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Enzymic action of thrombin on fibrinogen compared with that of trypsin
94
BIOCHIMICAET BIOPHYSICAACTA
BBA 35949 ENZYMIC ACTION OF THROMBIN ON F I B R I N O G E N COMPARED W I T H THAT OF T R Y P S I N
YUJI INADA, MASAYASUBANDO, IZUMI KOTOKU, AYAKO MATSUSHIMA AND JUN HIRANO Laboratory of Biological Chemistry, Tokyo Institute of Technology, Meguro-ku, Ookayama, Tokyo (Japan) (Received May 4th, 1971) (Revised manuscript received July I3th, 1971)
SUMMARY
Denaturation of fibrinogen (substrate) by hot alkali resulted in a partial or complete loss of susceptibility to the proteolytic or clotting action of thrombin (enzyme), while it markedly enhanced that to the proteolytic action of trypsin.
Thrombin is strikingly similar to trypsin with respect to its substrate specificity. In a previous report 1 it was suggested that the "rigid" structure of the thrombin molecule reflects its high specificity for substrate. The question arises whether the tertiary structure of fibrinogen is associated with the high specificity of the thrombinfibrinogen reaction or not. In order to clarify this point, a few comparative studies were performed on the action of thrombin and trypsin on native and denatured fibrinogen. Bovine fibrinogen was isolated from plasma according to the method of BLOMBACKAND BLOMBACK2 and was 94-97% clottable by thrombin. The fibrinogen preparation was dissolved in 0.95 % NaC1 and dialysed against the same NaC1 solution to remove free amino acids. Bovine thrombin and porcine trypsin were purchased from Mochida Seiyaku Co. and from NOVO Industri A/S, respectively. Experiments were carried out as follows: 21. 4 #M fibrinogen in 0.95 % NaC1 solution was incubated at pH 11.3, 70°; at each given incubation time 2-ml aliquots of the fibrinogen solution were taken out into a test tube containing 2 ml of 0.2 M acetate; and to the nfixture was added 0.5 ml of thrombin (9.9 units/ml) or 0. 5 ml of trypsin (63/~M). The pH value of the mixture was kept at 9.3 to avoid the appearance of fibrinogen precipitates. After 3-rain digestion at 2I °, the reaction mixture was subjected to measurement of proteolytic activity. Proteolytic activity was determined from the amount of amino acids produced by the alkali hydrolysis of the fibrinopeptides or tryptic peptides released from fibrinogen, according to the method described in a previous paper 1. The clotting activity of thrombin on fibrinogen was determined by measuring the clotting time according to the method of SEEGERS AND SMITH3 and was expressed as the reciprocal of clotting time (sec 1). The clots formed in the present study were Biochim. Biophys. Acta, 251 (I97 I) 94 95
Y. INADA et al.
95
[
i
200
=
/
~150
Ioo B
~n.-
o
50 C
/
o 20 40 60 80 incubation Time with Alkali in min
Fig. I. E n z y m i c a c t i o n o f t h r o m b i n or t r y p s i n on a l k a l i - d e n a t u r e d fibrinogen. F i b r i n o g e n w a s d e n a t u r e d u n d e r alkaline p H of 11.3 a t 7 o°. C u r v e A, p r o t e o l y t i c a c t i v i t y m e a s u r e d w i t h 9.5 # M fibrinogen a n d 7 # M t r y p s i n ; C u r v e B, p r o t e o l y t i c a c t i v i t y of t h r o m b i n m e a s u r e d w i t h 8/zM fibrinogen a n d i . i N . I . H . u n i t s / m l t h r o m b i n . D i g e s t i o n was carried o u t a t 21 °, pFI 9.3, for 3 m i n C u r v e C, c l o t t i n g a c t i v i t y m e a s u r e d u n d e r t h e s a m e e x p e r i m e n t a l conditions as t h o s e described for C u r v e B.
soluble in 5 M urea. Though it had been found that fibrinogen was denatured at temperatures as low as 47 ° (ref. 4) or at pH values outside the range of 5.5-1o.o (ref. 5), the denatured fibrinogen solution tends to precipitate when the pH or temperature is changed. To avoid the appearance of precipitates, it was decided on the basis of preliminary tests that the conditions for the denaturation experiment described here should be p H 11.3 and 7 o°. The change in proteolytic and clotting activities of thrombin was measured as a function of incubation time of fibrii,ogen and the results are shown by Curves B and C in Fig. I. A similar experiment was performed with trypsin instead of thrombin, which is shown by Curve A. The proteolytic activity of thrombin decreases gradually with increasing incubation time and reaches a level of 60% of the original activity, while the clotting activity decreases sharply and disappears completely at IO min incubation. On the other hand, the proteolytic activity of trypsin increases markedly with increasing incubation time and reaches 200% of the original level. It is natural that trypsin, as an important digestive enzyme, should exhibit such an avaricious capacity on denatured protein. It was surprising that the action of thrombin on fibrinogen disappears completely or partially with relatively slight denaturation of fibrinogen, and that the clotting process is more susceptible to denaturation than the proteolytic process. These observations may suggest that the native conformation of fibrinogen is absolutely essential for its physiologically significant susceptibility to thrombin, and that the subsequent clotting process requires a more subtle configuration than that of the initial proteolytic process. REFERENCES I I. KOTOKV, A. MATSUSmMA, M. BANDO AND Y. INADA, Biochim. Biophys. Acta, 214 (197 o) 49o. 2 t 3 . B L O M B ~ . C K A N D M . B L O M B ~ . C K , Arkiv. Kemi, I O ( 1 9 5 6 ) 4 1 5 . 3 W. H. SEEGERS AND H. P. SMITH, Am. J. Physiol., 137 (1942 ) 348 . 4 E. W. DAVIE AND O. D, RATNOFF, in H. NEURATH, The Proteins, Vol. 3, A c a d e m i c Press, N e w York, 1965, p. 366. 5 B. BLOMBACK, in W. H. SEEGERS, Blood Clotting Enzymology, A c a d e m i c Press, N e w Y o r k a n d L o n d o n , 1967, p. 187.
Biochim. Biophys. Acta, 251 (I971) 94--95
BIOCHIMICAET BIOPHYSICAACTA
BBA 35949 ENZYMIC ACTION OF THROMBIN ON F I B R I N O G E N COMPARED W I T H THAT OF T R Y P S I N
YUJI INADA, MASAYASUBANDO, IZUMI KOTOKU, AYAKO MATSUSHIMA AND JUN HIRANO Laboratory of Biological Chemistry, Tokyo Institute of Technology, Meguro-ku, Ookayama, Tokyo (Japan) (Received May 4th, 1971) (Revised manuscript received July I3th, 1971)
SUMMARY
Denaturation of fibrinogen (substrate) by hot alkali resulted in a partial or complete loss of susceptibility to the proteolytic or clotting action of thrombin (enzyme), while it markedly enhanced that to the proteolytic action of trypsin.
Thrombin is strikingly similar to trypsin with respect to its substrate specificity. In a previous report 1 it was suggested that the "rigid" structure of the thrombin molecule reflects its high specificity for substrate. The question arises whether the tertiary structure of fibrinogen is associated with the high specificity of the thrombinfibrinogen reaction or not. In order to clarify this point, a few comparative studies were performed on the action of thrombin and trypsin on native and denatured fibrinogen. Bovine fibrinogen was isolated from plasma according to the method of BLOMBACKAND BLOMBACK2 and was 94-97% clottable by thrombin. The fibrinogen preparation was dissolved in 0.95 % NaC1 and dialysed against the same NaC1 solution to remove free amino acids. Bovine thrombin and porcine trypsin were purchased from Mochida Seiyaku Co. and from NOVO Industri A/S, respectively. Experiments were carried out as follows: 21. 4 #M fibrinogen in 0.95 % NaC1 solution was incubated at pH 11.3, 70°; at each given incubation time 2-ml aliquots of the fibrinogen solution were taken out into a test tube containing 2 ml of 0.2 M acetate; and to the nfixture was added 0.5 ml of thrombin (9.9 units/ml) or 0. 5 ml of trypsin (63/~M). The pH value of the mixture was kept at 9.3 to avoid the appearance of fibrinogen precipitates. After 3-rain digestion at 2I °, the reaction mixture was subjected to measurement of proteolytic activity. Proteolytic activity was determined from the amount of amino acids produced by the alkali hydrolysis of the fibrinopeptides or tryptic peptides released from fibrinogen, according to the method described in a previous paper 1. The clotting activity of thrombin on fibrinogen was determined by measuring the clotting time according to the method of SEEGERS AND SMITH3 and was expressed as the reciprocal of clotting time (sec 1). The clots formed in the present study were Biochim. Biophys. Acta, 251 (I97 I) 94 95
Y. INADA et al.
95
[
i
200
=
/
~150
Ioo B
~n.-
o
50 C
/
o 20 40 60 80 incubation Time with Alkali in min
Fig. I. E n z y m i c a c t i o n o f t h r o m b i n or t r y p s i n on a l k a l i - d e n a t u r e d fibrinogen. F i b r i n o g e n w a s d e n a t u r e d u n d e r alkaline p H of 11.3 a t 7 o°. C u r v e A, p r o t e o l y t i c a c t i v i t y m e a s u r e d w i t h 9.5 # M fibrinogen a n d 7 # M t r y p s i n ; C u r v e B, p r o t e o l y t i c a c t i v i t y of t h r o m b i n m e a s u r e d w i t h 8/zM fibrinogen a n d i . i N . I . H . u n i t s / m l t h r o m b i n . D i g e s t i o n was carried o u t a t 21 °, pFI 9.3, for 3 m i n C u r v e C, c l o t t i n g a c t i v i t y m e a s u r e d u n d e r t h e s a m e e x p e r i m e n t a l conditions as t h o s e described for C u r v e B.
soluble in 5 M urea. Though it had been found that fibrinogen was denatured at temperatures as low as 47 ° (ref. 4) or at pH values outside the range of 5.5-1o.o (ref. 5), the denatured fibrinogen solution tends to precipitate when the pH or temperature is changed. To avoid the appearance of precipitates, it was decided on the basis of preliminary tests that the conditions for the denaturation experiment described here should be p H 11.3 and 7 o°. The change in proteolytic and clotting activities of thrombin was measured as a function of incubation time of fibrii,ogen and the results are shown by Curves B and C in Fig. I. A similar experiment was performed with trypsin instead of thrombin, which is shown by Curve A. The proteolytic activity of thrombin decreases gradually with increasing incubation time and reaches a level of 60% of the original activity, while the clotting activity decreases sharply and disappears completely at IO min incubation. On the other hand, the proteolytic activity of trypsin increases markedly with increasing incubation time and reaches 200% of the original level. It is natural that trypsin, as an important digestive enzyme, should exhibit such an avaricious capacity on denatured protein. It was surprising that the action of thrombin on fibrinogen disappears completely or partially with relatively slight denaturation of fibrinogen, and that the clotting process is more susceptible to denaturation than the proteolytic process. These observations may suggest that the native conformation of fibrinogen is absolutely essential for its physiologically significant susceptibility to thrombin, and that the subsequent clotting process requires a more subtle configuration than that of the initial proteolytic process. REFERENCES I I. KOTOKV, A. MATSUSmMA, M. BANDO AND Y. INADA, Biochim. Biophys. Acta, 214 (197 o) 49o. 2 t 3 . B L O M B ~ . C K A N D M . B L O M B ~ . C K , Arkiv. Kemi, I O ( 1 9 5 6 ) 4 1 5 . 3 W. H. SEEGERS AND H. P. SMITH, Am. J. Physiol., 137 (1942 ) 348 . 4 E. W. DAVIE AND O. D, RATNOFF, in H. NEURATH, The Proteins, Vol. 3, A c a d e m i c Press, N e w York, 1965, p. 366. 5 B. BLOMBACK, in W. H. SEEGERS, Blood Clotting Enzymology, A c a d e m i c Press, N e w Y o r k a n d L o n d o n , 1967, p. 187.
Biochim. Biophys. Acta, 251 (I971) 94--95