Refolding of trimeresurus flavoviridis phospholipases A2

Shinji Tsuno, Tomohisa Ogawa, Kin Ichi Nakashima, Naoko Oda, Sannumu Lee, Yasuyuki Shimohigashi, Haruhiko Aoyagi, Motonori Ohno

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Abstract

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 PLA2S by means of in vitro mutagenesis technique.

Original languageEnglish
Pages (from-to)2655-2659
Number of pages5
JournalBulletin of the Chemical Society of Japan
Volume65
Issue number10
DOIs
Publication statusPublished - Oct 1 1992

All Science Journal Classification (ASJC) codes

  • Chemistry(all)

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    Tsuno, S., Ogawa, T., Nakashima, K. I., Oda, N., Lee, S., Shimohigashi, Y., Aoyagi, H., & Ohno, M. (1992). Refolding of trimeresurus flavoviridis phospholipases A2. Bulletin of the Chemical Society of Japan, 65(10), 2655-2659. https://doi.org/10.1246/bcsj.65.2655