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Acid-Induced Unfolding of Champedak Galactose-Binding Lectin

[ Vol. 23 , Issue. 12 ]

Author(s):

Nurul Iman A. Kameel, Adawiyah S. Shuib and Saad Tayyab   Pages 1111 - 1117 ( 7 )

Abstract:


Acid denaturation of champedak galactose-binding (CGB) lectin was studied in the pH range, 7.0−1.0 using intrinsic fluorescence and ANS fluorescence measurements. The lectin remained stable up to pH 5.0 and showed local disordering in the vicinity of the protein fluorophores within the pH range, 5.0−3.5. Decrease in the pH from pH 3.5 to pH 2.5 led to structural transition, marked by the decrease in the intrinsic fluorescence and increase in the ANS fluorescence signals. This can be ascribed to the dissociation of the tetrameric lectin into monomeric forms. Further decrease in the pH up to pH 1.5 produced another transition, which specified the unfolding of monomers as reflected from the decrease in both intrinsic fluorescence and ANS fluorescence signals. Characterization of the conformational states obtained at pH 7.0, pH 2.5 and pH 1.5 based on intrinsic and ANS fluorescence spectra, gel chromatographic behavior and thermal denaturation confirmed the existence of folded monomeric forms at pH 2.5 and unfolded states at pH 1.5. However, the aciddenatured state of CGB lectin at pH 1.5 retained significant residual structure, as evident from the greater loss of both secondary and tertiary structures in the presence of 6 M guanidine hydrochloride at low pH values. Anion-induced refolding below pH 1.5 was also seen using ANS fluorescence measurements.

Keywords:

Acid denaturation, champedak galactose-binding lectin, intrinsic fluorescence, ANS fluorescence, far-UV circular dichroism, size exclusion chromatography, thermal denaturation.

Affiliation:

Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia.

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