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Journal of Biochemistry Advance Access published online on July 23, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm133
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© 2007 The Japanese Biochemical Society

The minimal structural requirement of concanavalin A that retains its functional aspects

Ejaz Ahmad1, Aabgeena Naeem2, Saleem Javed3, Savita Yadav4 and Rizwan Hasan Khan1,*

1Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India
2Department of Biochemistry, Aligarh Muslim University, Aligarh, 202002, India
3Department of Biochemistry, Faculty of Science, Jamia Hamdard, New Delhi, 110062, India
4Department of Biophysics, All India Institute of Medical Sciences, NewDelhi, 100016,India

*Corresponding Author, Rizwan H. Khan, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh - 202 002, INDIA, E-mail: rizwanhkhan{at}hotmail.com, rizwanhkhan1{at}yahoo.comTel: + 91-571-2720388, Fax: + 91-571-2721776

Received May 29, 2007; Accepted June 3, 2007


  Abstract

A systematic investigation of the effects of several commonly used detergents on the conformation and function of concanavalin A at pH 7 in solution form was made by using circular dichroism (CD), intrinsic fluorescence, 1-anilino 8-sulfonic acid (ANS) binding, dynamic light scattering (DLS) and sugar inhibition assay. In the presence of 6.0 mM sodium dodecyl sulphate (SDS), an anionic detergent, and 0.8 mM cetyl tri methyl ammonium bromide (CTAB), a cationic detergent, intermediate states of concanavalin A were obtained having a negative CD peaks at 222 and 208 nm respectively, a characteristic of {alpha}-helix. These states also retained tertiary contacts with altered tryptophan environment and high ANS binding (exposed hydrophobic area) which can be characterized as molten globule states. Concanavalin A in the presence of 5.0 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent, and 0.07 mM brij-35, a nonionic detergent, also exists in intermediate states. These intermediates (molten globules) had high ANS binding with native like secondary (inherent ß-sheet) and tertiary structure. The intermediate states were characterized further by means of dynamic light scattering measurements and kinetic data. To study the possible functional requirement of the minimum structure, the intermediate states characterized in the presence of detergents were shown to retain the activity with polysaccharide (dextran). The pattern of activity observed was brij-35>CHAPS>CTAB>SDS. The specific binding and activity of concanavalin A with ovalbumin was investigated as a function of time by turbidity measurements. Cationic and anionic detergents showed significant effects on the structure of concanavalin A as compared to zwitterionic and nonionic detergents.

Key Words: Circular dichroism, concanavalin A, detergents, molten globule, percent residual activit


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