Dissociation of pig liver carboxylesterase measured by quantitative micro-complement fixation

Abstract

Rate and apparent equilibrium constants for the dissociation of pig liver carboxylesterase into three subunit molecules have been determined by complement fixation. The dependence of the dissociation equilibria on pH are consistent with dissociation reactions involving the addition of two protons per subunit, a pH-independent dissociation, and a dissociation upon the loss of one proton per subunit. The rate constants for dissociation are consistent with terms first order in hydrogen and hydroxide ions and a pH-independent path. The equilibrium constants in the range 3–35 °C at pH 7.2 exhibit no dependence on temperature; the association reaction is entropy driven with ΔS = 68 cal mol −1 ∘ K −1 . The rate constants for the pH-independent dissociation follow ΔH ≠ ≅ 6 kcal mol −1. The order of effectiveness of concentrated salts in promoting denaturation is correlated with their effect on the activity coefficient of acetyltetraglycine ethyl ester and suggests that peptide groups become more exposed upon dissociation. The increased dissociation in the presence of urea derivatives containing alkyl substituents suggests exposure of hydrophobic regions upon dissociation; this is also consistent with ΔH = 0 for dissociation. It is likely that hydrophobic interactions contribute to the stability of the trimeric whole molecule.