The Trypsin-Inhibitory Efficiency of Human α2-Macroglobulin in the Presence of α1-Proteinase Inhibitor: Evidence for the Formation of an α2-Macroglobulin-α1-Proteinase Inhibitor Complex

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Abstract
The inhibition of bovine pancreatic trypsin was studied at pH 7, 25°C, using mixtures of purified human α2-macroglobulin (α2M) and α1-proteinase inhibitor (α1PI). 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 α2M-associated enzyme (α2M-E*) and the concentration of α1PI-associated, inactive enzyme (α1PI-E*) is given by the difference, [E]0— [α2M-E*].) The pattern of product distribution was found to be incompatible with an inhibitory model involving parallel, second-order reactions of E with α2M and α1PI. The data pointed to complex formation between the two inhibitors, limiting the level of α2M readily available for reaction with E. Analysis based on the binding equilibrium, α2M (dimeric unit) + α1PI⇆α2M —α1PI, yielded Kd= 2.1 ± 0.3 μM. Complex formation between α2M and α1PI was verified by gel permeation experiments. α2M was found to restrict the volume of distribution of α1PI in Sephadex G200 beds. Kd, deduced from gel permeation behaviour, was 0.8 ± 0.32 μM. Preliminary kinetic experiments with dialyzed plasma suggested that the α2M-α1PI interaction is effective also in vivo. Given Kd and the mean plasma levels of the two inhibitors ([α2M] = 2μM; [α1PI] = 36 μM), it was estimated that > 90% of α2M in human circulation must be complexed to α1PI and lack immediate antiproteinase activity.