title: Rapid method to separate the domains of soybean lipoxygenase-1: identification of the interdomain interactions.
abstract: Lipoxygenase-1 (LOX1) from soybeans was cleaved with chymotrypsin (Ramachandran et al., 31 (1992) 7700-7706). The domains were separated on a Sephadex G-50 column by minimising domain interactions at pH 4.0. The molecular weight and apparent homogeneity of the domains were established by SDS-PAGE. The solution conformation of the 60 kDa and 30 kDa fragments was compared with that of native LOX1. 1-Anilino-8-naphthalene sulphonate (ANS) binding measurements confirmed the exposure of large hydrophobic residues on the surface of the 60 kDa due to separation of the domains. The monomeric nature of the 60 kDa fragment was confirmed 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 cofactor iron was with the C-terminal domain. The attempts to resolve and reconstitute the catalytic activity of isolated domains were not successful.
authors.affiliation: Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore, India.
authors.forename: E
authors.initials: E
authors.lastname: Sudharshan
authors.forename: A G
authors.initials: AG
authors.lastname: Rao
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry; D006868:Hydrolysis; D008084:Lipoxygenase / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D013025:Glycine max / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D010446:Peptide Fragments; C027132:1-anilino-8-naphthalenesulfonate; D008084:Lipoxygenase