THROMBOSIS RESEARCH Printed in the United
vol.
6,
pp.
Pergamon
States
215-221, Press,
1975 Inc.
DISTRIBUTION OF PROTHROMBIN CARBOHYDRATE UNITS UPONTHROMBINACTIVATION Billy G. Hudsonl, Charles M. Heldebrant 2,3 , and Kenneth G. Mann’ 1 Present address: Department of Biochemistry, Kansas University Medical Center, Kansas City, Kansas 66103 2 Mayo Clinic and Mayo Foundation, Rochester, Minnesota 3 Present address: Department of Biochemistry, University of Washington, Seattle, Washington
(Received 14.1.1975; in Accepted by Editor
revised R.F.
form 27.1.1975. Doolittle)
ABSTRACT The number of heteropolysaccharide units of prothrombin has been reinvestigated and their distribution upon activation has been deterThe results of this study suggest that prothrombin contains mined. and that upon activation two units three heteropolysaccharide units, are present in the intermediate 3 portion of the molecule and one unit is present in the intermediate 1 portion. Subsequent activation of intermediate 1 yields intermediate 4, which is devoid of carbohydrate and intermediate 2, the immediate thrombin precursor, which contains one heteropolysaccharide unit. Cleavage of intermediate 2 to yield cr-thrombin is a conservative process with respect to carbohydrate, while the conversion of a-thrombin to g-thrombin results in the loss of a carbohydrate containing comprnent X.
INTRODUCTION Bovine prothrombin is a glycoprotein which has been reported to contain The monosaccharide constituents are dis9 to 15 percent carbohydrate (l-4). tributed about 4 heteropolysaccharide units which are composed of sialic acid, galactose, mannose, and N-acetylglucosamine in molar proportions of 2-3:2:3:2-4 2-4, respectively (4). These units are distributed unequally among the proteolytic products produced on thrombin activation of prothrombin as determined from the partial carbohydrate compositions of these intermediates (2,5). The intent of this paper is to determine the precise distribution of these heteropolysaccharide units based on the complete carbohydrate composition of prothrombin and the various intermediates and the carbohydrate composition of the heteropolysaccharide unit. METHODS Proteins Prothrombin was purified by the method of Bajaj and Mann (6). cy-Thrombin was purified by the modification of the method of Lundblad (7) described by Mann et al. (8) Prothrombin intermediates 1 and 3 were prepared and separated by used in this theTechniques described by Heldebrant et al. (5). The proteins study were analyzed electrophoretically and, by the techniques used (51, were effectively homogeneous. 215
PROTBROMBIN CARBOHYDRATE UNITS
216
Carbohydrate
vo1.6,No.3
Analyses
Neutral monosaccharides were identified and quantitated on a Technicon chromatographic system, as described by Lee et al. (6), after release from Glycoprotein saGl= were hydrolyzed with 2N sulprotein by acid hydrolysis. furic acid for 4 hours at 100’ in Tealed tubes, and the hydrolysate passed through a column of Uowex 50-X4 (H ) (200-400 mesh) coupled to a column of Dowex l-X8 (formate) (200-400 mesh) as described by Spiro (7). The effluent and wash were taken to dryness by lyophilization and then analyzed on the Corrections were made for hydrolytic destruction of monoTechnicon system. saccharides by the treatment of known quantities in the presence of bovine serum albumin under identical conditions of hydrolysis. Neutral monosaccharides were also released from the protein by hydrolysis with Dowex 50 ion-exchange resin according to the procedure of Lehnhardt and Winzler (11). The hexosamines, glucosamine and galactosamine, acid analyzer (12) after hydrolysis of the protein hours (10).
we,re determined on the amino with 4N HCl at 100’ for 6
Sialic acid was determined by the thiobarbituric method of Warren(13) hydrolysis of protein with O.lN HCl at 80’ for 1 hour. N-acetylneuraminic acid was used as a standard.
after
RESULTSAND DISCUSSION The carbohydrate compositions of prothrombin and its thrombin catalyzed proteolytic products are presented in Table 1. Intermediates 1 and 3 each contain all the monosaccharide constituents present in prothrombin, except galactosamine which is absent in the former. However, these intermediates differ in their total carbohydrate content. Intermediates 1 and 3 consist of 3.9 and 14 percent carbohydrate by weight, respectively, which accounts for 35 and 57 percent of the carbohydrate originally present in prothrombin. These latter values are in agreement with the percentages of 33 and 66 calculated from the percentage composition data reported by Owen et al. (2) for intermediates 1 and 3 respectively. The molar proportions of the individual monosaccharides are essentially the of glusame in both intermediates as well as prothrombin, with the exception cosamine and galactosamine, as seen from the calculations presented in Table 11. acid each occur in about a 1 to 1 ratio to galactose in Mannose, and sialic prothrombin and intermediates 1 and 3. In contrast, the amounts of glucosamine and galactosamine are enriched in intermediate 3. These comparisons suggest that the heteropolysaccharide units present in intermediates 1 and 3 have closely related structures but differ in the number of hexosamine residues. The distribution of the heteropolysaccharide units of prothrombin in the thrombin proteolytic products can be calculated on the basis of the composition data presented in Table land the composition of these units. Nelsestuen and Suttie (4) concluded that the carbohydrate of prothrombin is distributed about heteropolysaccharide units composed of sialic acid, galactose, mannose and Nacetylglucosamine in a molar proportion of 2-3:2:3:2-4, respectively. Therefore, it can be calculated, as given in Table 2 , that prothrombin contains 2 heteropolysaccharide units based on mannose content and 3 based on galactose or sialic acid. The lower number of units calculated on the basis of mannose reflects the lower amount of mannose relative to galactose or sialic acid reported here as compared to the value reported by Nelsestuen and Suttie (4).
Vo1.6,No.3
PROTHROMBIN
CARBOHYDRATE
217
UNITS
TABLE I Carbohydrate Composition Thrombin Catalyzed
Prothrombin
Monosaccharide
mg per 1oOmg proteina
Residues
Mannose
1.51
6.5
Galactose
1.35
of Prothrombin Proteolysis of
and Products Prothrombin
Intermediate
mg per 1oomg proteina
0.82
2.6
5.8
0.76
.05
0.2
Glucosaminea
2.45
Galactosamined Sialic
FucoseC
Total
Acid
Residues
Intermediate
perb mole
3
1+3
Residues
Residues
perb mole
perb mole
2.46
3.5
6.1
2.4
2.24
3.2
5.6
0.03
0.1
0.09
0.1
0.2
8.4
0.86
2.2
4.33
4.9
7.1
0.13
0.4
c.01
0.34
0.4
0.4
2.65
6.4
1.46
2.6
4.50
3.6
6.2
8.14
28.0
3.93
9.9
16.0
26.0
perb mole
mg per 1oOmg proteina
1
of
14.0
aValues expressed as residue weight per 1OOmg of glycoprotein. Protein content determined by quantitative amino acid analysis. b Based on molecular weight of 70,000, 51,000, and 23,000 for prothrombin, intermediate 1 and intermediate 3, respectively, as reported by Heldebrant --et al. (5) ‘Amount released on hydrolysis with d Expressed as N-acetyl derivative.
Dowex 50 resin.
Similar calculations (Table II> indicate that intermediate 1 contains 1 heteropolysaccharide unit based on mannose, galactose, or sialic acid, whereas intermediate 3 contains 1 unit based on mannose and 2 units based on galactose or sialic acid. it appears that the monosaccharide constituents of proHence, thrombin are distributed about 3 heteropolysaccharide units and on thrombin proteolysis, one of these units is associated with intermediate 1 and the other 2 are associated with intermediate 3. This distribution of units is consistent with the finding that about one-third of the total prothrombin carbohydrate by weight is accounted for by intermediate 1 and two-thirds by intermediate 3. The number of heteropolysaccharide units calculated above for prothrombin is inconsistent with the number reported by Nelsestuen and Suttie (4). They concluded that prothrombin contains 4 units, as opposed to 3, based on the findings of 7.5 galactose residues per mole of protein and 2 galactose residues They further calculated that 4 such units would reper mole of glycopeptide. present 15 percent of the prothrombin weight as carbohydrate based on the composi
PROTHROMBIN CARBOHYDRATE UNITS
218
Vo1.6,No.3
TABLE II Comparison of Monosaccharide Ratios and Number of Carbohydrate Units in Prothrombin and Products of Thrombin Catalyzed Proteolysis of Prothrombin
Residues/Galactose
Monolsaccharide
Pro thrombin
Residuea
Intermediate
1
Intermediate
Galactose
1.0
1.0
1.0
Nannose
1.1
1.1
1.1
Glucosamine
1.4
0.92
1.5
Galactosamine
0.1
0.0
0.1
1.1
1.1
1.2
Sialic
Basis
acid
+ fucose
of Calculation
Number of Carbohydrate
Units
per moleculeb
Mannose
2.2(2)
0.86(l)
1.2(l)
Galactose
2.9(3)
1.2
(1)
1.6(2)
Sialic
3.2(3)
1.3
(1)
1.8(2)
acid
3
aValues calculated from data given in Table I. b Values calculated from number of mannose , galactose and sialic acid residues per mole of protein (Table I) based on 3 mannose, 2 galactose, and 2 sialic acid residues per carbohydrate unit as reported by Nelsestuen and Suttie (4). Values based on sialic acid represent a maximum number since the unit contains 2 to 3 sialic acid residues. Values in parentheses rounded to nearest integer. tion of the glycopeptides. However, a comparison of their calculated composition data to other literature values indicates a large discrepancy as shown in Table III. The total carbohydrate composition of prothrombin reported herein and by several other investigators is quite close, 8.2 to 9.9 percent by weight, butprobably is much lower tlnn 15 percent. This difference is also prominent for each class of neutral sugar, hexosamine, and sialic acid. Thus, it appears more likely that prothrombin contains 3 heteropolysaccharide units rather than 4. An alternate explanation for this difference is that multiple forms of prothrombin occur which differ in their amounts of carbohydrate. Therefore, the results of this study indicate the overall distribution of the heteropolysaccharide units of prothrombin on thrombin activation to be as depicted schematically in Figure 1. The overall mechanism of the activation process was reported previously (5,15). Prothrombin (with 3 CHO units) is
Vo1.6,No.3
PROTHROMBIN CARBOHYDRATE UNITS
219
TABLE III Comparison
of
Literature Values for the Carbohydrate of Bovine Prothrombin
Data reported in Table I
Owen, -et al. (2)
Magnusson (1)
Composition
Tishkoff et al. -(3)
Nelsestuen 6 Suttie (4)
Carbohydrate Percentage
Weight
2.9
3.6a
3.0
3.8a
4. 5c
Glucosamine
2.5
2.5
1.8
3.3b
5.sc
Galactosamine
0.13
0
0.2
Sialic
acid
2.7
3.2
4.15
2.8
Total Carbohydrate
8.2
9.3
9.2
9.9
Neutral
sugar
aAmount of neutral color (7). b Amount determined
sugar
corrected
for
contribution
by Elson Morgan reaction
‘Values calculated from the amount of monosaccharide relative to galactose d Expressed as N-acetylglucosamine.
(11)
b
of
sialic
4.7c
15.0
acid
and represents
galactose in protein in glycopeptides.
0
to anthrone
total
hexosamine.
and the amount of
each
cleaved by thrombin to yield intermediate 3 ‘(with 2 CHO units) and intermediate 1 (with 1 CHO unit). Intermediate 1 is cleaved by factor Xa to yield intermediate 4 (no CHO unit) and intermediate 2 (with 1 CHO unit). Intermediate 2 is cleaved by Factor Xa to yield cu-thrombin (with 1 CHO unit). a-Thrombin is then converted to 8-thrombin by the excision of component x which bears the single CHO unit. In further support of this distribution of carbohydrate units, thrombin has been reported to contain a single unit (16). Moreover, the total carbohydrate content of thrombin of 5.3% as reported by Gwen et al. (2) and 5.0% as reported by Magnusson (17) agrees closely with the PredictedTezentage of 5 based on this reaction scheme and the composition of intermediate 1.
PROTHROMBIN CARBOHYDRATE UNITS
220
ALA
vo1.6,No.3
Prothrombin (70,000) , I I CHO CHO CHO I fhtombin +
Inl-3 (23,000) ALA-
Int -4 @l,OOO) +SER -x--
CHO CHO
+
x,
Int.-2(41,000) THR*-
Int-4 (13,000) SER-+
CHO
a- thrombin
(33,000) (6,000) THR T_~$~,I,,
(S,OOO-6,500) T Compon8nt CHO FIG.
X
1
Distribution of the heteropolysaccharide units of prothrombin on thrombin The sequence of events are as previously reported activation of prothrombin. and thrombins are represented schemThe prothrombin, intermediates, (5,15). CHO represents a heteropolysaccharide atically and molecular weights shown. is formed from cu-thrombin by unit. B-Thrombin, the last product identified, degradation of most of the A chain (6,000 daltons) and cleavage of the B chain as to delete the carbohydrate. (18)
so
ACKNOWLEDGEMENTS The authors would like to express their appreciation to Linda Wegener for her This investigation was supported by grants from the expert technical assistance. National Institutes of Health (Am-15112) and the Oklahoma Agricultural Experiment Station (Journal Article 2883) to Billy G. Hudson, and by grants from the National Institutes of Health (HL-15381-03 and HL-16150-01) to Kenneth G. Mann, and by general research support funds of the Mayo Clinic and Mayo Foundation.(Grant #5 SO1 RR 05530-10) REFERENCES 1.
Preparation MAGNUSSON,S. Ark Kemi 23, 285, 1965. --
and carbohydrate
analysis
of bovine
prothrombin.
vo1.6,~0.3
PROTHROMBIN
CARBOHYDRATE
UNITS
221
2.
OWEN, W.G., ESMON, C.T., AND JACKSON, C.M. The conversion of prothrombin to thrombin I. Characterization of the reaction products formed during the activation of bovine prothrombin. a-J Biol Chem 249,594,1974.
3.
TISHKOFF, G.H., WILLIAMS, L.C., AND BROWN, D.M. Preparation of highly purified prothrombin complex 1. Crystallization, biological activity and molecular properties. _-J Biol Chem 243,4151,1968.
4.
NELSESTUEN, G.L., AND SLJTTIE,J.W. The carbohydrate of prothrombin. Partial structural determination demonstrating the presence of o-galactose residues. J _--Biol Chem 247,6096,1972.
5.
HELDEBRANT, C.M., BUTKOWSKI, R.J., BAJAJ, S.P. AND MANN, K.G. The activation of prothrombin 11. Partial reactions, physical and chemical characterization of the intermediates. _-J Biol Chem 248,7149,1973.
6.
BAJAJ,S.P. AND MANN, K.G. Simultaneous purification of bovine prothrombin and factor X. Activation of prothrombin by trypsin-activated factor X. _J Biol Chem 248,7729,1973.
7.
LLJNDBLAD,R.L. A rapid method for the purification of thrombin and the inhibition of the purified enzyme with phenylmethylsulfonyl fluoride. Biochemistry 10,2501,1971.
6.
MANN, K.G., HELDEBRANT, C.M. AND FASS, D.N. Multiple active forms of thrombin I. Partial resolution, differential activities, and sequential formation. J Biol Chem 246,5994,1971. _--
9.
LEE, Y.C., MCKELVY, J.F. AND LANG,D. Rapid automatic analysis of sugar components of glycoproteins II. Neutral sugars. Anal Biochem 27,567,1969.
10.
SPIRO, R.G. The carbohydrates of glycoproteins: In: E.F. Neufeld (ed); Methods Enzymol 8,3,1966.
11.
LENHARDT, W.F., WINZLER, R.J. Determination of neutral sugars in glycoproteins by gas-liquid chromatography. J Chromatog 34,471,1968.
12.
GUIRE, P., RIQUETTI, P. AND HUDSON, B.G. An accelerated single column procedure for the programmed analysis of naturally occurring amino acids of collagen and basement membrane. J Chromatog 90,350,1974.
13.
WARREN, L. 1971,1959.
14.
J Biol Chem BOAS, N.F. Method for determination of hexosamines in tissues. _-204,553,1953.
15.
MANN, K.G., YIP, R., HELDEBRANT, C.M., FASS, D.N. Multiple active forms of Polypeptide chain location of active site serine and carbothrombin III. hydrate. _-J Biol Chem 248,1868,1973.
16.
MAGNUSSON, S. In: Structure-function relationships of proteolytic enzyme (Desnuelle, P., Neurath, H., and Ottesen, M.EDS) p.138, Munlcsgaard, Copenhagen, 1970.
17.
MAGNUSSON, S. in:
18.
LUNDBLAD, R.L., VOSEL, C.N., UHTES, L.C., NOYES, C., KINGDON, H.S. AND MANN, K.G. Manuscript in preparation.
The thiobarbituric acid assay of sialic acids. --J Biol Chem 234,
Enzymes (Bayer, P.D. EDS), Vol. 111,271,1971.
vol.
6,
pp.
Pergamon
States
215-221, Press,
1975 Inc.
DISTRIBUTION OF PROTHROMBIN CARBOHYDRATE UNITS UPONTHROMBINACTIVATION Billy G. Hudsonl, Charles M. Heldebrant 2,3 , and Kenneth G. Mann’ 1 Present address: Department of Biochemistry, Kansas University Medical Center, Kansas City, Kansas 66103 2 Mayo Clinic and Mayo Foundation, Rochester, Minnesota 3 Present address: Department of Biochemistry, University of Washington, Seattle, Washington
(Received 14.1.1975; in Accepted by Editor
revised R.F.
form 27.1.1975. Doolittle)
ABSTRACT The number of heteropolysaccharide units of prothrombin has been reinvestigated and their distribution upon activation has been deterThe results of this study suggest that prothrombin contains mined. and that upon activation two units three heteropolysaccharide units, are present in the intermediate 3 portion of the molecule and one unit is present in the intermediate 1 portion. Subsequent activation of intermediate 1 yields intermediate 4, which is devoid of carbohydrate and intermediate 2, the immediate thrombin precursor, which contains one heteropolysaccharide unit. Cleavage of intermediate 2 to yield cr-thrombin is a conservative process with respect to carbohydrate, while the conversion of a-thrombin to g-thrombin results in the loss of a carbohydrate containing comprnent X.
INTRODUCTION Bovine prothrombin is a glycoprotein which has been reported to contain The monosaccharide constituents are dis9 to 15 percent carbohydrate (l-4). tributed about 4 heteropolysaccharide units which are composed of sialic acid, galactose, mannose, and N-acetylglucosamine in molar proportions of 2-3:2:3:2-4 2-4, respectively (4). These units are distributed unequally among the proteolytic products produced on thrombin activation of prothrombin as determined from the partial carbohydrate compositions of these intermediates (2,5). The intent of this paper is to determine the precise distribution of these heteropolysaccharide units based on the complete carbohydrate composition of prothrombin and the various intermediates and the carbohydrate composition of the heteropolysaccharide unit. METHODS Proteins Prothrombin was purified by the method of Bajaj and Mann (6). cy-Thrombin was purified by the modification of the method of Lundblad (7) described by Mann et al. (8) Prothrombin intermediates 1 and 3 were prepared and separated by used in this theTechniques described by Heldebrant et al. (5). The proteins study were analyzed electrophoretically and, by the techniques used (51, were effectively homogeneous. 215
PROTBROMBIN CARBOHYDRATE UNITS
216
Carbohydrate
vo1.6,No.3
Analyses
Neutral monosaccharides were identified and quantitated on a Technicon chromatographic system, as described by Lee et al. (6), after release from Glycoprotein saGl= were hydrolyzed with 2N sulprotein by acid hydrolysis. furic acid for 4 hours at 100’ in Tealed tubes, and the hydrolysate passed through a column of Uowex 50-X4 (H ) (200-400 mesh) coupled to a column of Dowex l-X8 (formate) (200-400 mesh) as described by Spiro (7). The effluent and wash were taken to dryness by lyophilization and then analyzed on the Corrections were made for hydrolytic destruction of monoTechnicon system. saccharides by the treatment of known quantities in the presence of bovine serum albumin under identical conditions of hydrolysis. Neutral monosaccharides were also released from the protein by hydrolysis with Dowex 50 ion-exchange resin according to the procedure of Lehnhardt and Winzler (11). The hexosamines, glucosamine and galactosamine, acid analyzer (12) after hydrolysis of the protein hours (10).
we,re determined on the amino with 4N HCl at 100’ for 6
Sialic acid was determined by the thiobarbituric method of Warren(13) hydrolysis of protein with O.lN HCl at 80’ for 1 hour. N-acetylneuraminic acid was used as a standard.
after
RESULTSAND DISCUSSION The carbohydrate compositions of prothrombin and its thrombin catalyzed proteolytic products are presented in Table 1. Intermediates 1 and 3 each contain all the monosaccharide constituents present in prothrombin, except galactosamine which is absent in the former. However, these intermediates differ in their total carbohydrate content. Intermediates 1 and 3 consist of 3.9 and 14 percent carbohydrate by weight, respectively, which accounts for 35 and 57 percent of the carbohydrate originally present in prothrombin. These latter values are in agreement with the percentages of 33 and 66 calculated from the percentage composition data reported by Owen et al. (2) for intermediates 1 and 3 respectively. The molar proportions of the individual monosaccharides are essentially the of glusame in both intermediates as well as prothrombin, with the exception cosamine and galactosamine, as seen from the calculations presented in Table 11. acid each occur in about a 1 to 1 ratio to galactose in Mannose, and sialic prothrombin and intermediates 1 and 3. In contrast, the amounts of glucosamine and galactosamine are enriched in intermediate 3. These comparisons suggest that the heteropolysaccharide units present in intermediates 1 and 3 have closely related structures but differ in the number of hexosamine residues. The distribution of the heteropolysaccharide units of prothrombin in the thrombin proteolytic products can be calculated on the basis of the composition data presented in Table land the composition of these units. Nelsestuen and Suttie (4) concluded that the carbohydrate of prothrombin is distributed about heteropolysaccharide units composed of sialic acid, galactose, mannose and Nacetylglucosamine in a molar proportion of 2-3:2:3:2-4, respectively. Therefore, it can be calculated, as given in Table 2 , that prothrombin contains 2 heteropolysaccharide units based on mannose content and 3 based on galactose or sialic acid. The lower number of units calculated on the basis of mannose reflects the lower amount of mannose relative to galactose or sialic acid reported here as compared to the value reported by Nelsestuen and Suttie (4).
Vo1.6,No.3
PROTHROMBIN
CARBOHYDRATE
217
UNITS
TABLE I Carbohydrate Composition Thrombin Catalyzed
Prothrombin
Monosaccharide
mg per 1oOmg proteina
Residues
Mannose
1.51
6.5
Galactose
1.35
of Prothrombin Proteolysis of
and Products Prothrombin
Intermediate
mg per 1oomg proteina
0.82
2.6
5.8
0.76
.05
0.2
Glucosaminea
2.45
Galactosamined Sialic
FucoseC
Total
Acid
Residues
Intermediate
perb mole
3
1+3
Residues
Residues
perb mole
perb mole
2.46
3.5
6.1
2.4
2.24
3.2
5.6
0.03
0.1
0.09
0.1
0.2
8.4
0.86
2.2
4.33
4.9
7.1
0.13
0.4
c.01
0.34
0.4
0.4
2.65
6.4
1.46
2.6
4.50
3.6
6.2
8.14
28.0
3.93
9.9
16.0
26.0
perb mole
mg per 1oOmg proteina
1
of
14.0
aValues expressed as residue weight per 1OOmg of glycoprotein. Protein content determined by quantitative amino acid analysis. b Based on molecular weight of 70,000, 51,000, and 23,000 for prothrombin, intermediate 1 and intermediate 3, respectively, as reported by Heldebrant --et al. (5) ‘Amount released on hydrolysis with d Expressed as N-acetyl derivative.
Dowex 50 resin.
Similar calculations (Table II> indicate that intermediate 1 contains 1 heteropolysaccharide unit based on mannose, galactose, or sialic acid, whereas intermediate 3 contains 1 unit based on mannose and 2 units based on galactose or sialic acid. it appears that the monosaccharide constituents of proHence, thrombin are distributed about 3 heteropolysaccharide units and on thrombin proteolysis, one of these units is associated with intermediate 1 and the other 2 are associated with intermediate 3. This distribution of units is consistent with the finding that about one-third of the total prothrombin carbohydrate by weight is accounted for by intermediate 1 and two-thirds by intermediate 3. The number of heteropolysaccharide units calculated above for prothrombin is inconsistent with the number reported by Nelsestuen and Suttie (4). They concluded that prothrombin contains 4 units, as opposed to 3, based on the findings of 7.5 galactose residues per mole of protein and 2 galactose residues They further calculated that 4 such units would reper mole of glycopeptide. present 15 percent of the prothrombin weight as carbohydrate based on the composi
PROTHROMBIN CARBOHYDRATE UNITS
218
Vo1.6,No.3
TABLE II Comparison of Monosaccharide Ratios and Number of Carbohydrate Units in Prothrombin and Products of Thrombin Catalyzed Proteolysis of Prothrombin
Residues/Galactose
Monolsaccharide
Pro thrombin
Residuea
Intermediate
1
Intermediate
Galactose
1.0
1.0
1.0
Nannose
1.1
1.1
1.1
Glucosamine
1.4
0.92
1.5
Galactosamine
0.1
0.0
0.1
1.1
1.1
1.2
Sialic
Basis
acid
+ fucose
of Calculation
Number of Carbohydrate
Units
per moleculeb
Mannose
2.2(2)
0.86(l)
1.2(l)
Galactose
2.9(3)
1.2
(1)
1.6(2)
Sialic
3.2(3)
1.3
(1)
1.8(2)
acid
3
aValues calculated from data given in Table I. b Values calculated from number of mannose , galactose and sialic acid residues per mole of protein (Table I) based on 3 mannose, 2 galactose, and 2 sialic acid residues per carbohydrate unit as reported by Nelsestuen and Suttie (4). Values based on sialic acid represent a maximum number since the unit contains 2 to 3 sialic acid residues. Values in parentheses rounded to nearest integer. tion of the glycopeptides. However, a comparison of their calculated composition data to other literature values indicates a large discrepancy as shown in Table III. The total carbohydrate composition of prothrombin reported herein and by several other investigators is quite close, 8.2 to 9.9 percent by weight, butprobably is much lower tlnn 15 percent. This difference is also prominent for each class of neutral sugar, hexosamine, and sialic acid. Thus, it appears more likely that prothrombin contains 3 heteropolysaccharide units rather than 4. An alternate explanation for this difference is that multiple forms of prothrombin occur which differ in their amounts of carbohydrate. Therefore, the results of this study indicate the overall distribution of the heteropolysaccharide units of prothrombin on thrombin activation to be as depicted schematically in Figure 1. The overall mechanism of the activation process was reported previously (5,15). Prothrombin (with 3 CHO units) is
Vo1.6,No.3
PROTHROMBIN CARBOHYDRATE UNITS
219
TABLE III Comparison
of
Literature Values for the Carbohydrate of Bovine Prothrombin
Data reported in Table I
Owen, -et al. (2)
Magnusson (1)
Composition
Tishkoff et al. -(3)
Nelsestuen 6 Suttie (4)
Carbohydrate Percentage
Weight
2.9
3.6a
3.0
3.8a
4. 5c
Glucosamine
2.5
2.5
1.8
3.3b
5.sc
Galactosamine
0.13
0
0.2
Sialic
acid
2.7
3.2
4.15
2.8
Total Carbohydrate
8.2
9.3
9.2
9.9
Neutral
sugar
aAmount of neutral color (7). b Amount determined
sugar
corrected
for
contribution
by Elson Morgan reaction
‘Values calculated from the amount of monosaccharide relative to galactose d Expressed as N-acetylglucosamine.
(11)
b
of
sialic
4.7c
15.0
acid
and represents
galactose in protein in glycopeptides.
0
to anthrone
total
hexosamine.
and the amount of
each
cleaved by thrombin to yield intermediate 3 ‘(with 2 CHO units) and intermediate 1 (with 1 CHO unit). Intermediate 1 is cleaved by factor Xa to yield intermediate 4 (no CHO unit) and intermediate 2 (with 1 CHO unit). Intermediate 2 is cleaved by Factor Xa to yield cu-thrombin (with 1 CHO unit). a-Thrombin is then converted to 8-thrombin by the excision of component x which bears the single CHO unit. In further support of this distribution of carbohydrate units, thrombin has been reported to contain a single unit (16). Moreover, the total carbohydrate content of thrombin of 5.3% as reported by Gwen et al. (2) and 5.0% as reported by Magnusson (17) agrees closely with the PredictedTezentage of 5 based on this reaction scheme and the composition of intermediate 1.
PROTHROMBIN CARBOHYDRATE UNITS
220
ALA
vo1.6,No.3
Prothrombin (70,000) , I I CHO CHO CHO I fhtombin +
Inl-3 (23,000) ALA-
Int -4 @l,OOO) +SER -x--
CHO CHO
+
x,
Int.-2(41,000) THR*-
Int-4 (13,000) SER-+
CHO
a- thrombin
(33,000) (6,000) THR T_~$~,I,,
(S,OOO-6,500) T Compon8nt CHO FIG.
X
1
Distribution of the heteropolysaccharide units of prothrombin on thrombin The sequence of events are as previously reported activation of prothrombin. and thrombins are represented schemThe prothrombin, intermediates, (5,15). CHO represents a heteropolysaccharide atically and molecular weights shown. is formed from cu-thrombin by unit. B-Thrombin, the last product identified, degradation of most of the A chain (6,000 daltons) and cleavage of the B chain as to delete the carbohydrate. (18)
so
ACKNOWLEDGEMENTS The authors would like to express their appreciation to Linda Wegener for her This investigation was supported by grants from the expert technical assistance. National Institutes of Health (Am-15112) and the Oklahoma Agricultural Experiment Station (Journal Article 2883) to Billy G. Hudson, and by grants from the National Institutes of Health (HL-15381-03 and HL-16150-01) to Kenneth G. Mann, and by general research support funds of the Mayo Clinic and Mayo Foundation.(Grant #5 SO1 RR 05530-10) REFERENCES 1.
Preparation MAGNUSSON,S. Ark Kemi 23, 285, 1965. --
and carbohydrate
analysis
of bovine
prothrombin.
vo1.6,~0.3
PROTHROMBIN
CARBOHYDRATE
UNITS
221
2.
OWEN, W.G., ESMON, C.T., AND JACKSON, C.M. The conversion of prothrombin to thrombin I. Characterization of the reaction products formed during the activation of bovine prothrombin. a-J Biol Chem 249,594,1974.
3.
TISHKOFF, G.H., WILLIAMS, L.C., AND BROWN, D.M. Preparation of highly purified prothrombin complex 1. Crystallization, biological activity and molecular properties. _-J Biol Chem 243,4151,1968.
4.
NELSESTUEN, G.L., AND SLJTTIE,J.W. The carbohydrate of prothrombin. Partial structural determination demonstrating the presence of o-galactose residues. J _--Biol Chem 247,6096,1972.
5.
HELDEBRANT, C.M., BUTKOWSKI, R.J., BAJAJ, S.P. AND MANN, K.G. The activation of prothrombin 11. Partial reactions, physical and chemical characterization of the intermediates. _-J Biol Chem 248,7149,1973.
6.
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7.
LLJNDBLAD,R.L. A rapid method for the purification of thrombin and the inhibition of the purified enzyme with phenylmethylsulfonyl fluoride. Biochemistry 10,2501,1971.
6.
MANN, K.G., HELDEBRANT, C.M. AND FASS, D.N. Multiple active forms of thrombin I. Partial resolution, differential activities, and sequential formation. J Biol Chem 246,5994,1971. _--
9.
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10.
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11.
LENHARDT, W.F., WINZLER, R.J. Determination of neutral sugars in glycoproteins by gas-liquid chromatography. J Chromatog 34,471,1968.
12.
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16.
MAGNUSSON, S. In: Structure-function relationships of proteolytic enzyme (Desnuelle, P., Neurath, H., and Ottesen, M.EDS) p.138, Munlcsgaard, Copenhagen, 1970.
17.
MAGNUSSON, S. in:
18.
LUNDBLAD, R.L., VOSEL, C.N., UHTES, L.C., NOYES, C., KINGDON, H.S. AND MANN, K.G. Manuscript in preparation.
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