RAPID METHOD TO SEPARATE THE DOMAINS OF SOYBEAN LIPOXYGENASE-1 - IDENTIFICATION OF THE INTERDOMAIN INTERACTIONS

Lipoxygenase-1 (LOX1) from soybeans was cleaved with chymotrypsin (Ram achandran ct al., 31 (1992) 7700-7706). The domains were separated on a Sephadex G-50 column by minimising domain interactions at pH 4.0. Th e molecular weight and apparent homogeneity of the domains were establ ished by SDS-PAGE, The solution conformation of the 60 kDa and 30 kDa fragments,vas compared with that of native LOX1, 1-Anilino-8-naphthale ne sulphonate (ANS) binding measurements confirmed the exposure of lar ge hydrophobic residues on the surface of the 60 kDa due to separation of the domains. The monomeric nature of the 60 kDa fragment was confi rmed by HPLC gel filtration, The increased number of binding sites and magnitude of binding constant suggested the involvement of extensive hydrophobic interactions between the two domains, The essential cofact or iron was with the C-terminal domain, The attempts to resolve and re constitute the catalytic activity of isolated domains were not success ful. (C) 1997 Federation of European Biochemical Societies.