Refolding of trimeresurus flavoviridis phospholipases A₂

Trimeresurus flavoviridis (Habu snake) venom contains phospholipase A₂ (PLA₂) isozymes which are rich in disulfide bond and show diverse activities in spite of their highly homologous structures. When reduced form of Tyr(NO₂)-67-PLA₂, which is a mimic of Asp-49-PLA₂ mutant, was oxidized at pH 8.0 in the presence of 5 mM L-cysteine and 5 mM Ca²⁺, native form of Tyr(NO₂)-67-PLA₂ was produced as being judged from both activity regeneration and adequate HPLC profile. Reduced forms of two Lys-49-PLA₂ isozymes with extremely low lipolytic activity, which are called basic proteins I and II, also generated native forms of proteins when oxidized under the same conditions as above. Protein disulfide isomerase accelerated proper folding of reduced Tyr(NO₂)-67-PLA₂ at an initial phase of oxidation and was effective for rearrangement of incorrectly paired disulfide bonds of Asp-49-PLA₂ to native construction. However, this isomerase failed to convert reduced form of partially active L-fragment, Asp-49-PLA₂ lacking N-terminal octapeptide, to the native form, indicating that the entire sequence of protein in a nascent state is primarily required for proper folding. The present data together with the fact that reduced Asp-49-PLA₂ folded properly (S. Tanaka et al. J. Biochem., 96, 1443 (1984)) afforded a strong basis for the structure and function study of T. flavoviridis PLA_2S by means of in vitro mutagenesis technique.