Radiation-induced changes in purified prothrombin and thrombin

Biochimica et Biophysica Acta, 706 (1982) 1-8

1

Elsevier Biomedical Press BBA31258

RADIATION-INDUCED CHANGES IN PURIFIED PROTHROMBIN AND THROMBIN LATIKA P. CHANDERKAR and G.B. NADKARNI *

Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085 (India) (Received December 30th, 1981)

(Revised manuscript received April 27th, 1982)

Key words: y-Irradiation; Prothrombin; Thrombin; Clotting activity

The effect of v-irradiation on purified prothrombin and thrombin in aqueous solution has been assessed with reference to bifunctional activities, e.g., clotting and esterase functions, physieo-chemical changes in structure, and kinetics. The inactivation curves indicated that the clotting activity was more susceptible to T-radiation than the esterolytic function in both the proteins. Prothrombin was comparatively more sensitive to radiation than thrombin. The irradiation of prothrombin (100 kR) caused modifications in the protein resulting in reduced formation of thrombin after activation by Factor Xa. The modifications caused by irradiation were assessed in these proteins by changes in spectral characteristics, levels of tryptophan and disulphides, electrophoretic mobility and amino acid composition. Radiation-induced changes in thrombin were reflected in its kinetic behaviour. The dotting activity of thrombin was almost completely lost at 100 kR, while esterolysis was relatively less affected. The modification of tyrosine and tryptophan residues in thrombin influenced the dotting activity, while these were not involved for esterolysis. Histidine had involvement in both these activities.

Introduction In our earlier studies [1,2], structural and physicochemical alterations in fibrinogen exposed to y-irradiation were reported to result in incoagulability, thereby affecting the function of this protein as a substrate of thrombin. It is recognised that the conversion of prothrombin to thrombin is also an essential event in the process of blood coagulation [3]. Though haemorrhages are known to occur as a result of exposure of animals to ionizing radiations [4], the levels of prothrombin and coagulation accelerators in blood were not observed to be altered despite reduction in the number of circulating platelets [5,6]. The reported

* To whom correspondence should be addressed. Abbreviation: TAME, p-tosyl-L-arginine methyl ester. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

increase in fibrinolytic activity in irradiated dogs could not also be correlated with the concentrations of heparin or heparinoid substances [7]. Blood being essentially an aqueous system, the macromolecules contained in it are likely to be more susceptible to radiation [8]. Though several studies relating to the inactivation by radiation of enzymes having specific catalytic functions have been reported [9-12], there is not much information available on blood proteins. The present paper describes studies on purified thrombin and prothrombjn with reference to alterations caused by ~,-irradiation in the zymogen as well as the active enzyme. The changes observed in vitro using high doses of irradiation need not necessarily reflect the in vivo situations, though such attempts may provide useful information on the susceptible sites causing dysfunction of the proteins.

Materials and Methods

Materials. Bovine blood was obtained from a local abbatoir. Prothrombin was isolated and purified as described earlier [13] by a modified procedure of Moore et al. [14]. Bovine fibrinogen (Fraction I), thrombin, p-tosyl-L-arginine methyl ester (TAME), dithiothreitol, 2-hydroxyl-5-nitrobenzyl bromide, tetranitromethane and diethylpyrocarbonate were purchased from Sigma Chemical Co. All other chemicals used in these experiments were of analytical grade and obtained from standard sources. Irradiation of prothrombin and thrombin. The irradiations were performed in a Gamma-Cell 220 (AECL) with a dose rate of 11.4 k R / m i n with air as gas-phase. The aqueous solutions (1.5.10 -5 M) of prothrombin and thrombin in 0.1 M phosphate buffer, pH 6.0, or saline were irradiated under cold conditions (0-4°C). Prothrombin to thrombin conversion. The activation system for prothrombin to thrombin conversion has been described earlier [13]. The system contained, in a total volume of 2.6 ml, 2.0 ml prothrombin (2 mg/ml), 4 units of Factor Xa (0.1 ml), 0.1 ml 0.023 M CaC12, 0.1 ml bovine Factor V and 0.025 ml phospholipids containing 20 #g each of lysolecithin and phosphatidylserine in methanol [15]. The mixture was incubated for 24h at 37°C and thrombin formed was isolated by gel filtration on Sephadex G-100. The fractions were assayed for thrombin activity. Assay of thrombin. Clotting activity of thrombin was determined by measuring the clotting time of purified bovine fibrinogen as described earlier [13]. The system contained, in a total volume of 0.7 ml, 1.25 mg fibrinogen in saline, 0.2 ml 0.2 mM CaC12 and 5.0 #g thrombin. The activity has been expressed as NIH units [16]. One unit of thrombin activity has been expressed as the amount of thrombin which clotted 0.5 ml (1.25 mg) of purified fibrinogen [17] in 15 s at 28°C. Clotting activity using fibrinogen as the substrate was also determined by the procedure described by Kotoku et al. [18]. The esterase activity of thrombin was determined spectrophotometrically [19] using TAME as the substrate. The assay system comprised, in a total volume of 3ml, 0 . 5 . 1 0 - a M

TAME in 0.2 M Tris-HC1 buffer, pH 8.1, containing 0.01 M CaC12 and activated prothrombin (0.1 ml). The unit of activity has been expressed as #mol TAME hydrolysed per min at 25°C. Spectral studies. The absorption spectra of prothrombin in solution (1.5.10-SM), before and after irradiation, were obtained in the range of 220-360 nm, using a Perkin-Elmer DB Model-124 spectrophotometer equipped with a Hitachi Model 165 recorder. The protein samples were taken in a cuvette with a 1.0 cm lightpath after appropriate dilution (1:2). Fluorescence spectra were obtained in an Aminco-Bowman spectrophotofluorimeter between 260-460 nm, with an excitation wavelength of 278 nm. Prothrombin solution was diluted 1 : 10 for fluorescence measurements. Other estimations. Protein was estimated by the method of Lowry et al. [20], using bovine serum albumin as standard. Tryptophan content was determined colorimetrically using the procedure of Spies and Chambers [21]. Disulphide content of prothrombin was determined by the method of Iyer and Klee [22]. Prothrombin was hydrolysed with 6.0 M HC1 for 24h at l l0°C [23] and amino acids were estimated from the hydrolysate using an automatic amino acid analyzer. Polyacrylamide disc gel electrophoresis was performed by using the method of Davis [24]. Results

Effects of irradiation on bifunctional activities of thrombin The changes in the clotting and esterase activities of prothrombin and thrombin after irradiation are presented in Fig. 1. Though both the activities were reduced, concomitant with the dose of irradiation, the extent of reduction was not the same for these activities. The inactivation effect was more marked in prothrombin than in thrombin. The sensitivity to radiation of the clotting activity was observed to be more than that of esterolysis. Fig. 2 plots the profiles of the irradiated and unirradiated prothrombin on Sephadex G-100 gel filtration. The recovery of prothrombin after irradiation was lower. The protein recovered was 50 and 40%, respectively, when prothrombin was

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exposed to 100 and 200 kR, as c o m p a r e d to the unirradiated prothrombin. The irradiation of a protein in solution could cause either polymerization or degradation, depending on the dose [1 ]. N o

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12 20 28 FRACTION NUMBER Fig. 2. Gel filtration of irradiated and unirradiated prothrombin. 2 ml prothrombin (1 mg/ml) were loaded on a Sephadex G-100 column (l X60 cm) equilibrated with 0.05 M phosphate buffer, pH 6.0, containing 0.1 M NaCI. The flow rate was adjusted to 30 ml/h and 2.5-ml fractions were collected and estimated for protein. O, unirradiated; r-l, 25 kR; A, 50 kR; 0, 100 kR; ×, 200 kR.

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Fig. 3. Effect of y-irradiation (]00 kR) on protkrombin to thrombin conversion. The activated samp]e of prothrombin (6 nag) was applied on to a Sephadex G-100 column (l × 100 cm) equilibrated with 0.05 M phosphate buffer, pH 6.0, containing 0.1 M NaC1. The flow rate was adjusted to 30 ml/h and 2.0-ml fractions were collected for estimation of protein and the clotting activity. The elution profiles of the irradiated (×) and the unirradiated (O) protein are presented. The clotting activities in the fractions are shown in the inset.

protein appeared in the void volume and hence p r o t h r o m b i n was p r e s u m a b l y degraded into smaller fragments eluting b e y o n d the fractions collected. This would also reflect in the reduced levels of thrombin obtained after the activation, as observed in Fig. 3. It has been indicated earlier [13] that the thrombin formed after activation of p r o t h r o m b i n could be resolved into three forms. T h e formation of thrombin B was drastically reduced after irradiation, while thrombin A seemed to show an increase. The inset to Fig. 3 shows the distribution o f the clotting activity in the three forms of thrombin. It is apparent that fraction B had most of the activity, while A and C had low activities. The fraction A eluting after the activation of the irradiated p r o t h r o m b i n did not show any clotting activity, though the protein content was significantly increased. There were no measurable changes in fraction C.

Physico-chemical alterations The spectral changes in p r o t h r o m b i n exposed to various doses of y-radiation are shown in Fig. 4. Absorption spectra of the irradiated p r o t h r o m b i n showed a dose-dependent increase both in minim u m (249 nm) and m a x i m u m regions (278 nm). However, the changes observed in the emission spectra were not as striking as in the absorption

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Fig. 4. (left-hand figure) Increase in absorbance of prothrombin at 278 nm (O) and 249 nm (×) after exposure to y-radiation. Experimental details are given in text. Fig. 5. Decrease in fluorescence maxima of prothrombin exposed to T-irradiation. The excitation wavelength was 278 nm and the emission was measured at 339 nm. The reading with unirradiated protein was taken as control and the changes observed after irradiation were calculated as percent of the control. Fig. 6. Densitometric profile of polyacrylamide gel electrophoresis of irradiated (100 kR) prothrombin, indicating changes in mobility.

spectra, as shown in Fig. 5, though there was decrease in the fluorescence maxima. Table I gives the tryptophan and disulphide content in p r o t h r o m b i n exposed to different doses o f irradiation. The loss in tryptophan was not measurable up to 50 kR, though it showed reduction to 40% at 200 kR. It was observed that the accessibility of disulphide bonds to dithiothreitol was reduced to the extent of 60% at 200 kR. The amino acid composition of the irradiated (100 kR) and unirradiated p r o t h r o m b i n indicated that the main residues affected were aromatic

TABLE I EFFECT OF T-IRRADIATION ON TRYPTOPHAN AND DISULPHIDE CONTENTS OF PROTHROMBIN Dose of radiation (kR)

Tryptophan content (tool/tool)

Disulphide content (mol/mol)

0 25 50 ioo 200

1l.O 11.0 9.8 8.5 6.4

7.0 4.8 4.0 2.8 2.3

amino acids, along with half-cystine, methionine and histidine (in that order). The extent of reduction in these residues is shown in Table II, which also gives the reported values for a m i n o acid composition of p r o t h r o m b i n [25]. Methionine could be partially accounted for as methionine sulphone, while cysteic acid was not detectable. The rest of the amino acid residues did not show measurable changes. The electrophoretic patterns of the irradiated and unirradiated p r o t h r o m b i n were also altered, as can be seen in the densitometric profiles presented in Fig. 6. It was observed that the irradiated prothrombin m o v e d faster than the unirradiated protein, suggesting increase in the net charge on prothrombin.

Effect of 7-irradiation on clotting activity of thrombin The clotting activity was inhibited when thrombin was irradiated ( 0 - 1 0 0 kR). The release of fibrinopeptides decreased with the increase in the dose. The Lineweaver-Burk plots of the release of fibrinopeptides gave a pattern as in Fig. 7, which showed marked changes in K m, while Vm,x was relatively less affected. The dose effect on the

TABLE II A M I N O ACID COMPOSITION OF IRRADIATED (100 kR) A N D U N I R R A D I A T E D PROTHROMBIN Residues per mol were estimated on the basis of molecular weight of prothrombin as 68000, which was determined separately. Tryptophan was estimated ¢olorimetrically, as stated in the text. The results are expressed as per cent residues calculated after adding the residues/mol for all amino acids, including those not shown in the table. Amino acid

Prothrombin (reported values, Ref. 25)

Unirradiated prothrombin

Irradiated prothrombin (100 kR)

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1.4

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3.2

3.6 ± 0.3

TABLE III KINETIC CONSTANTS F O R CLOTTING OF FIBRINOGEN A N D HYDROLYSIS O F TAME WITH IRRADIATED THROMBIN Vm~, for fibrinogen (Fib) as substrate is expressed as ~tmol fibrinopeptides released/min per unit of thrombin, while that for TAME as substrate is expressed as/tmol ester hydrolysed/min per mg protein. Km(M ) (X 10 - s )

Vmax (X 10 - s )

Kcat(S- l ) Fib

Unirradiated 25 kR 50 kR 100 kR

Fib

TAME

Fib

TAME

0.25 0.5 0.8 1.9

22 22 22 25

1.6 1.3 1.1 0.8

1.2 1.2 1.1 !.0

0.5 0.4 0.3 0.2

C = Kcat / K m ( i - I s - I)X 103) TAME

0.4 0.4 0.4 0.3

Fib

TAME

210 82 40 10

0.2 0.2 0.2 0. !

TABLE IV EFFECT OF TRYPTOPHAN MODIFICATION ON CLOTTING A N D ESTEROLYTIC ACTIVITIES OF THROMBIN TO !.0 ml thrombin (1 m g / m l in 0.2 M acetate buffer, pH 4.0) was added 2-hydroxy-5-nitrobenzyl bromide in dimethylsulfoxide (1 rag/0.15 ml) at different concentrations. The reaction was carried out in the dark for 15 rain. Excess reagent was removed on Sephadex G-25 and the clotting activity was determined after adjusting the p H to 6.8 with 1 M NaOH. Molar excess 2-hydroxy5-nitrobenzyl bromide

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Percent inhibition Clotting activity

Esterase activity

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Vmax, gea t (i.e., V , , / E o) and C values (i.e., geat//Km) are listed in Table III. The affinity of thrombin for fibrinogen seemed to decrease with the dose of irradiation. The proteolytic activity was almost 1/10 of the control at 100 kR. However, esterase activity was not measurably altered. The kinetic constants for T A M E as the substrate are included in Table III.

Modifications of amino acid residues in thrombin In view of the changes observed above in the amino acid residues, their possible involvement in biological activity of thrombin was ascertained by using chemical modifiers. Thrombin was subjected to treatments with specific reagents causing chemical modifications and then clotting and esterase activities were measured. The binding of thrombin with tryptophan-specific 2-hydroxy-5-nitrobenzyl bromide [26] inhibited the clotting activity by about 85%, while esterase activity was reduced by only 20%, as shown in Table IV. The modification of tyrosine in thrombin by tetranitromethane (TNM) as described by Lundblad [27] reduced the dotting activity. It was observed that the clotting activity was almost completely inhibited (95%) at 5 . 1 0 - 3 M tetra-

nitromethane, while the esterase activity for T A M E was not measurably altered. The binding of thrombin with diethylpyrocarbonate (DEPC), which is known to modify histidine residues [28], reduced the proteolytic as well as esterolytic activities, as shown in Table V. The extent of inhibition was similar in respect of both the activities at 2 m M diethylpyrocarbonate. Discussion

Several studies on the conversion of prothrombin to thrombin by different activating systems have been reported [29-32]. Most of these studies relate to the structural changes and the formation of intermediate fractions, which point to the possible conformational changes in the protein caused during the activation. The conformation of prothrombin in aqueous solution is also known to be dependent on the presence of Ca 2+ . However, the procedure used in the present experiments for isolation of this protein excludes these ions, since the solutions used at all stages of purification contained E D T A [13,14]. Therefore, the changes observed could be attributed to the effects of radiation. After irradiation (above 100 kR) only 40-50% of the clotting activity could be obtained by thrombokinase activation. Thrombin formed was also markedly reduced, suggesting possible

degradation of available prothrombin due to irradiation. The spectral analysis suggested structural alterations around tryptophan residues, which seemed to be affected beyond 100 kR. The involvement of OH" radicals in causing this effect was shown earlier with fibrinogen [2]. The necessity for intact tryptophan in thrombin for clotting activity [26] and the changes around the aromatic residues during the activation of prothrombin have been observed. The reduced accessibility of disulphide bonds to dithiothreitol and the increase in the electrophoretic mobility of protein point to either formation of mixed disulphides or oxidation to cysteic acid, which could nevertheless influence the overall charge on the protein [33]. Amino acid composition indicates changes in half-cystine, methionine, tyrosine, phenylalanine, histidine and tryptophan. ~/-Glutamyl carboxyl groups in prothrombin, having a role in its conversion to thrombin [34,35], have been reported to be unstable to physical treatments such as heating [36,37]. However, glutamate levels did not seem to be altered after irradiation. Prothrombin being essentially the precursor of thrombin, which is a bifunctional enzyme, the radiation-induced alterations were also examined with references to the two activities. The exposure of thrombin to v-rays (100 kR) caused 25% loss of proteolytic activity, while esterolytic activity was not measurably altered. The affinity for the substrate for proteolysis was reduced by 50%, while that for esterolysis was altered only to the extent of 10%. Similarly, the K m value for fibrinogen as substrate was almost 22 times less than that for TAME. Bando et al. [38] had indicated that the affinity of thrombin for fibrinogen was 50-times more than that for TAME. The loss in clotting activity of thrombin exposed to 100 kR was only 25%, while with prothrombin irradiation the loss was 60% in clotting and 40% in esterolysis. These observations might suggest that prothrombin, the zymogen, was more sensitive to radiation. Similar observations have been made by Lynn with other serine proteases such as trypsin, chymotrypsin and their zymogens [39]. It is well-established that aromatic amino acids are sensitive to radiation [2,10,11]. The effect of chemical modification of tryptophan and tyrosine

residues in thrombin caused reduction in the clotting activity, while esterase activity was not altered. These observations suggest involvement of these amino acids in the interaction with fibrinogen [25,26], but not in the active site for esterolysis. The modification of histidine, on the other hand, reduced both these activities to the same extent, suggesting its common role in clotting activity as well as esterolysis. The involvement of histidine in both these activities was also suggested by Glover and Shaw [40]. Therefore the data obtained on the basis of esterolytic function alone may not adequately reflect the effects of radiation on the actual clotting mechanisms. References 1 Kamat, H.N. and Nadkarni, G.B. (1972) Radiat. Res. 49, 381-389 2 Chanderkar, L.P., Gurnani, S. and Nadkarni, G.B. (1976) Radiat. Res. 65, 283-291 3 Rosenberg, J.S., Belier, D.L. and Rosenberg, R.D. (1975) J. Biol. Chem. 250, 1607-1617 4 Mole, R.H. (1956) Br. J. Radiol. 26, 234-241 5 Jacobsen, L.O. (1954) in Radiation Biology, Vol. I, Part II (Hollander, A., ed.), pp. 1029-1090, McGraw-Hill, New York 6 Cohn, S.H. (1952) Blood 7, 225-234 7 Bacq, Z.M. and Alexander, P. (1961) Fundamentals of Radiobiology, 2nd exln., pp. 406-435, Pergamon Press, London 8 Dale, W.M. (1966) in Radiation Biology (Zuppinger, A., ed.), Vol. 1, pp. 1-38, Springer-Verlag, Berlin 9 Dale, W.M. (1966) In Radiation Biology (A. Zuppinger ed.) Vol. 1, pp. 214-235, Springer-Verlag, Berlin 10 Lynn, K.R. (1971) Radiat. Res. 46, 268-278 11 Lynn, K.R. and Raoult, A.P.D. (1973) Int. J. Radiat. Biol. 24, 25-31 12 Lynn, K.R. and Skinner, W.J. (1975) Radiat. Res. 63, 245-252 13 Chanderkar, L.P. and Nadkarni, G.B. (1979) Indian J. Biochem. Biophys. 16, 196-199 14 Moore, H.C., Lux, S.E., Malhotra, O.P., Bakerman, S. and Cartier,J.R. (1965) Bioehim. Biophys. Acta 1! 1, 174-180 15 Jobin, F. and Esnouf, M.P. (1967) Biochem. J. 102, 666-674 16 Shuiman, N.R. and Hearon, J.Z. (1963) J. Biol, Chem. 238, 155-164 17 Laki, K. (1951) Arch. Bioehem. Biophys. 32, 317-328 18 Kotoku, I., Matsushima, A., Bando, M. and Inada, Y. (1970) Bioehim. Biophys. Aeta 214, 490-497 19 Hummel, B.C.M. (1959) Can. J. Bioehem. Physiol. 37, 1393-1399 20 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275

21 Spies, J.R. and Chambers, D.C. (1948) Anal. Chem. 20, 30-39 (1949) 21, 1249-1266 22 lyer, S.K. and Klee, W.A. (1973) J. Biol. Chem. 248, 707-710 23 Moore, S. and Stein, W.H. (1963) Methods Enzymol. 6, 819-831 24 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 25 Owen, W.G., Esmon, C.T. and Jackson, C.M. (1974) J. Biol. Chem. 240, 594-605 26 Uhteg, L.C. and Lundblad, R.L. (1977) Bioehim. Biophys. Acta 491,551-557 27 Lundblad, R.L. and J.H. Harrison (1971) Biochem. Biophys. Res. Commun. 45, 1344-1349 28 Kelkar, S.M. and Nadkarni, G.B. (1977) Indian J. Bioehem. Biophys. 14, 305-309 29 Engel, A.M. and Alexander, B. (1973) Biochim. Biophys. Acta 320, 687-700 30 Mann, K.G., Heldebrant, C.M. and Fass, D.N. (1971) J. Biol. Chem. 246, 6106-6114

31 Seegers, W.H., Walz, D.A., Reuterby, J. and McCoy, L.E. (1972) Thromb. Res. 4, 829-860 32 Esmon, C.T., Owen, W.G. and Jackson, C.M. (1974) J. Biol. Chem. 249, 7798-7804 33 Pihl, A.L. and Sanner, T. (1963) Radiat. Res. 19, 27-41 34 Davie, E.W. and Hanahan, D.J. (1977) in The Plasma Proteins Vol. 3 (Putnam, F.W., ed.), pp. 421-544, Academic Press, New York 35 Nemerson, N.Y. and Furie, B. (1980) CRC Crit. Rev. Bioehem. 9, 45-85 36 Karpatkin, M. and Karpatkin, S. (1981) Ann. N.Y. Acad. Sci. 370, 281-290 37 Esmon, C.T., Sadowski, J.A. and Suttie, J.W. (1975) J. Biol. Chem. 250, 4744-4748 38 Bando, M., Matsushima, A., Hirano, J. and lnada, Y. (1972) J. Bioehem. (Tokyo) 71,897-899 39 Lynn, K.R. (1973) Int. J. Radiat. Biol. 23, 227-233 40 Glover, G. and Shaw, E. (1971) J. Biol. Chem. 246, 45944601