Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human α₂-macroglobulin, rat α₁-inhibitor-3 and chemically modified derivatives

The α-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal β-cysteinyl–γ-glutamyl thiol ester bonds are an important structural feature of most α-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH₃ or CH₃NH₂ in tetrameric human α₂-macroglobulin (α₂M) and monomeric rat α₁-inhibitor-3 (α₁ I₃). When employing NH₃ as the nucleophile, the thiol ester in α₁I₃ re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH₃NH₂ was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, α₂M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between α₂M and NH₃ or CH₃NH₂ was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for α₂M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native α₂M is restored when the proteinase-inhibitory capacity is recovered.