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Compact Acid-Induced State of Clitoria ternatea Agglutinin Retains Its Biological Activity

A. Naeem1,2, M. Saleemuddin1,2, and R. H. Khan1*

1Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India; fax: +91-571-2721776; E-mail: rizwanhkhan@hotmail.com

2Department of Biochemistry, Life Science, AMU, Aligarh 202002, India

* To whom correspondence should be addressed.

Received January 5, 2009; Revision received March 25, 2009
The effects of pH on Clitoria ternatea agglutinin (CTA) were studied by spectroscopy, size-exclusion chromatography, and by measuring carbohydrate specificity. At pH 2.6, CTA lacks well-defined tertiary structure, as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of 50% native-like secondary structure. The mean residue ellipticity at 217 nm plotted against pH showed a transition around pH 4.0 with loss of secondary structure leading to the formation of an acid-unfolded state. This state is relatively less denatured than the state induced by 6 M guanidine hydrochloride. With a further decrease in pH, this unfolded state regains ~75% secondary structure at pH 1.2, leading to the formation of the A-state with native-like near-UV CD spectral features. Enhanced 8-anilino-1-naphthalene-sulfonate binding was observed in A-state, indicating a “molten-globule” like conformation with exposed hydrophobic residues. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a compact conformation at low pH. Size-exclusion chromatography shows the presence of a compact intermediate with hydrodynamic size corresponding to a monomer. Thermal denaturation of the native state was cooperative single-step transition and of the A-state was non-cooperative two-step transition. A-State regains 72% of the carbohydrate-binding activity.
KEY WORDS: acid-induced unfolding, Clitoria ternatea agglutinin, carbohydrate binding, molten-globule state, thermal stability

DOI: 10.1134/S0006297909100046