Chemical denaturation of a homodimeric lysine-49 phospholipase A2: a stable dimer interface and a native monomeric intermediate

Bothropstoxin I (4BthTx-I) is a homodimeric lysine-49 (Lys49) phospholipase A2 isolated from Bothrops jararacussu venom, which damages liposome membranes via a Ca2+-independent mechanism. The stability of the BthTx-I homodimer was evaluated by equilibrium chemical denaturation with guanidinium hydrochloride monitored by changes in the intrinsic tryptophan fluorescence anisotropy, far-UV circular dichroism, dynamic light scattering, and 1-anilinonaphthalene-8-sulfonate binding. Unfolding of the BthTx-I dimer proceeds via a monomeric intermediate with native-like structure, with Gibbs free energy (ΔG0) values of 10.0 and 7.2 kcal mol−1 for the native dimer-to-native monomer and native-to-denatured monomer transitions, respectively. The experimentally determined ΔG0 value for the dimer-to-native monomer transition is higher than the value expected for an interaction dominated by hydrophobic forces, and suggests that an unusually high propensity of hydrogen-bonded side chains found at the BthTx-I homodimer interface make a significant contribution to dimer stability.