title: Conformational analysis of champedak galactose-binding lectin under different urea concentrations.
abstract: Conformational analysis of champedak galactose-binding (CGB) lectin under different urea concentrations was studied in phosphate-buffered saline (pH 7.2) using far-ultraviolet circular dichroism (far-UV CD), tryptophan (Trp) fluorescence and ANS fluorescence. In all cases, CGB lectin displayed a two-step, three-state transition. The first transition (from the native state to the intermediate state) started at ∼2.0 M urea and ended at ∼4.5 M urea, while the second transition (from the intermediate state to the completely denatured state) was characterized by the start- and end-points at ∼5.75 M and ∼7.5 M urea, respectively, when analyzed by the emission maximum of Trp fluorescence. A marked increase in the Trp fluorescence, ANS fluorescence and -CD values at 218 nm (-CD218 nm) represented the first transition, whereas a decrease in these parameters defined the second transition. On the other hand, emission maximum of the Trp fluorescence showed a continuous increase throughout the urea concentration range. Transformation of tetramer into monomer represented the first transition, whereas the second transition reflected the unfolding of monomer. Far-UV CD, Trp fluorescence and ANS fluorescence spectra were used to characterize the native, the intermediate and the completely denatured states of CGB lectin, obtained at 0.0 M, 5.0 M and 9.0 M urea, respectively. The intermediate state was characterized by the presence of higher secondary structures, increased ANS binding as well as increased Trp fluorescence intensity. A gradual decrease in the hemagglutination activity of CGB lectin was observed with increasing urea concentrations, showing complete loss at 4.0 M urea.
authors.affiliation: Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia. Electronic address: imankameel@gmail.com.
authors.forename: Nurul Iman Ahamed
authors.initials: NI
authors.lastname: Kameel
authors.affiliation: Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia. Electronic address: zack_wong123@yahoo.com.
authors.forename: Yin How
authors.initials: YH
authors.lastname: Wong
authors.affiliation: Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia. Electronic address: adawiyah@um.edu.my.
authors.forename: Adawiyah Suriza
authors.initials: AS
authors.lastname: Shuib
authors.affiliation: Biomolecular Research Group, Biochemistry Programme, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia. Electronic address: saadtayyab2004@yahoo.com.
authors.forename: Saad
authors.initials: S
authors.lastname: Tayyab
mesh_terms: D031622:Artocarpus / Q000737:chemistry*; D002942:Circular Dichroism; D005453:Fluorescence; D037161:Galectins / Q000737:chemistry* / Q000187:drug effects; D006384:Hemagglutination; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology*
chemical_list: D037161:Galectins; D014508:Urea
keywords: Champedak galactose-binding lectin; Conformational analysis; Far-UV CD; Hemagglutination; Tryptophan fluorescence; Urea denaturation