Functional consequences of tryptophan modification in human fibrinogen

70

Biochimica et Biophysica Acta, 536 (1978) 70--77 © Elsevier/North-Holland Biomedical Press

BBA 38006

F U N C T I O N A L CONSEQUENCES OF T R Y P T O P H A N MODIFICATION IN HUMAN F I B R I N O G E N YUICHI ISHIDA, HIDEFUMI TAKIUCHI, AYAKO MATSUSHIMA and YUJI INADA

Laboratory of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguroku, Tokyo 152 (Japan) (Received February 14th, 1978)

Summary When human fibrinogen was modified with H:O2, inter- and intra-molecular cross-links of fibrinogen were formed, accompanied with oxidation of tryptophan, methionine and tyrosine residues. These cross-links may be closely associated with oxidation of tryptophan residues. The polymerization activity of fibrinogen with thrombin was decreased markedly by this modification. Modification of tryptophan residues in fibrinogen was also performed with 2-hydroxy-5-nitrobenzyl bromide. Modification of two out of a total 78 tryptophan residues in the molecule with the reagent led to the intensification (1.7 times) of the polymerization activity with thrombin and further modification of the next two residues led to complete loss of the polymerization activity. The first two tryptophan residues to be modified are in Fragment D, and the next two occur in Fragment E.

Introduction In order to clarify the mechanism of polymerization of fibrinogen with thrombin, the primary structure of the fibrinogen molecule has been extensively studied and about 80% of the total amino acid sequence in the molecule was determined by Blomb~ck et al. [1] and by Doolittle et al. [2]. Blomb~/ck et al. [3] also found two complementary binding domains (N-DSKa and Fragment D), which may be importance for the initial alignment of the activated fibrinogen molecule to form fibrin. Recently, Inada et al. [4] reported that when human fibrinogen was illuminated for a short time in the presence of methylene blue, the polymerization activity of fibrinogen by the action of thrombin was completely lost, accompanied by the photooxidation of histidine and tryptophan residues. They also determined the states of tyrosine residues Abbreviation: SDS, sodium dodecyl sulfate.

71 in the fibrinogen molecule by a spectrophotometric m e t h o d [5]. Chemical modification studies on fibrinogen were carried out for tyrosine and/or lysine residues [6--9], and some insight has been obtained into their functional importance for polymerization. The present report deals with modification of fibrinogen using H202 in dioxane or with 2-hydroxy-5-nitrobenzyl bromide in relation to the functional tryptophan residues in the fibrinogen molecule. Experimental Human fibrinogen was supplied for Green Cross Company and its clottability with thrombin is 91.2%. Modification of fibrinogen with H202 in dioxane was as follows; to 4.5 ml 2.1 pM fibrinogen in 0.55 M bicarbonate buffer (pH 8.3), in the presence of 2.5 pM Mn 2÷, was added 0.5 ml H 2 0 : ( 0 - - 1 5 0 mM) in dioxane. The reaction mixture was incubated at 4°C for 120 min. The method for modification of proteins with H202 in dioxane was explored by Hachimori et al. [10] and they determined the state of tryptophan residues in proteins by measuring spectral changes. However, the present authors and Hachimori found that the oxidation of tryptophan residues proceeds only in the presence of Mn 2÷. The absorption spectra of native and modified fibrinogens were measured by a Shimazu recording spectrophotometer UV-200. The degree of oxidation of tryptophan residues in the molecule was determined spectrophotometrically, using the difference in the molar extinction coefficient (Ae) between nonoxidized and completely oxidized tryptophan, Ae = 3490 M -~. cm -1 at 282 nm [10]. Amino acid analysis was determined by a Hitachi amino acid analyzer KLA-3B, after hydrolysis of protein with 6 N HC1 at l l 0 ° C for 22 h. Gel electrophoresis was carried out with 3.5% acrylamide gel in the presence of 0.1% SDS for native and modified fibrinogens. The fibrinogens were reduced with 1% dithiothreitol with 1% SDS to split disulfide bonds in the fibrinogen molecule. The mixture of a, ~- and 3,-chains of the molecule was subjected to gel electrophoresis with 7% acrylamide gel containing 0.1% SDS [11]. The area of each band obtained by electrophoresis was measured by a Shimazu spectrophotometer MP8-50 attached with chromatogram scanner. Modification of tryptophan residues in fibrinogen was also carried out with 2-hydroxy-5-nitrobenzyl bromide by the method of Koshland et al. [12]. 10 ml 3 pM fibrinogen in 50 mM citrate buffer (pH 6.0) was added to 0.5 ml 2-hydroxy-5-nitrobenzyl bromide (0--105 mM) in acetone, and the mixture was incubated for 30 min at room temperature. The modified fibrinogens were dialyzed against 50 mM acetate buffer (pH 7.0) with 0.2 M NaC1. The degree of modification of tryptophan residues in the fibrinogen molecule with 2-hydroxy-5-nitrobenzyl bromide was determined by measuring the absorbance at 410 nm at pH 10.85, using the molar extinction coefficient of modified tryptophan, 1.8 • 104 M -~ • cm -~ [13]. 50 mg modified fibrinogen was digested with 3.8 mg plasmin overnight at room temperature [14]. Fragments thus obtained were separated by chromatography with DEAE-cellulose to obtain Fragments E and D and others [15]. The polymerization activity of fibrinogen with thrombin was determined by measuring the absorbance change due to the

72 increase of turbidity [4]. Protein concentration was estimated by the method of Lowry et al. [16]. Results and Discussion

Modification o f fibrinogen with H202 in dioxane in the presence of Mn 2+ Fig. 1 shows the spectral change of fibrinogen by the modification of tryptophan residue with H202 in dioxane. The absorption spectrum of native fibrinogen has an absorption band with a peak position at 280 nm corresponding to tryptophan and tyrosine residues (curve A). By increasing the H20~ concentration, the peak position of the absorption band was shifted to a longer wavelength, with isosbestic points at 296 nm and 274 nm. The new band with a peak position at 320 nm appears as shown by curves B (1.0 mM H202), C (2.0 mM H=O2) and D (10 mM H202) and is responsible to the formation of N'-formylkynurenine which has an absorption band with a peak position at 320 nm [17]. Curve E shows the absorption spectrum of fibrinogen oxidized with 10 mM H202 and hydrolyzed with 1 N HC1 for 20 min and has a band with a peak at 360 nm, which agrees with the peak position of kynurenine [17]. Production of N'-formylkynurenine from tryptophan by oxidation with H202 was also confirmed by paper chromatography. Fig. 2. shows the decrease of polymerization activity of fibrinogen and degrees of oxidation of amino acid residues in the fibrinogen molecule during the process of the oxidation with various concentrations of H202. Oxidation

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of tryptophan residues proceeds rapidly and, at 2.0 mM H202, about 40% of the 78 residues in the molecule [18] are oxidized. Amino acid analyses of oxidized fibrinogen revealed that only tyrosine and methionine residues were oxidized and the other amino acid residues including histidine were not. 30% tyrosine residues and 15% methionine residues were oxidized with 2.0 mM H202. The polymerization activity of fibrinogen was reduced markedly with increasing H202 concentration and was lost almost completely at 1.0 mM. This indicates that tryptophan residues in the fibrinogen molecule may play in important role in the polymerization activity with thrombin. Gel disc electrophoresis of fibrinogen modified with various concentrations of H202 was carried out using 3.5% acrylamide gel and their patterns are shown in by panel (a) in Fig. 3. The band of native fibrinogen (a-I) is reduced in area by increasing the H202 concentration; a-II for 1.0 mM H202 and a-III for 2.0 mM H202. Conversely, the new band appears in the region corresponding to a molecular weight higher than that of fibrinogen (340 000). Panel (b) in Fig. 3 represents the gel electrophoretic patterns carried out with 7% acrylamide gel after splitting disulfide bonds of native (b-I) and oxidized fibrinogen (b-II and b-III) with H202. Each band of a,/3- and 7-chains in fibrinogen disappears by increasing the H202 concentration; b-II for 1.0 mM H202 and b-III for 2.0 mM H202. Conversely the new bands with molecular weight higher than that of the a-chain are formed during the oxidation. Fig. 4 represents plots of the area of each chain (a-, ~- and 7-chains) obtained from gel electrophoretic patterns against H202 concentration. As seen in the figure, the amounts of a-(curve A) and/3-chain (curve B) decrease by increasing the H~O2 concentration, while that of the 7-chain (curve C) keeps a constant

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level at lower H2O 2 concentrations and then decreases rapidly at concentrations ranging from 1--2 mM. Curve D shows the change in the amount of the fibrinogen molecule. These results indicate intra-molecular cross-links between each chain in the fibrinogen molecule and also inter-molecular cross-links between the molecules. At lower H202 concentrations, the 7-chain does not participate in cross links although a- and ~-chains do. During blood coagulation the active form of Factor XIII catalizes the reaction of the inter-molecular cross-links between 7-chains of the fibrin monomer [19]. In the present experiment, the authors found that the inter- and intramolecular cross-links take place when fibrinogen was modified with H~O~ in dioxane in the presence of Mn ~+. During the oxidation of fibrinogen, tryptophan residues change to N'-formylkynurenine. Pirie [17] reported that illumination on tryptophan residues of proteins and free tryptophan by sunlight causes splitting of the indole ring and formation of N'-formylkunurenine. Dilley [20] also demonstrated that tryptophan residues in lysozyme were photooxidized by sunlight and accompanied by the formation of a covalently cross-linked polymer.

TABLE I CROSS LINKS OF FRIBRINOGEN, OXIDATIONOF AMINO ACID RESIDUES AND THE DECREASE OF THE POLYMERIZATIONACTIVITY WITH THROMBIN DURING THE OXIDATION OF FIBRINOGEN WITH HYDROGEN PEROXIDE IN DIOXANE IN THE PRESENCE OF Mn2+ H202 conen, (mM)

0 0.4 1.0 2.0 4.0

Polymerization activity (relative) %

Amino acid composition (mol/molecule)

100 11 3 2 --

78 66 52 46 38

TrP

Tyr

101 -84 74 --

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64 -57 55 --

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Reduced

Monomer

Polymer

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102 81 Sl 54 43

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34 35 27 9 3

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38 40 80 112 iii

75 Table I summarizes the degree of cross-linking of fibrinogen during the process of oxidation with H202, accompanied by oxidation of amino acid residues and reduction of polymerization activity with thrombin. One of the authors demonstrated earlier that histidine residues in one of the binding domains, N-DSK, play an important role in polymerization of fibrin m o n o m e r [4]. Therefore, a decrease of the polymerization activity obtained in the present study may be due to the oxidation of a few of the histidine residues in fibrinogen which could n o t be detected by amino acid analysis.

Modification of fibrinogen with 2-hydroxy-5-nitrobenzyl bromide The modification reagent, 2-hydroxy-5-nitrobenzyl bromide, is specific for tryptophan residues in proteins in acidic and neutral solution where a free sulfhydryl group is absent [12]. Human fibrinogen was modified with various concentrations of 2-hydroxy-5-nitrobenzyl bromide, and the polymerization activity of modified fibrinogen by the action of thrombin and the degree of modification of tryptophan residues in the molecule were measured. The results are shown in Fig. 5. The reaction of tryptophan residues with the reagent proceeds into three steps (curve A); the first two residues in the molecule react easily at reagent concentrations ranging between 0 and 0.5 mM and the next two residues become reactive at concentrations between 1 and 2 mM. At more than 2 mM 2-hydroxy-5-nitrobenzyl bromide, the reactivity of tryptophan residues in fibrinogen to the reagent increases markedly. The polymerization activity of fibrinogens with thrombin was changed by the modification of fibrinogen with the reagent. The modification of the first two tryptophan residues in the fibrinogen molecule causes the intensification of the polymerization activity (1.7 times} compared with the activity of the native fibrinogen with thrombin. When the next two tryptophan residues are modified, the polymerization activity decreased sharply with increasing the reagent concentration, as seen in curve B. The marked decrease of the polymerization activity

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76 T A B L E II P O S I T I O N O F T R Y P T O P H A N R E S I D U E S IN T H E F I B K I N O G E N M O L E C U L E M O D I F I E D W I T H 2H Y D R O X Y - 5 - N I T R O B E N Z Y L BROMID]~ ON F R A G M E N T S D I G E S T E D W I T H P L A S M I N Concn. of HNB * (mM)

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2.30

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with thrombin may be due to the modification of the functional tryptophan residues in one of two domains of N-DSK or Fragment D. In order to identify the position of tryptophan residues modified with 2-hydroxy-5-nitrobenzyl bromide on fragments digested with plasmin, the modified fibrinogens, in which approx. 2, 4 and 8 tryptophan residues in the molecule were modified with 0.23, 1.15 and 2.30 mM 2-hydroxy-5-nitrobenzyl bromide, were digested with plasmin. Their fragments were separated by DEAE-cellulose chromatography and the degree of modification in each fragment, Fragments D and E, were determined (Table II). As is clear from Table II, the first two tryptophan residues are located in Fragment D and the next two may be present in Fragment E. The fibrinogen molecule consists of one Fragment E and two Fragments D and other peptides [19]. From the results obtained in the present study, it may be concluded that one of tryptophan residues in Fragment D, which is accessible to the reagent, is closely associated with the intensification of the polymerization activity of fibrinogen by the action of thrombin, and the two tryptophan residues in Fragment E play an important role in the initial alignment of activated fibrinogen molecule to form fibrin. The position of tryptophan residues in Fragment E is either T r p ( 3 3 ) o r Trp(41) in the a-chain in the fibrinogen molecule, judging from the primary structure of Fragment E [19]. Modification of the tryptophan residues in fibrinogen with 2-hydroxy-5nitrobenzyl bromide did not cause cross-linking of fibrinogen. References 1 Blomb~/ck, B., Hessel, B. and H o g g , D. ( 1 9 7 6 ) T h r o m b o s i s Res. 8 , 6 3 9 - - - 6 5 8 2 D o o l i t t l e , R . F . , W a t t , K . W . K . , C o t t r e n , B.A. a n d T a k a g i , T. ( 1 9 7 8 ) I n t e r n a t i o n a l S y r u p . o n P r o t e i n , 1 9 7 8 , A c a d e m i c Press, in the press 3 Blomb~'ck, B., H o g g , D.H., G ~ r d l u n d , B., Hessel, B. a n d K u d r y k , B. ( 1 9 7 6 ) T h r o m b o s i s Res., Suppl. If, 8 , 3 2 9 - - 3 4 6 4 I n a d a , Y., Hessel. B. a n d Blomb~/ck, B. ( 1 9 7 8 ) B i o c h i m . B i o p h y s . A c t a 532, 1 6 1 - - 1 7 0 5 I n a d a , Y. a n d Blomb~/ck, B. ( 1 9 7 S ) B i o c h i m . B i o p h y s . A c t a 5 3 3 , 7 4 - - 7 9 6 L a k i , D. a n d Mihalyi, E. ( 1 9 4 9 ) N a t u r e 1 6 3 , 66 7 C a s p a r y , E . A . ( 1 9 5 6 ) B i o c h e m . J. 6 2 , 5 0 7 - - 5 1 2 8 Phillips, H.M. a n d Y o r k , J . L . ( 1 9 7 3 ) B i o c h e m i s t r y 12, 3 6 3 7 - - 3 6 4 2 9 Mihalyi, E. and A l b e r t , A. ( 1 9 7 1 ) B i o c h e m i s t r y 10, 2 3 7 - - 2 4 2

77

10 Hachimori, Y., Horinishi, H., Kurihara, K. and Shibata, K. (1964) Biochim. Biophys. Acta 93, 346-36O 11 McDonagh, J., Messle, H., McDonagh, R.P., Murano, G. and Blomb//ck, B. (1972) Biochim. Biophys. Acta 2 5 7 , 1 3 5 - - 1 4 2 12 Koshland, D.E., Karkharis, Y.D. and Latham, H.G. (1964) J. Am. Chem. Soc. 86, 1 4 4 8 - - 1 4 5 0 13 Barman, T.E. and Koshland, D.E. (1967) J. Biol. Chem, 242, 5771--5776 o 14 Gardlund, B., Kowalska-Loth, B., GrSndahl, N.J. and Blomb~/ck, B. (1972) Thrombosis Res. 1 , 3 7 1 - 388 15 Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J. and Takagi, T. (1977) Biochemistry 16, 1710--1714 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - - 2 7 5 17 P~rie, A. (1971) Biochem. J. 125, 203--208 18 Triantaphyllopoulos, E. and Triantaphyllopoulos, D.C. (1967) Blochem. J. 105, 393--400 19 Doolittle, R.F. (1975) in The Plasma Proteins (Putnam, F.W., ed.), Vol. II, pp. 109--161, Academic Press, New York 20 Dilley, K.J. (1973) Biochem. J. 1 3 3 , 8 2 1 - - 8 2 6