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Rapid method to separate the domains of soybean lipoxygenase-1: identification of the interdomain interactions.

Sudharshan, E and Rao, A. G. A. (1997) Rapid method to separate the domains of soybean lipoxygenase-1: identification of the interdomain interactions. FEBS letters, 406 (1-2). pp. 184-8. ISSN 0014-5793

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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.

Item Type: Article
Uncontrolled Keywords: Lipoxygenase-1 domain separation domain interaction surface hydrophobicity circular dichroism fluoroscence
Subjects: 600 Technology > 08 Food technology > 22 Legumes-Pulses > 05 Soya bean
500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 27 Nov 2008 05:32
Last Modified: 30 Jun 2015 12:49
URI: http://ir.cftri.com/id/eprint/2218

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