THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc

VoI. 259, No. 9, Issue of May 10, pp. 5691-5697,1984 Printed in V. S.A.

Covalent Structures of ,t? and y Autolytic Derivatives of Human a-Thrombin”

(Received for publication, August 18, 1983)

Xavier Bichat, Universite Paris 7, Paris, France

Nonclotting 8- and y-thrombins have been prepared by autolysis of human a-thrombin at pH 8.6 in the presence of 0.4 M NaCl and purified on BioRex 70. Reduced and carbamidomethylated A and B chains fragments were separated by gel filtration and reverse phase high performance liquid chromatography. Structural characterization of these fragments dem- onstrated that a to B conversion results from two cleav- ages at Arg 62 and Arg 73 in the B chain, releasing an intact 11-residue peptide. B to y conversion corre- sponds to the additional loss of a fragment of the B chain stretching from IIe 124 to Lys 154. Autolysis is not accompanied by cleavages in the A chain. Loss of clotting activity is therefore related solely to the exci- sion of residues 63 to 73 in the B chain. With the exception of cleavage at Arg 73, these results differ from a proposed model for a to y conversion of bovine thrombin.

In the final stages of blood clotting, prothrombin activation produces a two-chain a-thrombin molecule which converts fibrinogen into fibrin. Due to its interactions with numerous other protein and cellular substrates, thrombin is a central regulator of the hemostatic process (1). Its amino acid se- quence has been determined in both bovine (2) and human (3) species and recently confirmed by the sequences of bovine ( 4 ) and human prothrombin cDNAs and the partial sequence of the human gene (5). Thrombin is a serine protease and, like trypsin, cleaves peptide bonds involving basic amino acid residues, most often arginine. However, unlike trypsin, it exhibits a marked specificity in cleaving only a very limited number of bonds in macromolecular substrates. Although thrombin has been crystallized (6, 7), no structural data are available yet which could provide molecular bases for its high degree of specificity. In this regard however, tentative three- dimensional models of t,hrombin, built on the basis of homo- logies with other serine proteases, have brought up some information (2, 8-10).

* This research was supported by grants from the Centre National de la Recherche Scientifique. (GBnie Biomi.dical81), from the Institut National de la SantB et de la Recherche MBdicale (CRL 81-5-OZZ), and from the Fondation pour la Recherche MBdicale. A preliminary report of this study has been presented at the IXth International Congress on Thrombosis and Haemostasis, Stockholm, July 1983 (19). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 Supported by a fellowship from the French MinistGre de la Recherche et de I’Industrie.

1) To whom reprint requests may be addressed.

Degradation of a-thrombin, either autolytically or by treat- ment with plasmin, factor Xa, or trypsin, leads to at least two definite forms, referred to as the 0- and y-derivatives, with a drastic reduction in clotting activity. Amidolytic activity on small synthetic substrates, however, is only slightly altered, suggesting that thrombin-fibrinogen interaction is specifically impaired. Therefore, these derivatives have been extensively used as models to study structure-function relationships in the thrombin molecule. Covalent structures of bovine p- and y-thrombins have been reported (11, 12), but only limited information is available concerning their human counterparts. NH2-terminal sequence studies on mixtures enriched in either the 0 or y forms are consistent with a three-chain structure for the P form and a four-chain structure for the y form, resulting from at least two cleavages, at Arg 73 and Lys 154, in B chain (8, 13).

The purpose of this work was to obtain definite information as to the exact regions of the polypeptide chain that are cleaved off in human p- and y-thrombins.

MATERIALS AND METHODS’

RESULTS

Autolytic Degradation of Human Thrombin-Purified hu- man a-thrombin (3,000 NIH units/mg) shows a single band M, = 35,500) by DodS04-polyacrylamidez gel electrophoresis under nonreducing conditions. The kinetic data of autolytic degradation were followed both by DodS04-polyacrylamide gel electrophoresis and by analytical chromatography on BioRex 70 (Fig. 1). In this latter procedure, a-thrombin (time zero) is eluted as a single peak (peak 1) at 0.75 M NaCI. Incubation of a-thrombin in high Na‘ alkaline buffer gives rise to autolytic degradation as evidenced by the results at 72 and 144 h of incubation. The starting material (peak 1) progressively decreases, giving rise to two more anionic species (peaks 2 and 3) eluted at 0.30 M NaCl. At 144 h, peak 3 is the major constituent. The sequence of degradation is clearly 1+ 2-3 and one may reasonably predict that peaks 2 and 3 correspond, respectively, to P- and y-thrombins as defined by

Portions of this paper (including “Materials and Methods,” Figs. A-D, Tables 1-8, and additional references) are presented in mini- print at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-2409, cite the authors, and include a check or money order for $6.40 per set of photocopies. Fullsize photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

The abbreviations used are: DodSO,, sodium dodecyl sulfate; RP- HPLC, reverse phase high performance liquid chromatography.

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5691

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5692 Structure of Human ,8- and y-Thrombins

Time = 144h 72 h 0

ilrl a/ -

6 20 i o t irne trnin,

1, 20 40 timelrninl

FIG. 1. Autolytic degradation of human a-thrombin. a, poly- acrylamide gel electrophoresis of the autolytic product of human a- thrombin. Autolysis was performed as described under “Methods.” Samples were taken for analysis at 0, 72, and 144 h. Electrophoresis was carried out according to Laemmli. The concentration of poly- acrylamide was 15% and the acrylamide/bisacrylamide ratio 30:l. b, analytical ion exchange chromatography of the thrombin forms, obtained after 0, 72, and 144 h of autolysis. 50-pg samples were analyzed on BioRex 70 as described under “Methods.”

Fenton et al. (13). Some material eluted in the void volume of the BioRex column (peak 4) also increases with time.

Isolation and Identification of the Various Thrombin Deriv- atives-BioRex 70 chromatography was used on a preparative scale to obtain the various autolytic thrombin derivatives. Material eluted at the positions of peaks 2 and 3 (Fig. 1) was reduced and carbamidomethylated. Definite identification of the nature of these two thrombin derivatives was first pro- vided by analysis of their polypeptide chain composition by RP-HPLC on Lichrosorb RP-8 (Fig. A, Miniprint). Material eluted in peak 2 has a three-chain structure, and material in

peak 3 a four-chain structure. These two compositions corre- spond, respectively, to those described for @- and y-thrombin. Although positive identification of the exact nature of each peak on the HPLC chromatograms was only made aposteriori, the peptide chain nomenclature proposed by Fenton et al. (13) (A, B1, B2, B3, and B4 chains) will be used from now on for clarity in the description of the steps.

Polypeptide Chain Purification-The chains of reduced and carbamidomethylated a-, @-, and y-thrombins were first frac- tionated on a preparative scale by gel filtration on Sephadex G-75 (Fig. B).

Clearly, a-thrombin B chain ( M , = 31,000) is split into two separate components B1 ( M , = 9,000) and B2 (MI = 21,000) in P-thrombin. In y-thrombin, B2 is further cleaved into B3 (Mr = 6,000) and B4 ( M , = 12,000). Thrombin A chain does not absorb significantly at 280 nm and the last fractions of the chromatograms were collected (Fig. B) to recover the respective A chains. The presence of peptide material in these fractions was confirmed by RP-HPLC on Lichrosorb RP-8 (Fig. C) which provided pure material for structural studies.

A peptide present in the dialysis bath of a 74-h autolysate was concentrated and purified by RP-HPLC on pBondapack C-18 and later referred to as the autolytic @ peptide (Fig. D). Dialyzable material from a 144-h autolysate is a highly het- erogeneous mixture of peptides unsuitable for structural stud- ies.

Structural Characterization of the Different Polypeptide Fragments-Purified polypeptide fragments were character- ized by their NHz- and COOH-terminal sequences (see de- tailed data in the Miniprint). Results are summarized in Fig. 2 for comparison to the amino acid sequence of human a- thrombin (3).

“ A chains purified from a-, p-, and y-thrombins are iden- tical. This can be stated because of: 1) identical behavior on RP-HPLC; 2) similar amino acid compositions (Table 1, Miniprint); and 3) identical NHz- and COOH-terminal se- quences (Fig. 2; Tables 2 and 3).

Based on the same criteria, B1 chains isolated from p- and y-thrombins exhibit strict identity. Their NHz-terminal se- quence is that of a-thrombin B chain (Table 4) and their COOH-terminal residue corresponds to Arg 62 (Table 5). The B2 chain isolated from @-thrombin has the same COOH- terminal sequence as a-thrombin B chain and corresponds to residues 74 to 259 (Tables 4 and 6). Cleavages at Arg 62 and Arg 73 during the a to @ conversion are confirmed by the recovery of the intact 11-residue @ autolytic peptide extending from residue 63 to Arg 73 (Tables 6-8).

In y-thrombin, the B3 and B4 chains derive, respectively, from the NH2- and the COOH-terminal parts of @-thrombin B2 chain (Tables 4 and 5). B3 chain terminates at Arg 123 and the first residue of the B4 chain corresponds to Gly 155. The intermediate portion of the peptide chain extending from residue 124 to Lys 154 could not be recovered as an intact peptide and several other cleavages in this region probably do occur during the P to y conversion.

DISCUSSION

Data presented in this paper provide full structural char- acterization of the autolytic @ and y derivatives of human a- thrombin. Only thrombin B chain is affected by autolysis. a to @ conversion corresponds to two cleavages at Arg 62 and Arg 73, giving rise to the B1 and B2 fragments and to a dialyzable 11-residue peptide. @ to y conversion results from cleavages a t Arg 123 and Lys 154 (and probably other clevages in the 124-154 region), producing the B3 and B4 chains. These conclusions are schematized in Fig. 3 and compared to

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Structure of Human p- and y-Thrombins

A chain- _----- - -------- 01 Y __-____-- - - - """---OR B

VpOm -___-___-___- -__ on a 1101.1--C-- - - - - - - - -S~IDOR 1 d 0 38

Bchain- 1 43 64 72

9: 119 124 I54 205 259 ~ ~ o ~ w - - - . ~ - + a r ~ v ~ ~ ~ ~ ~ ~ ~ ~ K ~ ~ . - - - ~ - - - - - i r m r l r ~ ~ - - - - - - - - - - w ~ ~ ~ ~ ~ ~ - - - - - E - - ~ - - - ~ ~ w ~ lvl-0 - - . . . - - ._-___--_______._________________---------~-.____________..__________________________._______-w E a

61 IVtO" "" ""

81 1Vl.S"""- """ I V R

U L V R " _ ""

82 wI#Kls - . - - - - - . - - - - - - - _ - - - - - - - -. - _ _ - - - - - - ~. - - - - - - - _ _ - ~ _ _ - - 101 B W*OlVk Il0r-S- -

" _ ""

83 M)lK _--._-__---____ L C M B4 O Q C S - - - - - - - - - - - - - _ ~ _ _ _ _ FOE y

FIG. 2. NHa- and COOH-terminal sequence data obtained on the various polypeptide fragments. The central sequence is that reported by Butkowski et al. (3). Numbering is that of the A and B chains of human thrombin, respectively. All residues were identified twice. NHZ-terminal sequences were determined by: 1) manual Edman degradation, followed by identification of the PTH-derivatives by HPLC on pBondapak C-18, and 2) the manual DABITC-PITC procedure, DABTH-derivatives being identified by two-dimensional thin layer chromatog- raphy. COOH-terminal sequences were determined by digestion of carboxypeptidases B and Y. Released amino acids were identified by HPLC and thin layer chromatography after conversion to PTH- and DABTH-derivatives, respectively.

-.-

5693

I 17 1 3 6 - :,IT " + &-I- -L W CYD i 2S9 MCYO S

I 17 36

M c m

-5 :.- I73 U7 4 2 7 ; A(", &, S 259 "

61 74 123 155 - - Y bovine human

FIG. 3. Comparison between human and bovine thrombins. Right, schematic polypeptide structures of human a-, j3-, and y-thrombins. Lit, schematic polypeptide structures of bovine a-, j3-, and y-thrombins (Refs. 11 and 12). Numbering of the residues is that of human thrombin A and B chains, respectively. In the B chain, H , D, and S correspond to the catalytic site residues, His 43, Asp 97, and Ser 205. CHO indicates the carbohydrate moiety linked to Asn 53. Thrombin contains four disulfur bridges shown: one between the A and B chains and three in the B chain.

the proposed model for autolytic degradation of bovine throm- bin (11, 12).

Asp at positions 6 and 122 in the B chain has been previ- ously reported as Asn by Butkowski et al. (3); but our present results are in agreement with those of Walz et al. (14) and Thompson et al. (15) and consistent with the DNA sequence (5).

Autolytic cleavage sites described here include the two minimal cleavages already reported at Arg 62 and Lys 154, as deduced from NH2-terminal sequence analysis of mixtures of human p- and y-thrombins obtained by mild tryptic digestion (8, 13).

Differences between the human and bovine systems are obvious from Fig. 3. Only in bovine thrombin is the A chain affected by autolysis. Human A chain is 13 residues shorter than bovine A chain on the NH2-terminal side, due to a Pro- Arg (-1)-Thr (1) sequence in human prothrombin (versus Glu- Lys-Thr in bovine) that is readily cleaved by thrombin during human prothrombin activation. Autolysis of bovine thrombin results in cleavages at Lys -1 and Lys 17 in the A chain. Despite complete identity of the amino acid sequence sur- rounding Lys 17 in the two species, we did not observe autolytic cleavage at this site in the human system. The "A" chain of human p- and y-thrombins is therefore 19 residues longer on the COOH-terminal side than its bovine counter-

part. Autolytic cleavages in the B chain are also different in the two species. In human P-thrombin, the region extending from residue 63 to 73 is cleaved off, as compared to 66 to 73 in bovine B chain. Recovery of the peptide 63-73 in the human system eliminates the possibility of an initial cleavage at Lys 65, even though the sequence including Arg 62 (human cleavage site) and Lys 65 (bovine cleavage site) is strictly identical in both species. ,8 to y conversion seems totally different in the two species. In bovine B2 chain cleavages are thought to occur in the portion of the chain separating the two cysteines of the 173-187 disulfide bridge, leading to the formation of B3 and B4 chains covalently linked together by this disulfide bridge. In human thrombin, proteolytic degra- dation of the 124-154 region confers to y-thrombin a structure in which the B1, B3, and B4 fragments are bound only by noncovalent interactions. Therefore, despite a 84% degree of identity between human and bovine thrombins, surprisingly, only one autolytic cleavage site (at Arg 73) is identical in both species. One should note however that the region which is cleaved off in human y-thrombin contains the highest inci- dence of substitution between the two species (38.7% versus 14.7% for the entire B chain).

From comparisons with other serine proteases, speculations have been made concerning the direct or indirect involvement of at least two regions of thrombin in fibrinogen recognition

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5694 Structure of Human p- and y-Thrombins

(2, 8-10). The first of them is the COOH-terminal portion of the A chain. Its deletion in bovine p- and y-thrombins is suggestive of the importance of this region for the expression of full thrombin activity. The fact that we found it to be retained in human p- and y-thrombins, however, rules out this possibility. The second region is the portion of the peptide chain surrounding the site of attachment of the carbohydrate moiety (Asn 53 in the B chain). Note that in the human system loss of clotting activity is related solely to the excision of the p autolytic peptide (residues 63-73 in B chain), which is located in the vicinity of this region.

If one assumes for thrombin, as for all serine proteases, a two-P barrel structure, then the p autolytic peptide would be located in the NH2-terminal barrel and its deletion in p- thrombin could result in a severe alteration of the structure of this molecular domain. On the other hand, peptide lost during the p to y conversion would affect the interdomain region, where in chymotrypsin cleavages occur between the B and C chains without major changes in activity.

Finally, having in mind the proposed relation between exons and functional domains in proteins (17, 18), it is of interest that two introns of the prothrombin gene, the 0.4- and 0.6-kilobase pairs introns (5), are located, respectively, in the ,6 autolytic peptide and in the region of B chain cleaved off in y-thrombin.

Acknowledgment-We would like to thank Dr. Steinbuch from the Centre National de Transfusion Sanguine (Orsay, France) for kindly supplying the PPSB fraction.

REFERENCES

1. Fenton, J. W., I1 (1981) Ann. N. Y. Acad. Sci. 370,468-495 2. Magnusson, S., Peterson, T. E., Sottrup-Jensen, L., and Claeys,

H. (1974) in Proteases and Biological Control (Reich, E., Rifkin, D. B., and Shaw, E., eds) pp. 123-149, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

3. Butkowski, R. J., Elion, J., Downing, M. R., and Mann, K. G.

(1977) J. Biol. Chem. 252, 4942-4957 4. McGillivray, R. T. A., Friezner Degen, S. J., Chandra, T., Woo,

S. L. C., and Davie, E. W. (1980) Proc. Natl. Acad. Sci. U. S. A.

5. Friezner Degen, S. J., McGillivray, R. T. A., and Davie, E. W. (1983) Biochemistry 22,2087-2097

6. Tsernoglou, D., Walz, D. A., McCoy, L. E., and Seegers, W. H. (1974) J. Biol. Chem. 249,999

7. McKay, D. B., Kay, L. M., and Stroud, R. M. (1977) in Chemistry and Biology of Thrombin (Lundblad, R. L., Fenton, J. W., 11, and Mann, K. G., eds) pp. 113-121, Ann Arbor Sci. Publ. Inc., Ann Arbor, MI

8. Elion, J., Downing, M. R., Butkowski, R. J., and Mann, K. G. (1977) in Chemistry and Biology of Thrombin (Lundblad, R. L., Fenton, J. W., 11, and Mann, K. G., eds) pp. 97-111, Ann Arbor Sci. Publ. Inc., Ann Arbor, MI

9. Hewett-Emmett, D., Czelusniak, J., and Goodman, M. (1981) Ann. N. Y. Acad. Sci. 370,511-527

10. Furie, B., Bing, D. H., Feldmann, R. J., Robison, D. J., Burnier, J. P., and Furie, B. C. (1982) J. Biol. Chem. 257,3875-3882

11. Kingdon, H. S., Noyes, C. M., and Lundblad, R. L. (1977) in Chemistry and Biology of Thrombin (Lundblad, R. L., Fenton, J. W., 11, and Mann, K. G., eds) pp. 91-96, Ann Arbor Sci. Publ. Inc., Ann Arbor, MI

12. Lundblad, R. L., Noyes, C. M., Mann, K. G., and Kingdon, H. S. (1979) J. Biol. Chem. 254,8524-8528

13. Fenton, J. W., 11, Landis, B.H., Walz, D. A., and Finlayson, J. S. (1977) in Chemistry and Biology of Thrombin (Lundblad, R. L., Fenton. J. W.. 11. and Mann. K. G., eds) pp. 43-70, Ann Arbor

77,5153-5157

Sci. Publ. Inc:, Ann Arbor, M I "

14. Walz. D. A,. and Seeaers, W. H. (1974) Biochem. Biophvs. Res. Commun. 60,717-722 '

15. Thompson, A. R., Enfield, D. L., Ericsson, L. M., Legaz, M. E., and Fenton, J. W., I1 (1977) Arch. Biochem. Biophys. 178 ,

16. Downing, M. R., Butkowski, R. J., Clark, M. M., and Mann, K. G. (1975) J. Biol. Chem. 250 , 8897-8906

17. Jung, A., Sippel, A. E., Grez, M., and Schutz, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 7 7 , 5759-5763

18. Go, M. (1981) Nature (Lond.) 291,90-92 19. Boissel, J. P., Rabiet, M. J., Labie, D., and Elion, J. (1983)

Thrombos. Huemostasis 50,265 Additional references are found on p. 5695.

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Structure of Human p- and y-Thrombins 5695 Coon-terminal seauence determlnatlon. COOH-termlnal ~equences have

been Obtalned by dlgestlon ulth carboxypeptldases 8 and/or Y. Volatlle

0 20 4 0 60 min

Fv&&. Analysis o f t h r w i n derivatives polypptide chain ccqositlon by

was reduced and carbmidmethylated. Sampler (?. 50 pg) !we applied to a llchmsorb RP 8 column developed I S described in this section.

Hatenal in Peak 2 ( a ) and 3 (b) f m the Biorex 70 separation ( F t g . 1)

Z 19

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5696 Structure of Human p- and y-Thrombins

I I 600 700 a& ml

r

NQ2 3 , 3 1.5

6.2 2.8

1.1 4.3 0.9 0.9 4.8 0 .1 2.0 3 .2 2.8

0 E.. PI

4 ,autolytic

peptide N

scrip ,_... -20

/'"".'

..-

032.. /'

0

/

6 10 30 ' min

1 1 Thr H.TLC 2 2 Phe H.TLC

4 4 3 3 g; 7 5 5 m y n N$

@ I ' h m a ! P : 1 1 mr 2 2 P k

H.nc H.TLC

mr HJLC nf phc Gly H;TLC

H . l L C 5

1 ~ Tw sequence deteninat ions were p e r f o m d ; th+ f i r s t by mwtl f h n &gradatton using PITC and the second M by DIBITC-PITC pmcedure.

2 - o f A Chain 3 - Rn abbreviation% am : H.HPLC Of PTH-MinO acid i TCL. thin l W W

4 - The y i e l d i n n - m l was detenined fm HPLC of PM-mino acid and 11s chmutography o f OIIBTH-mim acid.

m u n h d to the n a r e r t i n t a t r . 5 - NQ : not quantitated.

'I 60 mne B 60 Arg 36

B t Y 15 A r g 36 H.TLC W A r g 36 H.TLC 0.9

TLC 3.1 5 . 2

G b 35 H.TLC

6" " O W

3 - The abbreviations am : H,HPLC of PTH-mino acid ; TLC. th in layer 2 - of A chain

4 - The y i e l d i n n ml was detenined flm nPLC Of PM-amino acid. chrmatography o f DMm-amino acid.

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Structure of Human p- and y-Thrombins TABLE 6

TABLE 4 Ammno acid campOrition O f the aUtOlYtlC 0 Peptide'

5697

0.6 0.7 1.1 1 . 3 0.9 0.8 0.7

2.7 1.2

cycle nMber

H,TLC H.TLC

H.TLC H.TLC H .TLC

H . n c

15 13 8

1 I l e H.TLC 2

7 VI1 H.TLC

3 9

Glu n,nc 5 2 3 4 4 n 2

H . n c 6 n.nc 11 n.nc 8 H . n t 5 H H 4 5

74 75

As"

76 61 u 11e

77 78

US 79 Ser

1lC

H, TLC H. TLC H , TLC

H

4 4 2 H.nt 12

H.nt 12 H.nt 8 u.nt H . n c &

% r r a p n t 71 AS" 75 76

1 l e 61"

77 Lys

B4 r n p e n t 155 156 157 158

2 PIV Ser

1.3.4 see table 2 H.TLt 6

H.TLC 11 H.nc 13

n.nc 5

H.TLC 9 n.nc 12 H.nt H.TLC 4 5

1.3.4.5 : see table 2 2 : o f 8 chain

B chain 1.3.4 lee table 3

15 30

60

30 60 15

30

60

15

30

60

60 60

30

50

60 60 15

30

60

15

30

60

61 Y 259 G l u 259 G ~ Y 258 G l u 259 G ~ Y 258 Phc 257

H.nt n.nc H . n c w.nc w.nc H.nC

H.TLC n c

H.TLC H .nc H.TLC

n.nc H .TLC

H.TLC n.nc n.nc H

H,TLC H H.nC H.TLt H.TLC H , n C nc

H,TLC TLC

H.TLC H.TLC

TLC TLC

TLC

TLC H.TLC H,TLC H,TLC H.TLC H.TLC H,TLC H H,TLC H,TLC H.TLC H.TLC

TLC

H,TLC TLC

H,TLC H H,TLC H.TLC H,TLC

1.6 2.2 0.5 2.4 1.3 0.7

2.7 1.9

4 . 2 1.4

3.6 3.9 4.8 4.4 6.5 2.1

1.0 0.5 1.6 1.3 1.7 1.4

2.0 1.6 1.7

0.7 0.7 0.5 1.3

0.7 1.1

0.4 1.6

0.9 1.3

0.8

0.9 0.4 0.3 1.4

0.7 1.1

B~ f n g l n t

A r g ..

3 kl a

62 62 61

Le" 59-60 62

Le" 61

59-60 62 61

LeU ASD

59-60 58

8, f""t 'I 61"

G ~ Y 259

G l u 258 259 258 259

257

:b 6 2 258

51 f r a p n t

A r g none

62 62 61

62 61

3 Le" 59-60 A r g Val Le" 59-60

8) fragnent

none A r g A r g

123

ASP 123 122

P I V A r g

121

ASP 123

PrO 122 121

A r g Le" 120

ASP 123

P I 0 122

Le" 121 17n

61 u 259

G l u 259 258 257 259 258 257

G ~ Y 258

::: GI" 61 y Phe

1.3.4 : see table 3 2 : o f 8 chain

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