##### Abstract

The inhibition of bovine pancreatic trypsin was studied at pH 7, 25 degrees C, using mixtures of purified human alpha(2)-macroglobulin (alpha(2)M) and alpha(1)-proteinase inhibitor (alpha(1)PI). The partitioning of the enzyme between the two inhibitors was determined by comparing control esterase activity, assayed with N-benzoyl-L-arginine ethyl ester as substrate, with that remaining after incubation with inhibitory mixtures. (At [I](0) > [E](0), remaining esteratic activity reflects the concentration of alpha(2)M-associated enzyme (alpha(2)M-E*) and the concentration of alpha(1)PI-associated, inactive enzyme (alpha(1)PI-E*) is given by the difference, [E](0) - [alpha(2)M-E*].) The pattern of product distribution was found to be incompatible with an inhibitory model involving parallel, second-order reactions of E with alpha(2)M and alpha(1)PI. The data pointed to complex formation between the two inhibitors, limiting the level of alpha(2)M readily available for reaction with E. Analysis based on the binding equilibrium, alpha(2)M (dimeric unit) + alpha(1)PI reversible arrow alpha(2)M - alpha(1)PI, yielded K-d = 2.1 +/- 0.3 mu M. Complex formation between alpha(2)M and alpha(1)PI was verified by gel permeation experiments. alpha(2)M was found to restrict the volume of distribution of alpha(1)PI in Sephadex G200 beds. K-d, deduced from gel permeation behaviour, was 0.8 +/- 0.32 mu M. Preliminary kinetic experiments with dialyzed plasma suggested that the alpha(2)M-alpha(1)PI interaction is effective also in vivo. Given Kd and the mean plasma levels of the two inhibitors ([alpha(2)M] = 2 mu M; [alpha(1)PI] = 36 mu M), it was estimated that > 90% of alpha(2)M in human circulation must be complexed to alpha(1)PI and lack immediate antiproteinase activity.