Chemically modified bovine prothrombin as a substrate in studies of activation kinetics and fluorescence changes...

The activation of bovine prothrombin is known to be accompanied in purified systems by proteolytic reactions catalyzed by the product, thrombin. These reactions, which are directed principally towards the prothrombin substrate and the Factor V cofactor, are eliminated if the lysine residues of prothrombin are chemically modified beforehand with methyl acetimidate. Amidinated prothrombin in which the usual lysine content has been reduced by 75% is cleaved completely by Factor Xa to give thrombin which has little or no activity towards fibrinogen, the thrombin-sensitive bond in prothrombin, or Factor V, but with normal activity towards the synthetic chromogenic substrate D-Phe-Pipecolyl-L-Arg-p-nitroanilide. The formation of thrombin could therefore be studied spectrophotometrically by discontinuous assays of thrombin without complication by the proteolytic feedback activity of this enzyme. Such assays showed that the rate of appearance and yield of thrombin is the same whether from native or amidinated prothrombin, in spite of the lack of proteolytic activity in the product from the latter. Native and amidinated prothrombin have identical fluorescence emission spectra (lambda ex = 280 nm) which, upon activation, show a broadening and a red shift of the peak of emission from 330 to 336 nm. This change is different from the quenching known to occur when prothrombin binds Ca2+ and is contingent upon cleavage by Factor Xa. When monitored at the 370 nm band, the shift is seen as an increase in fluroescence intensity which, when the concentrations of Factor Xa and Factor V are adjusted appropriately, has the same general appearance as a progress curve obtained by discontinuous assay of thrombin activity. However, the curves obtained with the native zymogen appear to contain a component due to proteolysis by the accumulating product. in the case of amidinated zymogen, this is not longer so: the curves reflect only proteolysis by Factor Xa. In addition, a comparison of the fluorescence shift with the time course of thrombin appearance shows that the shift results mainly from the cleavage of intact prothrombin by Factor Xa, with little or no contribution from later events in the activation pathway. Modified Stern-Volmer plots for the quenching of fluorescence (lambda ex = 295 nm) of the intact amidinated zymogen and its activation products by sodium iodide allow the conclusion that activation results in the exposure to solvent of tryptophan residues that were previously sheltered. However, there is a variation in the pattern of quenching with the protein concentration, suggesting that these residues may be sheltered in the zymogen by intermolecular rather than intramolecular interactions.