Fluorescence Monitoring of the Conformational Change in α_2-Macroglobulin Induced by Trypsin Under Second-Order Conditions: The Macroglobulin Acts Both as a Substrate and a Competitive Inhibitor of the Protease

Abstract

The reaction of bovine pancreatic trypsin with human plasma α₂-macroglobulin (α₂M) was studied at 25°C, using equimolar mixtures of E and I in 50 mM potassium phosphate buffer, pH 7. The conformational change in α₂M was monitored through the increase in protein fluorescence at 320 nm (exc λ, 280 nm). At [α₂M]₀ = [E]₀ = 11.5-200 nM, the fluorescence change data fit the integrated second-order rate equation, (F_∞ - F₀)/(F_∞ - F₁) = 1 + k_i,obsd [α₂M]₀t, indicating that cleavage of the bait region in α₂M was the rate-determining step.

The apparent rate constant (k_i,obsd) was found to be inversely related to reactant concentration. The kinetic behavior of the system was compatible with a model involving reversible, non-bait region binding of E to α₂M, competitively limiting the concentration of E available for bait region cleavage. The intrinsic value of k_i was (1.7±0.24) × 10⁷ M^-l s ^-1. K_p, the inhibitory constant associated with peripheral binding, was estimated to be in the submicromolar range.

The results of the present study point to a potential problem in interpreting kinetic data relating to protease-induced structural changes in macromolecular substrates. If there is nonproductive binding, as in the case of trypsin and α₂M, and the reactions are monitored under pseudo first-order conditions ([S]₀ ≫ [E]₀), an intrinsically second-order process (such as the rate-limiting bait region cleavage in α₂M) may become kinetically indistinguishable from an intrinsically first-order process (e.g. rate-limiting conformational change). Hence an excess of one component over the other should be avoided in kinetic studies addressing such systems.

Key Words:

• α₂-Macroglobulin
• Protease-induced conformational change
• Protein fluorescence