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On the structure of the carbohydrate chains of the blood-group substance (A + H)
CarIutp.drate Research Ekvier PublishingCompany,
Printed
in Belgium
437
Amsterdam
ON THE STRUCTURE OF THE CARBOHYDRATE BLOOD-GROUP SUBSTANCE (A+H)
CHAINS
OF
THE
N. K. KOCHETKOV, V. A. DEREVITSKAYA,L. M. LICHOSHERSTOV, M. D. MARTYNOVA, AND S. N. SENCHENKOYA
Institttte of Organic Chemistry,
U. S. S.
R. Academy
of
Sciences,
Moscow
((I.
S. S. R.)
(Received August 4th, 1969)
ABSTRACT
The structure of the carbohydrate chains of blood-group substance (A + H) (BGS) in the region adjacent to the peptide backbone has been investigated. Two approaches were used: (1) a study of the degradation of BGS by a combination of chemical and enzymic (preparation from Clostridiurnperfingens) methods, and (2) a study of the alkaline degradation of BGS by measurement of the accumulated products of degradation of N-acetylhexosamines (3-acetamido-5-dihydroxyethylfuran) and D-galactose (metasaccharinic acid and S-hydroxymethyl-2-furaldehyde). It has been shown that the carbohydrate-peptide linkage-unit contains a 2-acetamido2-deoxy-D-galactose residue. Directly adjacent to this region is a chain of several iV-acetylhexosamine
residues bound by (1~3)
linkages
and partially
branched
at C-6.
INTRODUCTION
Recent structural studies of the blood-group substance (BGS) have shown the presence of a peptide backbone with carbohydrate chains attached by glycosidic bonds to serine and threonine residuesXd3. The elucidation of the structure of some oligosaccharides released on degradation of BGS yielded information on the structure of the terminal part of the carbohydrate chains 3-6 . The structure of the carbohydrate chains in the region adjacent to the peptide backbone is unknown. We have developed two new routes for approaching this problem. Firstly, we have studied the degradation of BGS (A + H), isolated from the lining of pig stomachs, by an enzyme preparation from
CZostridiumperfingeens, together
with some chemical
methods.
Secondly,
we
have investigated the structure of the carbohydrate chains by estimation of the alkaline-degradation products of N-acetyl hexosamines and D-galactose. The latter approach, which has not been applied hitherto in studies of glycoprotein structure, may be of general value. RESULTS AND DISCUSSION
Degradatioiz of BGS by an enzyme
preparation
from
Clostridium
perfringens.
After treatment of BGS with the enzyme preparation, ca. 45% of the substance was split off as a fraction of low molecular weight; fucose (7.5%), and galactose Carbohyd.
Res.,
12 (1970) 437-447
438
N. K. KOCHETKOV et al.
and N-acetylhexosamines (-35%) were released’. The peptide part of the polymer was largely unaffected, and the fragment (1) of high molecular weight was stable to repeated treatment with the enzyme preparations. Since 1 still contains -65% of carbohydrate, a more extensive degradation of BGS was sought by a combination of chemical and enzymic methods. BGS was treated in sequence with periodate and borohydride, and the resulting polyol (BGS I) was subjected to acid hydrolysis (pH 1.5, 3 h, 100”). The overall breakdown of BGS in this procedure is ca. 50%, whereas that of amino acids is less than 0.5%. The fragment (2) of high molecular weight subsequently isolated by gel filtration was then treated with the enzyme preparation, which resulted in cleavage of ca. 45% of the carbohydrate content. Further gel filtration gave a new fragment (3) of high molecular weight, which retained the amino acids characteristic of BGS, but in increased percentage (58%, CJ 16% for BGS) (see Table I). Fraction 3 contains ca. 40% of carbohydrate, no fucose, and + 6% of galactose. The remainder of the carbohydrate content is ZV-acetylhexosamine. TABLE
I
CONTENT
OF AMINO
ACIDS
AND
HEXOSAMINE.5
IN
BGS
AND
ITS
FRAGMENTSa
Preparation
Amino acids % (a)
N-Acet& hexosantitws % (b)
a/b
2-Acetamido-2-deo.q= D-galactose (pmolesjb (c)
2-Acetamido-I-deoxyD-glucose @moles)b (n)
c/d
BGS (A + H) 3 3 treated with NazCOa
16.0 58.2 35.8
41.0 28.9 15.8
0.39 2.0 2.26
1.13 2.29 0.82
1.45 0.30 0.16
0.8 7.6 5.1
=Determined by using an amino acid analyser after hydrolysis with 4~ HCi for 24 h at 100” in cacao. bReferred to 1 mp of substance.
The ratio of 2-acetamido-2-deoxy-D-galactose to 2-acetamido-2-deoxy-o-glucose (GluNac) in 3 is 7.6, whereas in the original BGS it is only 0.8. The preponderance of 2-acetamido-2-deoxy-D-galactose in 3 suggests that this hexosamine could constitute the sugar residues at the reducing end of the oligosaccharide chains and thereby be involved in the carbohydrate-peptide linkage, as well as forming part of the carbohydrate chains adjacent to the peptide backbone. The D-galactose and GiuNac are mainIy in the periphery of the molecule, since they are more readily split off during the enzymic and chemical degradation of BGS. This view was supported by the data obtained from the alkaline treatment (0.05~ Na,CO,, IOO”, 15 min) of 3. The monosaccharides which are gIycosidicalIy linked to serine and threonine are the first to be destroyed. Analysis of the mixture of alkaline-degradation products of 3 showed the decrease of 2-acetamido-2-deoxy-D-galactose (I .47 pmoles, Table I) to be 10.5 times that of Z-acetamido-2-deoxy-D-gIucose (0. I4 pmole). Carboh-vd. Res., 12 (1970) 437447
BLOOD-GROUP
439
SUBSTANCE
The degradation of BGS
with alkali
/&Elimination of the carbohydrate chains from the serine and threonine units of the peptide chain is known to proceed upon treatment of BGS with alkali. The carbohydrate chains liberated are then eroded from the reducing end to form a series of oligosaccharides that are characteristic of the terminal sequence of the monosaccharides
(cl: refs. 4 and 6).
The rapid degradation of the carbohydrate chains at the reducing end, which is observed on treating BGS with alkali, is characteristic of polysaccharides having (1+3) linkagesg. The degradation of such chains should result in the formation of 3-acetamido-5-dihydroxyethylfuran and, probably, 2-acetamido-2,3-dideoxy-D-hex2-enofuranose from N-acetylhexosamine residues. These compounds give the direct Ehrlich reaction, and the presence of chromogens in the mixture of alkaline-degradation products of BGS was reported earlier”. Metasaccharinic acid should be formed from the residues of gaiactose substituted at C-3, and isosaccharinic acid” from galactose residues substituted at C-4. A preliminary study of the degradation of BGS in 0.05~ Na,CO, has shown that the accurate determination of the amount of metasaccharinic and isosaccharinic acids by periodate oxidation is complicated by the sensitivity of BGS to periodate. Therefore, both BGS and the product (BGS I) obtained after periodate oxidation and borohydride reduction were subjected to alkaline degradationr2. The alkaline conditions chosen were based on model experiments with 2-acetamido-2-deoxy-D-glucose which was heated in 0.05M Na2C0, at 50, 70, and 100” (Fig. 1); 70” proved to be optimal, and, at this temperature, chromogen forms rapidly and is stable upon prolonged
heating.
Absorbance
Y 1
2
3
h
Fig. 1. The rate of formation of chromogen from 2-acetamido-2-deoxy-n-glucose on treatment with 0.05hc NaoCOa as determined by the Ehrlich reaction: 1, 50”; 2, 70’; 3, 100”. Chromogen
and metasaccharinic
acid are formed upon treating
BGS and BGS
I
in 0.05n1 Na,CO, at 70”. Isosaccharinic acid was not detected”, even after acid hydrolysis (0.5N HCI, lOO”, 12 h) of the alkaline-degradation products of BGS. This indicates the absence of 4- and 4,6-substituted galactose residues in the eroded parts of the carbohydrate chains. Curbohyd Res., 12 (1970) 437-447
N. K. KOCHETKOV et d.
Upon treatment of BGS and BGS I with aqueous Na,CO, (Fig. 2, Table II), chromogen was liberated rapidly; its release was markedly decreased in 4 h. Metasaccharinic acid formation began 7 min after treatment of BGS I with alkali, and the amount increased very slowly, corresponding, after 4 h, to -0.5% of galactose (of BGS) destruction. During this time, 10.5% of the N-acetylhexosamines were cleaved (Table II). The rates of &elimination reactions of 2-acetarnido-2-deoxy-D-glucosyl and D-galactosyl derivatives of the methylamide of N-benzyloxycarbonyl-L-serine were Absorbance 1 2
0.4-
x52 Absorbonce
0.33 0.2-
3 0.05
O-lk’
;L
2
1
4
3
10
20
30
min
5 ----z-
Fig. 2. The rate of formation of chromogen and metasaccharinic acid from BGS and BGS I on treatment with 0.05~ NaaCOa at 70”. 1, BGS; 2, BGS I (by Ehrlich reaction); 3, BGS I (by reaction with thiobarbituric acid). TABLE
II
THE NUMBER
OF DEGRADED
[CALCULATED ANALYSIS
FROM (AAA)
THE
DATA]
UNITS
AND
OF
5-HYDROXYMETHYL-kURLLDEHYDE Na2C03
AT
OF THE
WEIGHT
OF
BGS
GALACTOSE (III)
[~ALCULAT=D FORMED]
UPON
BGS I)
AND (I)
moht
THE
TREAThlENT
OF N-ACETYLHEXOSAMINES
FdRMED
AND
OF
BGS
AND
AMINO
FROht
hfa4sAccH~xr4rc
ACID
(II)
ACID AND
BGS I WrTH o.oi?ihl
70”.
Time of the treatment with NazC03 (min)
7 14 30 60 120 240 360 480
(%
3-ACETAhlIDO-S-DIHYDROXYFeTHYLFURAN
BGP
BGS Ib
N-Acetylhexosanrines from I
from III
0.52 1.14 2.1 3.3 5.2 6.5 6.6 -
0.02 0.14 0.31 0.50 0.72 0.71 0.71
D-
Galaciose
N-acetylhexosamines
D-Galactose
From I
From aaa
From II
From III
0.52
-
1.15 3.3 4.9 6.1 6.2 -
10.4 10.6 -
0.00 0.03 0.10 0.17 0.31 0.48 -
0.16 0.29 0.49 0.61 0.62 0.62
0.01
=The percentage of N-acetylhexosamines, galactose, and fucose in the original BGS is 43,22, and IO%, respectively. bathe percentage of N-acetylhexosamines, galactose, and fucose in original BGS I is 26, 10, and O%, respectively. Carbohyd. Res., 12 (1970) 43747
441
BLOOD-GROUP SJBSTANCE
approximately equal (Table III). Therefore, the difference in the rates of release of metasaccharinic acid and chromogen cannot be accounted for on this basis. Nor can the absence of metasaccharinic acid at the beginning of the degradation be accounted for by the differences in the rate of formation of metasaccharinic acid and chromogen. The degradation13 of laminarin, having a known number of reducing ends (kindly provided by Professor D. G. Manners), upon treatment with 0.05~ Na&O, at 70” results in rapid formation of metasaccharinic acid (Fig. 3). Thus, 3 moles of metasaccharinic acid are released in 7 min from each reducing end of laminarin, under conditions where no metasaccharinic acid was formed from BGS. The decrease in the glucose content (anthrone method) in laminarin after alkaline degradation corresponds precisely to the amount of metasaccharinic acid liberated. TABLE
III
DEGRADATION
OF THE
DE~x~-/LD-~LU~~SYL 0.05hl
TREATMENTWITH
GLYCOSIDIC
LINKAGE
(6) DERIVATIVES NazC03 AT 70".
OF
OF 3-m
O-/I-D-GALACTOSYL
(5)
N-BENZYLOXYCARBDNYL-L-SERINE
Trearment time (min)
Cleacage of 5 (% of sample)
Cleacage of 6 (% of sample)
1.5 30 60
77 96 100
72 97 100
AND
0-2-ACETAMIDO-2-
hmnw_.4hwx
UPON
Absqraance
20
60
120
180 min
Fig. 3. The rate of formation of metasaccharinic acid from BGS I and laminarin on treatment with 0.05~ NaaCOs at 70”. I, BGS I; 2, laminarin (by reaction with thiobarbituric acid).
Thus, the data obtained from the treatment of BGS and BGS I with 0.05~ Na,CO, at 70” indicate that (1+3)-linked galactose residues (as well as those” branched at C-6) do not contribute to the carbohydrate-peptide linkage and are essentially absent from the parts of the carbohydrate chains immediately adjacent to the peptide backbone. Apparently, these portions are made up of N-acetylhexosamine residues. The possible presence of a small number of (143)-linked galactose residues branched at C-4 or C-2 in this part of the BGS molecule is not excluded by this evidence. (1+3)-Linked galactose residues that are branched at C-Q are known to Carbohyd.
Res.. 12 (1970) 437-447
442
N. K. KOCHETKOV
et al.
afford 4-substituted metasaccharinic acid in alkaline medium, which cannot be directly determined l1 . However, after acid hydrolysis (0.5N HCl, lOO”, 12 h) of the alkaline-degradation products of BGS, there was no increase in the yield of metasaccharinic acid. Hence, the degraded chains do not contain any 3,4_disubstituted galactose residues. By analogy with glucose derivatives, 2,3-disubstituted galactose residues should he readily converted by alkali into 2-substituted 3-deoxyhex-2-enose derivatives which, under mild, acidic conditions, should yield 5-hydroxymethyl-2-furaldehyder4. Actually, 5-hydroxymethyl-2-furaldehyde is formed on heating 2,3-di-U-methyl-Dgalactose in 0.05~ Na,CO, for 7 min or 2 h at 70”, followed by acidic treatment, to extents corresponding to 9 or 58% degradation. Similar treatment of n-galactose gave no 5-hydroxymethyl-2-furaldehyde. To detect 2,3-disubstituted galactose residues in the cleaved parts of the carbohydrate chains, BGS and BGS 1 were treated with aqueous NaJO, at 70”, and then with acid at 50”. The absorption measured at 284 nm then corresponds to 5-hydroxymethyl-2-furaldehyde’4. The amount of 2-substituted 3-deoxyhex-2-enose formed and determined from 5-hydroxymethyl-Zfuraldehyde increased slowly and, after 4 h, corresponded to O&0.7% of galactose (of BGS). This, however, cannot radically change the conclusions that have been previously drawn, since the possible presence of 2,3-disubstituted galactose in the carbohydrate-peptide linkage unit has been ruled out. The presence of small proportions of 3- and 2,3_substituted galactoses (ca. 1.0% from BGS) in the reducing end of the carbohydrate chain has not been explained so far, but it might indicate the inhomogeneity of the BGS carbohydrate chains. All of these data support the conclusion that there are 2-acetamido-2-deoxy-Dgalactose residues in the region of the glycosylpeptide linkage of BGS. After 4 h of alkaline degradation of BGS, chromogen formation ceases (Fig. 2), indicating completion of the process. The alkaline-degradation products obtained after 4 h of treatment of the BGS I with Na,C03 were separated on Biogel P-6 into two fractions (Fig. 4). Almost all of the amino acids were in the fraction (4) of high molecular weight. The amino acid content of the low molecular fraction was 0.5% of BGS I (Table IV). The yield of 4 is ca. 50%, and, hence, the alkaline treatment results in cleavage of ca. 60% of the carbohydrate residues of BGS. The carbohydrate remaining bound to the peptide skeleton of the glycoprotein is not split off under the alkaline conditions chosen. The anaIysis of the chromogens formed provides further information concerning the carbohydrate portion of BGS which is degraded by alkali. Chromogens are formed only from 3- and 6-monosubstituted and 3,6_disubstituted N-acetylhexosamines15. An unsubstituted chromogen should form from the residues of (l-*3)linked N-acetylhexosamines which arose from alkaline degradation of BGS carbohydrate chains: (1+6)-linked N-acetylhexosamines or (1+3)-linked units branched at C-6 should yield a substituted chromogen. Since (l-+4)-linked residues are stable to alkali, the N-acetylhexosamine residues at the reducing end of BGS I carbohydrate chains should be bound by (143) links, with or without branches at C-6. Gel-filtraCarbolaycf. Res., 12 (1970)
437-447
BLOOD-GROUP
TABLE AMINO
443
SUBSTANCE
IV
ACIDa
CONTENT
OF
BGS I, AND FRACTIONS
4,
AND
LMF 4
Amino ncia!s
ZIGS Z (%)
4 (%)
LMF 4 (%)
Asparaginic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine x Amino acids
0.31 5.09 2.20 0.76 3.10 0.37 0.91 0.92 0.15 0.64 14.45
0.57 5.48 1.43 1.38 6.78 0.56 1.75 1.47 0.23 1.12 20.77
0.06 0.13 0.13 0.16 0.13 0.10 0.11 0.11 0.04 0.07 1.04
%xcluding
Abr
the basic amino acids. Absorbance
iar bance
I
1 lb
5.0
Go
2%
60
ml
140
200
260
ml
Fig. 4. Gel-filtration of products from alkaline hydrolysis of BGS I (0.05hl NaeCO3, 70”, 4 h) on a column (1.5 x 60 cm) of Biogel P-6 in 0.1~ CHsCOOH. Fractions were assayed by 1. absorbance at 232 nm; 2, by the phenol-sulphuric acid method: and 3, by the Ehrlich reaction. Fig. 5. Gel-filtration of LMF 4 (0.05&t Na2C03, 70”, 4 h) on a column (1.5 x 60 cm) of Biogel P-2 in 0.1~ CH&OOH. Fractions were assayed by: 1, absorbance at 232 nm; 2. by the phenol-sulphuric acid method; and 3, by the Ehrlich reaction. tion
on
Biogel
P-6 (Fig.
ated solely in the fraction
4) shows that the chromogen
of low moIecular
formed from BGS I accumulweight (LMF 4). The fractionation of
LMF 4 on BiogeI P-2 (Fig. 5) gave two chromogen peaks Cr and II) which probably correspond to a substituted and an unsubstituted chromogen. Further evidence for the presence of two types of chromogen in LMF 4 was CurEohyd. Res., 12 (1970) 437447
N. K. KOCHETKOV
444
et al.
based on periodate oxidation of the unsubstituted chromogen to form 3-acetamidoZ-furaldehyde, according to the scheme: CH,OH
&$H,oH
zL”l,,,:“‘_OHC~NH*c
NHAc
2-Acetamido-2-deoxy-D-glucose and its 3,6-dimethyl ether were used as model compounds. After heating in 0.05~ Na,C03 at 70”, with subsequent periodate oxidation, the chromogens formed were determined by the direct Ehrlich reaction. 3-Acetamido-2-furaldehyde reacted rapidly, but the resulting colour was unstable and disappeared in 15 min. 3-Acetamido-5-(1-hydroxy-2-methoxyethyl)furan was not oxidized with periodate, and colour formation in the Ehrlich reaction reached a maximum in 15-20 min. This distinctive behaviour of the chromogens allowed their separate determination. Thus, analysis of fractions I and II (Fig. 5) obtained from LMF 4 by this method confirmed that it is mainly the substituted chromogen which is formed from the fraction of higher molecular weight, whereas the fraction of low molecular weight aIIords the unsubstituted chromogen. The absorbances of chromogen reaction-products formed from equimolecular amounts of 3-substituted and 3,6disubstituted 2-acetamido-2-deoxy-D-glucose in the Ehrlich reaction are nearly equal15. The ratio of the absorbances of the reaction products from fractions I and Ii (Fig. 5) is ca. 3:1, and, hence, most of the hexosamines constituting the reducing end of carbohydrate chains of BGS I are bound by (l-+3) links and are branched at position 6. This conclusion is supported by the fact that experiments (A. V. Sharova) on the acid hydrolysis of BGS give oligosaccharides containing only hexosamine residues. EXPERIMENTAL
BGS (A + H) from pig-stomach linings (at pH 3) and BGS I l2 were obtained by literature procedures. The enzyme preparation was isolated7 from culture liquid of Clostridium perfiingens (type A). Neutral monosaccharides were determined by the phenol-sulphuric acid” and anthrone methodsi*, fucose by the Dische methodrg, and hexosamines by the Elson-Morgan method after preliminary hydrolysis with 2~ HCl for 2 h at loo”. Amino acids and hexosamines were determined on an amino acid analyser after hydrolysis with 4N HCl for 24 h at 100” in uacuo. Paper chromatography was carried out with 6:4:3 butyl alcohol-pyridine-water and detection with alkaline silver nitrate. Acid hydrolysis of BGS I. - A solution of BGS I (1.6 g) in 96 ml of diluted HCl (pH 1.5) was heated for 3 h at lOO”, neutralized with Dowex-2 (CHsCOO-), and loaded onto a column (69 x 3.9 cm) of Sephadex G-50. Fractions (40 ml) were collected, and their absorbance at 232 nm was measured. The sugars were determined by the phenol-sulphuric acid method. The fractions corresponding to the high Carbohyd.
Res.. 12 (1970)
437-447
BLOOD-GROUP
445
SUBSTANCE
molecular weight substance were collected, evaporated, and freeze-dried to yield 720 mg of HMF 2. Emymic hydrozysfs of 2. - To a solution of 715 mg of 2 in 71.5 ml .of 0.01~ phosphate buffer (pH 6.9), a 0.1% aqueous solution (36 ml) of theenzymepreparation was added, and the mixture was incubated for 18 h at 37”. Low molecular weight products were separated by gel-filtration on Sephadex G-50, and the high molecular weight substance was freed from enzyme by gel-filtration on Sephadex G-200. Yield: 400 mg of 3. The enzymic hydrolysis of BGS (A + H) was performed in a manner analogous to that described above. Alkaline degradation of BGS (A 4- Hj and BGS 1: - BGS or BGS I (50 mg) was dissolved in 10 ml of 0.05~ Na,CO, and heated at 70”. AIiquots were taken for determination of chromogen (0.5 ml), metasaccharinic acid (2 x 0.2 ml), galactose (0.1 ml), and 5-hydroxymethyl-2-furaldehyde (0.5 ml), after 7, 15, 30, 60, 120, 240, 360, and 480 min. The quantity of chromogen formed from the N-acetylhexosamines was determined by using Ehrlich’s reagent14. Water (0.5 m1) and 1 ml of p-dimethylaminobenzaldehyde solution (10 mg/ml of 50% CH,COOH) were added to the aliquot with shaking, and then 0.2 ml of cont. HCl was added. Absorbance was measured at 540 nm after IO-15 min. Free and 6-substituted metasaccharinic acids and isosaccharinic acid were determined by the method of Barker et al.“, and 4- and 4,6-substituted metasaccharinic acids and 5-substituted isosaccharinic acid were determined” after preliminary hydrolysis of the alkaline-degradation products with 0.5~ HCI for 12 h at 100”. 5-Hydroxymethyl-Z-furaldehyde (7) was determined by the following procedure: 0.1~ HCl (0.54 ml) was added to the aliquot (O-5 ml) which was then diluted with water to 4 ml. The absorbance of the solution was measured at 284 nm, the solution was then heated at 50’ for 12,15,18, and 24 h, and the absorbance was remeasured. The quantity of 7 was calculated by using the molar extinction coefficientsX4 (Table II). The reaction mixture was extracted twice with butyl alcohol, and the u. v. spectrum of the extract was identical to that of 7 (ref. 14). No 7 was found when D-galactose was treated with 0.05M Na,CO, at 70”. Alkaline degradation of 2-acetamido-2-deoxy-D-glucose. - A solution of 5 mg of the sugar in 5 ml of 0.05M Na,CO, was heated at 50,70, and 100”. Aliquots (0.5 ml) were taken after 5,15,30,60,120, and 180 min, and the quantity of chromogen formed was determined by the aforementioned method (Fig. 3). Alkaiine degradation of laminarin. -This was accomplished under the conditions used for BGS degradation, with laminarin concentrations of 0.1 and 0.05 mg/ml. Aliquots (0.2 ml) were taken from the reaction mixture after 3, 7, 10,20,40, 60, 120, and 180 min, and the metasaccharinic acid was determined” (Fig. 2). Glucose was determined by the anthrone method in aliquots taken from the original reaction mixture and after heating for 120 min. Alkaline degradation of 2,3-di-0-methyl-D-galactose. The sugar (2 mg) Carbohyd. Res., 12
(1970) 437447
444
N. K. KOCHETKOV
et al.
was dissolved in 40 ml of 0.05~ Na2CQ, and heated at 70”. Aliquots (1 ml) were taken after 7, 15, 30, 60, 90, and 120 min, 0.1~ HCl (1.03 ml) was added, the solution was diluted with water to 3 ml (final HCl concentration, 0.01~), and the u. v. spectrum of the mixture was recorded after heating for 24 h at 50”. AZkaZine degradation of O-j?-D-galactosyl and O-(2-acetamido-2-deoxy-B_DgZucosyZ) derivatives of N-benzyloxycarbonyl-L-serine methylamide. - The reaction was performed under the same conditions as for the degradation of BGS, using a 0.5 mg/ml solution of glycosides. Aliquots (0.2 ml) were taken after 15, 30, 60, and 120 min. The determination of the degree of destruction (see Table III) was based on the estimation of formaldehyde” after periodate oxidation. The amount of formaldehyde is equivalent to that of free monosaccharides formed from glycosides upon /?-elimination. The separation of products of alkaline degradation of BGS. - BGS I (200 mg) was dissolved in 40 ml of 0.05~ Na,CO, and heated for 4 h at 70”. The solution was neutralized with Amberlite IR-120 (Hf) batchwise, concentrated under diminished pressure, loaded onto a column (60 x 1.5 cm) of Biogel P-6, and eluted with 0.1~ CH,COOH. Fractions (6 ml) were collected, and the absorbance was measured at 232 nm. Fractions were assayed for sugar content by the phenol-sulphuric acid method, and chromogen was determined by the method mentioned above. Fig. 4 shows the gel-filtration pattern. The fractions corresponding to the fragment (4) of
high molecular weight (fraction 1) and that (LF 4) of low molecular weight (fraction 2) were concentrated and freeze-dried to give 4 (92 mg) and LF 4 (76 mg). LF 4 (76 mg) was dissolved in 2 ml of 0.1~ CHsCOOH, loaded onto 2 column (60 x 1.5 cm) of Biogel P-2, and eluted with 0.1~ CH,COOH. Fractions (6 ml) were collected, their absorbance at 232 nm was measured, and the content of sugars and chromogen was determined in aliquots. Fig. 5 shows the gel-filtration pattern. The fractions corresponding to fragments of high molecular weight (fraction l) and low molecular weight (fraction II) were concentrated and freeze-dried to give fraction I (42 mg) and fraction II (20 mg). Determination of the substituted chromogen in the presence of unsubstituted one. - N210, solution (O-2 ml; 0.0618~ NaIO, in 3~ H,PO,) was added to 0.5 ml of the solution under investigation, and the mixture was shaken and left at room temperature for 20 min. A portion (1 ml) of a Na,AsO, solution (10% of Na,AsO, in 0.5~ Na,SO,) was added, and the solution was shaken until the brown colour disappeared (2-3 min) and then diluted with water to 2 ml. Then 2 ml of a solution containing 10 mg of p-dimethylaminobenzaldehyde per ml of 50% CH,COOH and 0.4 ml of cont. HCl were added, and the absorbance at 540 nm was measured after 15-20 min. REFERENCES 1 N. K. KOCHETKOV, V. A. DEREWTSKAYA, Ah?, S. G. KARA-MURZA, Carbohyd. Res., 3 (1967) 403. 2 B. ANDERSON. N. SENO, P. SAMPSON, I. G. RIBY, P. HOFFMAN, AND K. MEYER, J. Biol. Chem., 239 (1964) PC 2716. 3 K. LLOYD,E. A.KABAT,E.LAGUG,AND F. GRKJEZO, Biochemistry,5 (1966)1489.
Carbokyd. Res., 12 (1970) 437-447
BLOOD-GROUP
447
SUBSTANCE
4 K. 0. LLOYD, E. A. KABAT, AND E. LICARIO, Biochemistry, 7 (1968) 2976.
5 W. M. WATKINS, Science, 152 (1966) 172. A. S. R. DONALD, AND W. T. J. MORGAN, Biochem. Biophys. Res. Commun., 30 (1968) 1; 33 (1968) 508; BJocJzem. J., 107 (1968) 861. 7 L. M. LICHOSHERSTOV,M. D. MARTYNOVA, AND V. A. DEREVIWKAYA, Biokhimia, 33 (1968) 1135. 8 N. K. KOCHETKOV, V. A. DEREVITXAYA, L. M. LICHOSHER~TOV, AND M. D. MARTYE~OVA,Dokl. Akud. Nuuk SSSR, 186 (1969) 216. 9 R. L. WHISTLER AND J. N. BEMILLER, Adcan. Curbohyd. Chem., 13 (1958) 289. 10 K. W. KNOX AND W. T. J. MORGAN, Biochem. J., 58 (1954) V. 11 S. A. BARKER, A. R. LAW, P. J. SOARERS, AND M. STACEY, Carbohyd. Res., 3 (1967) 435. 12 N. K. KOCHETKOV, G. S. Kxor, L. S. BOGDASHOVA, L. M. LICHOSHER~TOV, AND V. A. DEREVITSKAYA, I.v. Akad. Nauk SSSR, Ser. Kilim., 9 (1968) 2095. 13 W. D. ANNAN, E. L. HIRST, AND D. J. MANNERS, J. Chem. Sot., (1965) 885. 14 E. F. L. J. ANET, Chem. 2nd. (London), (1963) 1035. 15 D. AhlINOFF, W. T. J. MORGAN, AND W. M. WATKINS, Biochem. J., 51 (1952) 379. 16 L. M. LICHOSHERSTOV,V. A. DEREVITSKAYA, AND V. 1. FEDOROVA, Biokhimiya, 34 (1969) 45. 17 M. DUBOIS, K. A. GILLE.T,J. K. HAMILTON, P. A. REBERS, AND F. ShmH, Anal. Chem., 28 (1956) 350. 18 G. N. ZAIT~EVA AND T. N. AFANASYEVA, Biokhimiyo, 22 (1957) 1035. 19 2. DISCHE AND L. B. SHEITLES, J. Biol. CJlem., 175 (1948) 595. 20 T. NASH, Biorlrem. J.. 55 (1953) 416.
6 W. P. AXON,
Carboltyd. Res., 12 (1970) 437-447
Printed
in Belgium
437
Amsterdam
ON THE STRUCTURE OF THE CARBOHYDRATE BLOOD-GROUP SUBSTANCE (A+H)
CHAINS
OF
THE
N. K. KOCHETKOV, V. A. DEREVITSKAYA,L. M. LICHOSHERSTOV, M. D. MARTYNOVA, AND S. N. SENCHENKOYA
Institttte of Organic Chemistry,
U. S. S.
R. Academy
of
Sciences,
Moscow
((I.
S. S. R.)
(Received August 4th, 1969)
ABSTRACT
The structure of the carbohydrate chains of blood-group substance (A + H) (BGS) in the region adjacent to the peptide backbone has been investigated. Two approaches were used: (1) a study of the degradation of BGS by a combination of chemical and enzymic (preparation from Clostridiurnperfingens) methods, and (2) a study of the alkaline degradation of BGS by measurement of the accumulated products of degradation of N-acetylhexosamines (3-acetamido-5-dihydroxyethylfuran) and D-galactose (metasaccharinic acid and S-hydroxymethyl-2-furaldehyde). It has been shown that the carbohydrate-peptide linkage-unit contains a 2-acetamido2-deoxy-D-galactose residue. Directly adjacent to this region is a chain of several iV-acetylhexosamine
residues bound by (1~3)
linkages
and partially
branched
at C-6.
INTRODUCTION
Recent structural studies of the blood-group substance (BGS) have shown the presence of a peptide backbone with carbohydrate chains attached by glycosidic bonds to serine and threonine residuesXd3. The elucidation of the structure of some oligosaccharides released on degradation of BGS yielded information on the structure of the terminal part of the carbohydrate chains 3-6 . The structure of the carbohydrate chains in the region adjacent to the peptide backbone is unknown. We have developed two new routes for approaching this problem. Firstly, we have studied the degradation of BGS (A + H), isolated from the lining of pig stomachs, by an enzyme preparation from
CZostridiumperfingeens, together
with some chemical
methods.
Secondly,
we
have investigated the structure of the carbohydrate chains by estimation of the alkaline-degradation products of N-acetyl hexosamines and D-galactose. The latter approach, which has not been applied hitherto in studies of glycoprotein structure, may be of general value. RESULTS AND DISCUSSION
Degradatioiz of BGS by an enzyme
preparation
from
Clostridium
perfringens.
After treatment of BGS with the enzyme preparation, ca. 45% of the substance was split off as a fraction of low molecular weight; fucose (7.5%), and galactose Carbohyd.
Res.,
12 (1970) 437-447
438
N. K. KOCHETKOV et al.
and N-acetylhexosamines (-35%) were released’. The peptide part of the polymer was largely unaffected, and the fragment (1) of high molecular weight was stable to repeated treatment with the enzyme preparations. Since 1 still contains -65% of carbohydrate, a more extensive degradation of BGS was sought by a combination of chemical and enzymic methods. BGS was treated in sequence with periodate and borohydride, and the resulting polyol (BGS I) was subjected to acid hydrolysis (pH 1.5, 3 h, 100”). The overall breakdown of BGS in this procedure is ca. 50%, whereas that of amino acids is less than 0.5%. The fragment (2) of high molecular weight subsequently isolated by gel filtration was then treated with the enzyme preparation, which resulted in cleavage of ca. 45% of the carbohydrate content. Further gel filtration gave a new fragment (3) of high molecular weight, which retained the amino acids characteristic of BGS, but in increased percentage (58%, CJ 16% for BGS) (see Table I). Fraction 3 contains ca. 40% of carbohydrate, no fucose, and + 6% of galactose. The remainder of the carbohydrate content is ZV-acetylhexosamine. TABLE
I
CONTENT
OF AMINO
ACIDS
AND
HEXOSAMINE.5
IN
BGS
AND
ITS
FRAGMENTSa
Preparation
Amino acids % (a)
N-Acet& hexosantitws % (b)
a/b
2-Acetamido-2-deo.q= D-galactose (pmolesjb (c)
2-Acetamido-I-deoxyD-glucose @moles)b (n)
c/d
BGS (A + H) 3 3 treated with NazCOa
16.0 58.2 35.8
41.0 28.9 15.8
0.39 2.0 2.26
1.13 2.29 0.82
1.45 0.30 0.16
0.8 7.6 5.1
=Determined by using an amino acid analyser after hydrolysis with 4~ HCi for 24 h at 100” in cacao. bReferred to 1 mp of substance.
The ratio of 2-acetamido-2-deoxy-D-galactose to 2-acetamido-2-deoxy-o-glucose (GluNac) in 3 is 7.6, whereas in the original BGS it is only 0.8. The preponderance of 2-acetamido-2-deoxy-D-galactose in 3 suggests that this hexosamine could constitute the sugar residues at the reducing end of the oligosaccharide chains and thereby be involved in the carbohydrate-peptide linkage, as well as forming part of the carbohydrate chains adjacent to the peptide backbone. The D-galactose and GiuNac are mainIy in the periphery of the molecule, since they are more readily split off during the enzymic and chemical degradation of BGS. This view was supported by the data obtained from the alkaline treatment (0.05~ Na,CO,, IOO”, 15 min) of 3. The monosaccharides which are gIycosidicalIy linked to serine and threonine are the first to be destroyed. Analysis of the mixture of alkaline-degradation products of 3 showed the decrease of 2-acetamido-2-deoxy-D-galactose (I .47 pmoles, Table I) to be 10.5 times that of Z-acetamido-2-deoxy-D-gIucose (0. I4 pmole). Carboh-vd. Res., 12 (1970) 437447
BLOOD-GROUP
439
SUBSTANCE
The degradation of BGS
with alkali
/&Elimination of the carbohydrate chains from the serine and threonine units of the peptide chain is known to proceed upon treatment of BGS with alkali. The carbohydrate chains liberated are then eroded from the reducing end to form a series of oligosaccharides that are characteristic of the terminal sequence of the monosaccharides
(cl: refs. 4 and 6).
The rapid degradation of the carbohydrate chains at the reducing end, which is observed on treating BGS with alkali, is characteristic of polysaccharides having (1+3) linkagesg. The degradation of such chains should result in the formation of 3-acetamido-5-dihydroxyethylfuran and, probably, 2-acetamido-2,3-dideoxy-D-hex2-enofuranose from N-acetylhexosamine residues. These compounds give the direct Ehrlich reaction, and the presence of chromogens in the mixture of alkaline-degradation products of BGS was reported earlier”. Metasaccharinic acid should be formed from the residues of gaiactose substituted at C-3, and isosaccharinic acid” from galactose residues substituted at C-4. A preliminary study of the degradation of BGS in 0.05~ Na,CO, has shown that the accurate determination of the amount of metasaccharinic and isosaccharinic acids by periodate oxidation is complicated by the sensitivity of BGS to periodate. Therefore, both BGS and the product (BGS I) obtained after periodate oxidation and borohydride reduction were subjected to alkaline degradationr2. The alkaline conditions chosen were based on model experiments with 2-acetamido-2-deoxy-D-glucose which was heated in 0.05M Na2C0, at 50, 70, and 100” (Fig. 1); 70” proved to be optimal, and, at this temperature, chromogen forms rapidly and is stable upon prolonged
heating.
Absorbance
Y 1
2
3
h
Fig. 1. The rate of formation of chromogen from 2-acetamido-2-deoxy-n-glucose on treatment with 0.05hc NaoCOa as determined by the Ehrlich reaction: 1, 50”; 2, 70’; 3, 100”. Chromogen
and metasaccharinic
acid are formed upon treating
BGS and BGS
I
in 0.05n1 Na,CO, at 70”. Isosaccharinic acid was not detected”, even after acid hydrolysis (0.5N HCI, lOO”, 12 h) of the alkaline-degradation products of BGS. This indicates the absence of 4- and 4,6-substituted galactose residues in the eroded parts of the carbohydrate chains. Curbohyd Res., 12 (1970) 437-447
N. K. KOCHETKOV et d.
Upon treatment of BGS and BGS I with aqueous Na,CO, (Fig. 2, Table II), chromogen was liberated rapidly; its release was markedly decreased in 4 h. Metasaccharinic acid formation began 7 min after treatment of BGS I with alkali, and the amount increased very slowly, corresponding, after 4 h, to -0.5% of galactose (of BGS) destruction. During this time, 10.5% of the N-acetylhexosamines were cleaved (Table II). The rates of &elimination reactions of 2-acetarnido-2-deoxy-D-glucosyl and D-galactosyl derivatives of the methylamide of N-benzyloxycarbonyl-L-serine were Absorbance 1 2
0.4-
x52 Absorbonce
0.33 0.2-
3 0.05
O-lk’
;L
2
1
4
3
10
20
30
min
5 ----z-
Fig. 2. The rate of formation of chromogen and metasaccharinic acid from BGS and BGS I on treatment with 0.05~ NaaCOa at 70”. 1, BGS; 2, BGS I (by Ehrlich reaction); 3, BGS I (by reaction with thiobarbituric acid). TABLE
II
THE NUMBER
OF DEGRADED
[CALCULATED ANALYSIS
FROM (AAA)
THE
DATA]
UNITS
AND
OF
5-HYDROXYMETHYL-kURLLDEHYDE Na2C03
AT
OF THE
WEIGHT
OF
BGS
GALACTOSE (III)
[~ALCULAT=D FORMED]
UPON
BGS I)
AND (I)
moht
THE
TREAThlENT
OF N-ACETYLHEXOSAMINES
FdRMED
AND
OF
BGS
AND
AMINO
FROht
hfa4sAccH~xr4rc
ACID
(II)
ACID AND
BGS I WrTH o.oi?ihl
70”.
Time of the treatment with NazC03 (min)
7 14 30 60 120 240 360 480
(%
3-ACETAhlIDO-S-DIHYDROXYFeTHYLFURAN
BGP
BGS Ib
N-Acetylhexosanrines from I
from III
0.52 1.14 2.1 3.3 5.2 6.5 6.6 -
0.02 0.14 0.31 0.50 0.72 0.71 0.71
D-
Galaciose
N-acetylhexosamines
D-Galactose
From I
From aaa
From II
From III
0.52
-
1.15 3.3 4.9 6.1 6.2 -
10.4 10.6 -
0.00 0.03 0.10 0.17 0.31 0.48 -
0.16 0.29 0.49 0.61 0.62 0.62
0.01
=The percentage of N-acetylhexosamines, galactose, and fucose in the original BGS is 43,22, and IO%, respectively. bathe percentage of N-acetylhexosamines, galactose, and fucose in original BGS I is 26, 10, and O%, respectively. Carbohyd. Res., 12 (1970) 43747
441
BLOOD-GROUP SJBSTANCE
approximately equal (Table III). Therefore, the difference in the rates of release of metasaccharinic acid and chromogen cannot be accounted for on this basis. Nor can the absence of metasaccharinic acid at the beginning of the degradation be accounted for by the differences in the rate of formation of metasaccharinic acid and chromogen. The degradation13 of laminarin, having a known number of reducing ends (kindly provided by Professor D. G. Manners), upon treatment with 0.05~ Na&O, at 70” results in rapid formation of metasaccharinic acid (Fig. 3). Thus, 3 moles of metasaccharinic acid are released in 7 min from each reducing end of laminarin, under conditions where no metasaccharinic acid was formed from BGS. The decrease in the glucose content (anthrone method) in laminarin after alkaline degradation corresponds precisely to the amount of metasaccharinic acid liberated. TABLE
III
DEGRADATION
OF THE
DE~x~-/LD-~LU~~SYL 0.05hl
TREATMENTWITH
GLYCOSIDIC
LINKAGE
(6) DERIVATIVES NazC03 AT 70".
OF
OF 3-m
O-/I-D-GALACTOSYL
(5)
N-BENZYLOXYCARBDNYL-L-SERINE
Trearment time (min)
Cleacage of 5 (% of sample)
Cleacage of 6 (% of sample)
1.5 30 60
77 96 100
72 97 100
AND
0-2-ACETAMIDO-2-
hmnw_.4hwx
UPON
Absqraance
20
60
120
180 min
Fig. 3. The rate of formation of metasaccharinic acid from BGS I and laminarin on treatment with 0.05~ NaaCOs at 70”. I, BGS I; 2, laminarin (by reaction with thiobarbituric acid).
Thus, the data obtained from the treatment of BGS and BGS I with 0.05~ Na,CO, at 70” indicate that (1+3)-linked galactose residues (as well as those” branched at C-6) do not contribute to the carbohydrate-peptide linkage and are essentially absent from the parts of the carbohydrate chains immediately adjacent to the peptide backbone. Apparently, these portions are made up of N-acetylhexosamine residues. The possible presence of a small number of (143)-linked galactose residues branched at C-4 or C-2 in this part of the BGS molecule is not excluded by this evidence. (1+3)-Linked galactose residues that are branched at C-Q are known to Carbohyd.
Res.. 12 (1970) 437-447
442
N. K. KOCHETKOV
et al.
afford 4-substituted metasaccharinic acid in alkaline medium, which cannot be directly determined l1 . However, after acid hydrolysis (0.5N HCl, lOO”, 12 h) of the alkaline-degradation products of BGS, there was no increase in the yield of metasaccharinic acid. Hence, the degraded chains do not contain any 3,4_disubstituted galactose residues. By analogy with glucose derivatives, 2,3-disubstituted galactose residues should he readily converted by alkali into 2-substituted 3-deoxyhex-2-enose derivatives which, under mild, acidic conditions, should yield 5-hydroxymethyl-2-furaldehyder4. Actually, 5-hydroxymethyl-2-furaldehyde is formed on heating 2,3-di-U-methyl-Dgalactose in 0.05~ Na,CO, for 7 min or 2 h at 70”, followed by acidic treatment, to extents corresponding to 9 or 58% degradation. Similar treatment of n-galactose gave no 5-hydroxymethyl-2-furaldehyde. To detect 2,3-disubstituted galactose residues in the cleaved parts of the carbohydrate chains, BGS and BGS 1 were treated with aqueous NaJO, at 70”, and then with acid at 50”. The absorption measured at 284 nm then corresponds to 5-hydroxymethyl-2-furaldehyde’4. The amount of 2-substituted 3-deoxyhex-2-enose formed and determined from 5-hydroxymethyl-Zfuraldehyde increased slowly and, after 4 h, corresponded to O&0.7% of galactose (of BGS). This, however, cannot radically change the conclusions that have been previously drawn, since the possible presence of 2,3-disubstituted galactose in the carbohydrate-peptide linkage unit has been ruled out. The presence of small proportions of 3- and 2,3_substituted galactoses (ca. 1.0% from BGS) in the reducing end of the carbohydrate chain has not been explained so far, but it might indicate the inhomogeneity of the BGS carbohydrate chains. All of these data support the conclusion that there are 2-acetamido-2-deoxy-Dgalactose residues in the region of the glycosylpeptide linkage of BGS. After 4 h of alkaline degradation of BGS, chromogen formation ceases (Fig. 2), indicating completion of the process. The alkaline-degradation products obtained after 4 h of treatment of the BGS I with Na,C03 were separated on Biogel P-6 into two fractions (Fig. 4). Almost all of the amino acids were in the fraction (4) of high molecular weight. The amino acid content of the low molecular fraction was 0.5% of BGS I (Table IV). The yield of 4 is ca. 50%, and, hence, the alkaline treatment results in cleavage of ca. 60% of the carbohydrate residues of BGS. The carbohydrate remaining bound to the peptide skeleton of the glycoprotein is not split off under the alkaline conditions chosen. The anaIysis of the chromogens formed provides further information concerning the carbohydrate portion of BGS which is degraded by alkali. Chromogens are formed only from 3- and 6-monosubstituted and 3,6_disubstituted N-acetylhexosamines15. An unsubstituted chromogen should form from the residues of (l-*3)linked N-acetylhexosamines which arose from alkaline degradation of BGS carbohydrate chains: (1+6)-linked N-acetylhexosamines or (1+3)-linked units branched at C-6 should yield a substituted chromogen. Since (l-+4)-linked residues are stable to alkali, the N-acetylhexosamine residues at the reducing end of BGS I carbohydrate chains should be bound by (143) links, with or without branches at C-6. Gel-filtraCarbolaycf. Res., 12 (1970)
437-447
BLOOD-GROUP
TABLE AMINO
443
SUBSTANCE
IV
ACIDa
CONTENT
OF
BGS I, AND FRACTIONS
4,
AND
LMF 4
Amino ncia!s
ZIGS Z (%)
4 (%)
LMF 4 (%)
Asparaginic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine x Amino acids
0.31 5.09 2.20 0.76 3.10 0.37 0.91 0.92 0.15 0.64 14.45
0.57 5.48 1.43 1.38 6.78 0.56 1.75 1.47 0.23 1.12 20.77
0.06 0.13 0.13 0.16 0.13 0.10 0.11 0.11 0.04 0.07 1.04
%xcluding
Abr
the basic amino acids. Absorbance
iar bance
I
1 lb
5.0
Go
2%
60
ml
140
200
260
ml
Fig. 4. Gel-filtration of products from alkaline hydrolysis of BGS I (0.05hl NaeCO3, 70”, 4 h) on a column (1.5 x 60 cm) of Biogel P-6 in 0.1~ CHsCOOH. Fractions were assayed by 1. absorbance at 232 nm; 2, by the phenol-sulphuric acid method: and 3, by the Ehrlich reaction. Fig. 5. Gel-filtration of LMF 4 (0.05&t Na2C03, 70”, 4 h) on a column (1.5 x 60 cm) of Biogel P-2 in 0.1~ CH&OOH. Fractions were assayed by: 1, absorbance at 232 nm; 2. by the phenol-sulphuric acid method; and 3, by the Ehrlich reaction. tion
on
Biogel
P-6 (Fig.
ated solely in the fraction
4) shows that the chromogen
of low moIecular
formed from BGS I accumulweight (LMF 4). The fractionation of
LMF 4 on BiogeI P-2 (Fig. 5) gave two chromogen peaks Cr and II) which probably correspond to a substituted and an unsubstituted chromogen. Further evidence for the presence of two types of chromogen in LMF 4 was CurEohyd. Res., 12 (1970) 437447
N. K. KOCHETKOV
444
et al.
based on periodate oxidation of the unsubstituted chromogen to form 3-acetamidoZ-furaldehyde, according to the scheme: CH,OH
&$H,oH
zL”l,,,:“‘_OHC~NH*c
NHAc
2-Acetamido-2-deoxy-D-glucose and its 3,6-dimethyl ether were used as model compounds. After heating in 0.05~ Na,C03 at 70”, with subsequent periodate oxidation, the chromogens formed were determined by the direct Ehrlich reaction. 3-Acetamido-2-furaldehyde reacted rapidly, but the resulting colour was unstable and disappeared in 15 min. 3-Acetamido-5-(1-hydroxy-2-methoxyethyl)furan was not oxidized with periodate, and colour formation in the Ehrlich reaction reached a maximum in 15-20 min. This distinctive behaviour of the chromogens allowed their separate determination. Thus, analysis of fractions I and II (Fig. 5) obtained from LMF 4 by this method confirmed that it is mainly the substituted chromogen which is formed from the fraction of higher molecular weight, whereas the fraction of low molecular weight aIIords the unsubstituted chromogen. The absorbances of chromogen reaction-products formed from equimolecular amounts of 3-substituted and 3,6disubstituted 2-acetamido-2-deoxy-D-glucose in the Ehrlich reaction are nearly equal15. The ratio of the absorbances of the reaction products from fractions I and Ii (Fig. 5) is ca. 3:1, and, hence, most of the hexosamines constituting the reducing end of carbohydrate chains of BGS I are bound by (l-+3) links and are branched at position 6. This conclusion is supported by the fact that experiments (A. V. Sharova) on the acid hydrolysis of BGS give oligosaccharides containing only hexosamine residues. EXPERIMENTAL
BGS (A + H) from pig-stomach linings (at pH 3) and BGS I l2 were obtained by literature procedures. The enzyme preparation was isolated7 from culture liquid of Clostridium perfiingens (type A). Neutral monosaccharides were determined by the phenol-sulphuric acid” and anthrone methodsi*, fucose by the Dische methodrg, and hexosamines by the Elson-Morgan method after preliminary hydrolysis with 2~ HCl for 2 h at loo”. Amino acids and hexosamines were determined on an amino acid analyser after hydrolysis with 4N HCl for 24 h at 100” in uacuo. Paper chromatography was carried out with 6:4:3 butyl alcohol-pyridine-water and detection with alkaline silver nitrate. Acid hydrolysis of BGS I. - A solution of BGS I (1.6 g) in 96 ml of diluted HCl (pH 1.5) was heated for 3 h at lOO”, neutralized with Dowex-2 (CHsCOO-), and loaded onto a column (69 x 3.9 cm) of Sephadex G-50. Fractions (40 ml) were collected, and their absorbance at 232 nm was measured. The sugars were determined by the phenol-sulphuric acid method. The fractions corresponding to the high Carbohyd.
Res.. 12 (1970)
437-447
BLOOD-GROUP
445
SUBSTANCE
molecular weight substance were collected, evaporated, and freeze-dried to yield 720 mg of HMF 2. Emymic hydrozysfs of 2. - To a solution of 715 mg of 2 in 71.5 ml .of 0.01~ phosphate buffer (pH 6.9), a 0.1% aqueous solution (36 ml) of theenzymepreparation was added, and the mixture was incubated for 18 h at 37”. Low molecular weight products were separated by gel-filtration on Sephadex G-50, and the high molecular weight substance was freed from enzyme by gel-filtration on Sephadex G-200. Yield: 400 mg of 3. The enzymic hydrolysis of BGS (A + H) was performed in a manner analogous to that described above. Alkaline degradation of BGS (A 4- Hj and BGS 1: - BGS or BGS I (50 mg) was dissolved in 10 ml of 0.05~ Na,CO, and heated at 70”. AIiquots were taken for determination of chromogen (0.5 ml), metasaccharinic acid (2 x 0.2 ml), galactose (0.1 ml), and 5-hydroxymethyl-2-furaldehyde (0.5 ml), after 7, 15, 30, 60, 120, 240, 360, and 480 min. The quantity of chromogen formed from the N-acetylhexosamines was determined by using Ehrlich’s reagent14. Water (0.5 m1) and 1 ml of p-dimethylaminobenzaldehyde solution (10 mg/ml of 50% CH,COOH) were added to the aliquot with shaking, and then 0.2 ml of cont. HCl was added. Absorbance was measured at 540 nm after IO-15 min. Free and 6-substituted metasaccharinic acids and isosaccharinic acid were determined by the method of Barker et al.“, and 4- and 4,6-substituted metasaccharinic acids and 5-substituted isosaccharinic acid were determined” after preliminary hydrolysis of the alkaline-degradation products with 0.5~ HCI for 12 h at 100”. 5-Hydroxymethyl-Z-furaldehyde (7) was determined by the following procedure: 0.1~ HCl (0.54 ml) was added to the aliquot (O-5 ml) which was then diluted with water to 4 ml. The absorbance of the solution was measured at 284 nm, the solution was then heated at 50’ for 12,15,18, and 24 h, and the absorbance was remeasured. The quantity of 7 was calculated by using the molar extinction coefficientsX4 (Table II). The reaction mixture was extracted twice with butyl alcohol, and the u. v. spectrum of the extract was identical to that of 7 (ref. 14). No 7 was found when D-galactose was treated with 0.05M Na,CO, at 70”. Alkaline degradation of 2-acetamido-2-deoxy-D-glucose. - A solution of 5 mg of the sugar in 5 ml of 0.05M Na,CO, was heated at 50,70, and 100”. Aliquots (0.5 ml) were taken after 5,15,30,60,120, and 180 min, and the quantity of chromogen formed was determined by the aforementioned method (Fig. 3). Alkaiine degradation of laminarin. -This was accomplished under the conditions used for BGS degradation, with laminarin concentrations of 0.1 and 0.05 mg/ml. Aliquots (0.2 ml) were taken from the reaction mixture after 3, 7, 10,20,40, 60, 120, and 180 min, and the metasaccharinic acid was determined” (Fig. 2). Glucose was determined by the anthrone method in aliquots taken from the original reaction mixture and after heating for 120 min. Alkaline degradation of 2,3-di-0-methyl-D-galactose. The sugar (2 mg) Carbohyd. Res., 12
(1970) 437447
444
N. K. KOCHETKOV
et al.
was dissolved in 40 ml of 0.05~ Na2CQ, and heated at 70”. Aliquots (1 ml) were taken after 7, 15, 30, 60, 90, and 120 min, 0.1~ HCl (1.03 ml) was added, the solution was diluted with water to 3 ml (final HCl concentration, 0.01~), and the u. v. spectrum of the mixture was recorded after heating for 24 h at 50”. AZkaZine degradation of O-j?-D-galactosyl and O-(2-acetamido-2-deoxy-B_DgZucosyZ) derivatives of N-benzyloxycarbonyl-L-serine methylamide. - The reaction was performed under the same conditions as for the degradation of BGS, using a 0.5 mg/ml solution of glycosides. Aliquots (0.2 ml) were taken after 15, 30, 60, and 120 min. The determination of the degree of destruction (see Table III) was based on the estimation of formaldehyde” after periodate oxidation. The amount of formaldehyde is equivalent to that of free monosaccharides formed from glycosides upon /?-elimination. The separation of products of alkaline degradation of BGS. - BGS I (200 mg) was dissolved in 40 ml of 0.05~ Na,CO, and heated for 4 h at 70”. The solution was neutralized with Amberlite IR-120 (Hf) batchwise, concentrated under diminished pressure, loaded onto a column (60 x 1.5 cm) of Biogel P-6, and eluted with 0.1~ CH,COOH. Fractions (6 ml) were collected, and the absorbance was measured at 232 nm. Fractions were assayed for sugar content by the phenol-sulphuric acid method, and chromogen was determined by the method mentioned above. Fig. 4 shows the gel-filtration pattern. The fractions corresponding to the fragment (4) of
high molecular weight (fraction 1) and that (LF 4) of low molecular weight (fraction 2) were concentrated and freeze-dried to give 4 (92 mg) and LF 4 (76 mg). LF 4 (76 mg) was dissolved in 2 ml of 0.1~ CHsCOOH, loaded onto 2 column (60 x 1.5 cm) of Biogel P-2, and eluted with 0.1~ CH,COOH. Fractions (6 ml) were collected, their absorbance at 232 nm was measured, and the content of sugars and chromogen was determined in aliquots. Fig. 5 shows the gel-filtration pattern. The fractions corresponding to fragments of high molecular weight (fraction l) and low molecular weight (fraction II) were concentrated and freeze-dried to give fraction I (42 mg) and fraction II (20 mg). Determination of the substituted chromogen in the presence of unsubstituted one. - N210, solution (O-2 ml; 0.0618~ NaIO, in 3~ H,PO,) was added to 0.5 ml of the solution under investigation, and the mixture was shaken and left at room temperature for 20 min. A portion (1 ml) of a Na,AsO, solution (10% of Na,AsO, in 0.5~ Na,SO,) was added, and the solution was shaken until the brown colour disappeared (2-3 min) and then diluted with water to 2 ml. Then 2 ml of a solution containing 10 mg of p-dimethylaminobenzaldehyde per ml of 50% CH,COOH and 0.4 ml of cont. HCl were added, and the absorbance at 540 nm was measured after 15-20 min. REFERENCES 1 N. K. KOCHETKOV, V. A. DEREWTSKAYA, Ah?, S. G. KARA-MURZA, Carbohyd. Res., 3 (1967) 403. 2 B. ANDERSON. N. SENO, P. SAMPSON, I. G. RIBY, P. HOFFMAN, AND K. MEYER, J. Biol. Chem., 239 (1964) PC 2716. 3 K. LLOYD,E. A.KABAT,E.LAGUG,AND F. GRKJEZO, Biochemistry,5 (1966)1489.
Carbokyd. Res., 12 (1970) 437-447
BLOOD-GROUP
447
SUBSTANCE
4 K. 0. LLOYD, E. A. KABAT, AND E. LICARIO, Biochemistry, 7 (1968) 2976.
5 W. M. WATKINS, Science, 152 (1966) 172. A. S. R. DONALD, AND W. T. J. MORGAN, Biochem. Biophys. Res. Commun., 30 (1968) 1; 33 (1968) 508; BJocJzem. J., 107 (1968) 861. 7 L. M. LICHOSHERSTOV,M. D. MARTYNOVA, AND V. A. DEREVIWKAYA, Biokhimia, 33 (1968) 1135. 8 N. K. KOCHETKOV, V. A. DEREVITXAYA, L. M. LICHOSHER~TOV, AND M. D. MARTYE~OVA,Dokl. Akud. Nuuk SSSR, 186 (1969) 216. 9 R. L. WHISTLER AND J. N. BEMILLER, Adcan. Curbohyd. Chem., 13 (1958) 289. 10 K. W. KNOX AND W. T. J. MORGAN, Biochem. J., 58 (1954) V. 11 S. A. BARKER, A. R. LAW, P. J. SOARERS, AND M. STACEY, Carbohyd. Res., 3 (1967) 435. 12 N. K. KOCHETKOV, G. S. Kxor, L. S. BOGDASHOVA, L. M. LICHOSHER~TOV, AND V. A. DEREVITSKAYA, I.v. Akad. Nauk SSSR, Ser. Kilim., 9 (1968) 2095. 13 W. D. ANNAN, E. L. HIRST, AND D. J. MANNERS, J. Chem. Sot., (1965) 885. 14 E. F. L. J. ANET, Chem. 2nd. (London), (1963) 1035. 15 D. AhlINOFF, W. T. J. MORGAN, AND W. M. WATKINS, Biochem. J., 51 (1952) 379. 16 L. M. LICHOSHERSTOV,V. A. DEREVITSKAYA, AND V. 1. FEDOROVA, Biokhimiya, 34 (1969) 45. 17 M. DUBOIS, K. A. GILLE.T,J. K. HAMILTON, P. A. REBERS, AND F. ShmH, Anal. Chem., 28 (1956) 350. 18 G. N. ZAIT~EVA AND T. N. AFANASYEVA, Biokhimiyo, 22 (1957) 1035. 19 2. DISCHE AND L. B. SHEITLES, J. Biol. CJlem., 175 (1948) 595. 20 T. NASH, Biorlrem. J.. 55 (1953) 416.
6 W. P. AXON,
Carboltyd. Res., 12 (1970) 437-447