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

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 K_d= 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. K_d, 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 K_d 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.

Key Words:

• α₂-Macroglobulin
• α₁-Proteinase inhibitor
• Protein-protein interaction