MICHAEL LASKOWSKI, J R . , S.EIIRENPREIS, T. H . DONNELLY AND H . A . SCHERAGA
1340
I'd. SP
[COiXTRIBUTIC)N FROM 1 ITE D E P A R T M E S T OF C H E h f I S l R Y , C0RNEI.L UNIVERSIT\ ]
Equilibria in the Fibrinogen-Fibrin Conversion. V. Reversibility and Thermodynamics of the Proteolytic Action of Thrombin on Fibrinogen',' B Y M I C H A E L LASKOWSKI, JR. , 3 S E Y M O U R E I I R E N P R E I S ,
THOMAS H. DONNELLY AND
H.4ROLD
A.
SCHERAGA
RECEIVEDAUGUST3, 1959 The equilibrium of the proteolytic step (step 1) of the thrombin-fibrinogen reaction has been investigated in 1 M NaBr :it PH R.3 at three temperatures. Esseiitiallp the same equilibrium position for this proteolytic reaction is attained in both the forward and reverse directions. A:i analyiis of the thermodynamic parameters derived from the equilibrium constant atid its temperature dependence leads to the following mechanism for the step 1 equilibrium: Hydrolysis of the peptide bond, connecting t h e peptide P to the protein core, also involves the rupture of some secondary bonds, probably a carboxyl. . . carhoxyl, acetic acid dimer type hydrogen bond. After hydrolysis, P is still bound t o the protein core by additional secondary (hydrogen) bonds. At pH 5.3,essentially no dissociation of t h e peptide-core complex takes place. If t h e reaction mixture is dialyzed or if the pH is raised thereby breaking the hydrogen bonds, then the complex dissociates, and step 1 goes t o completion. I n this respect, this proteolytic reactioii differs from t h a t involving simple peptides, since enzymatic hydrolysis of t h e latter is generally complete. T h e groups unmasked in the dissociation of P from the protein core play a role in the subsequent polymerization of fibrin nionomer in step 2 .
Introduction Paper IVs of this series was preliminary to the present one; the former considered the kinetics of the thrombin-catalyzed conversion of fibrinogen, F, to fibrin monomer, f , in 1 AI NaBr a t p H 6.3, while the present paper is concerned with the reversibility and thermodynamics of the same reaction. A review of the pertinent literature will be found in the Introduction of paper I V 6 and in ref. 5 . Also, many of the experimental details pertaining to the work presented in this paper will be found in paper IV.6 The reaction under consideration here is step 1 of the scheme T
Proteolysis
P,'f+P
Polymerization
nf
Clotting
mf,
----f f-
(1)' fn
(2)
fibrin
(3)
where T is thrombin, P represents peptide and carbohydrate material,' fn designates intermediate polymers and n and m are variable numbers. The equilibrium of step 1 has been investigated over a range of concentrations and temperatures (under conditions where steps 2 and 3 are inhibited) by determining the concentration of f in the reaction mixture. Thermodynamic data have been obtained for the equilibrium and compared with corresponding data for the hydrolysis of a lowmolecular-weight peptide. It would appear that the proteolysis catalyzed by thrombin is anomalous in the sense that the reaction does not go to com(1) This investigation was supported by research grant No. H-1662 from the National Heart Institute of the National Institutes of Health, Public Health Service. (2) Various aspects of this work have been presented before the Division of Biological Chemistry at several meetings of the American Chemical Society: 122nd meeting, Atlantic City, New Jersey, September, 1952; 124th meeting, Chicago, Illinois, September, 1953; 128th meeting, Minneapolis, Minnesota, September, 1955; 131st meeting, Miami, Florida, April, 1957. A preliminary summary was given in'paper 11' of this series and also in a recent review6 of the fibrinogen-fibrin conversion. (3) Research Fellow of the National Heart Institute, United States Public Health Service, 1952-1956. (4) T . H. Donnelly, M. Laskowski, Jr., N. Notley and H . A. Scheraga, Arch. Biochem. and Bio.phyr., 6 6 , 369 (1955). (5) H. A. Scheraga and A t . Laskowski, Jr., A d v . in Protein Chem., 12, 1 (1957). (6) S. Ehrenpreis, M. Laskowski, Jr., T. H. Donnelly and H . A. Scheraga, THISJOURNAL, 80, 4255 (l9A8). (7) Since P may represent several species, equation 1 is an o v c r simplified forniulatiou of the proteolytic rwrtiou. Viirtllci
