Plasmin was immobilized on collagenous substrates using carbodiimide as a linking agent. The kinetics of soluble and immobilized plasmin were monitored by reacting them with the chromogenic substrate S-2251 (H-D-Val-Leu-Lys-pNA) in the presence and absence of a2-antiplasmin (a2-PI). The ability of immobilized plasmin to lyse synthetic clots formed from fibrinogen and thrombin was determined by detecting the formation of fibrin degradation products (FDP). The activity of immobilized plasmin was 0.02 casein units (CU)/mg of collagen. The kinetic analysis of soluble and immobilized plasmin in the presence and absence of a2-PI shows that while soluble plasmin activity was inhibited by the presence of a2-PI, the plasmin inhibitor did not interfere with the ability of immobilized plasmin to attack fibrin. In the absence of a2-PI, the ability of the immobilized plasmin to lyse synthetic clots was the same as that of soluble plasmin. In the presence of a2-PI, immobilized plasmin produced twice the amount of FDP as did soluble plasmin. The immobilized plasmin activity was stable for a period of at least 3 months.

No abstract

A least square estimation of the Michaelis-Menten kinetic parameters from the direct linear plot was obtained using a minimization procedure developed to produce explicit expressions for K m and V m ull as a function of substrate concentration and measured reaction rate. This was accomplished by minimizing the distance between a point in parameter space representing the optimal parameter values and all the lines obtained from the direct linear plot. Under certain limiting conditions, the developed generalized expressions simplify to the least square estimates of Eadie-Hofstee and inverse Hanes plots. The expressions developed are simple to use and can accommodate replicate data, producing less conservative error bounds for the parameters than those obtained by other techniques.