double reduction would yield a 3-deoxy-N-acetylglycosaminitol. A similar reduction of one of the double bonds in chromogen III would yield a dihydro- furan. The base-catalyzed epimerization followed by base-elimination and reduction can produce the epimers of the reduced chromogens, 0-acetylation of the reaction mixtures containing the final products or their intermediates can give rise to the number of peaks found on glc analysis.

It is interesting to note that with @Gal (1 --) 3) DG~cNAc increasing base concentration results in a higher yield of a peak which chromatographs with N-acetylmannosaminitol. An equilibrium mixture of GlcNAc and ManNAc in alkali is approximately 85: 15 (30) ; a similar equilibrium mixture was found after hydrolysis of @nGal (1 + 6) DG~cNAc treated with 0.5 M NaBH4 in 0.5 N NaOH. The greater amount of N-acetylmannosaminitol formed (Table II) with higher concentrations of base must be due to the greater base stability of S-O-substituted ManNAc compared to the GlcNAc derivative.

It is clear from these results that the 1 M NaBH4 in 0.05 N NaOH conditions of Iyer and Carlson (18) can be used as the first step in the isolation of complete oligosaccharide chains from the blood group glycoproteins. The gas chromatographic method

of assay for free or bound degradation products is suitable for monitoring the reaction and provides a quantitation of the maximum amount of chain degradation. The application of this technique and the fractionation, isolation, and characterization of the non- degraded oligosaccharides will allow a more precise definition of the heterogeneity of the blood group carbohydrate chains and will provide information about the specificity of biosynthesis of these biologically active prosthetic groups.

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immunochemical studies on blood groups: liii. a study of various conditions of alkaline borohydride...

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