Estimation of Trypsin by Fibrinolysis

Abstract

It has been pointed out that the actions of crystalline trypsin on blood-clotting systems are: 1. thromboplastic, 1 2. prothrombinolytic 2 and thrombinolytic, 3 3. fibrinogenolytic and fibrinolytic. 4 Preliminary tests with varying amounts of crystalline trypsin (X.T. = preparation of Dr. T. E. Weichselbaum, Washington University, St. Louis) and commercial trypsin (F. T. = Fairchild Bros, and Foster) established the fact that fibrinogen and fibrin are more sensitive than thrombin to the lytic action of the protease. This is a serious handicap in attempts to assay trypsin by the thrombinolytic method previously suggested. 3 However, fibrinolysis offers an alternative and simpler technic for the enzyme assay, which may be made especially accurate if the fibrinolysis is followed by relative turbidimetry (cf. 4 ) using the Evelyn photoelectric colorimeter.

Method. Essential reagents include a stable fibrinogen (F) 4 and a stable thrombin (e.g. T_G = 1:100 dilution of “rabbit clotting globulin,” kindly supplied by Dr. I. A. Parfentjev, 5 Lederle Labs., N. Y.). In each of a series of enzyme dilutions, 1 cc trypsin is added to 5 cc F + 3 cc T_G + 1 cc saline (= control for experiments, other than those cited, in which it is desired to test effects of inhibitors, etc.), a few sec prior to the onset of clotting. Control of clotting conditions must include temperature and pH. Relative turbidity is measured at intervals by the galvanometer scale deflection of the Evelyn apparatus. Data are plotted in the form of a series of curves corresponding to the various dilutions of trypsin used. It is not the shape of the curves which is significant but their relative positions along the time axis. Hence, conversion of the galvanometer reading (G) into “photometric density” (L), by means of the formula L = 2-log G, is an unnecessary refinement of the method described.