title: Purification and physicochemical characterization of two galactose-specific isolectins from the seeds of Trichosanthes cordata.
abstract: A galactose-specific lectin has been purified from the seeds of Trichosanthes cordata by affinity chromatography on crosslinked guar gum. The affinity-eluted lectin could be resolved into two isolectins, TCA-I and TCA-II by ion-exchange chromatography on DEAE cellulose. The molecular weights of the isolectins were determined as 59 and 52 kDa by SDS-PAGE. TCA-I is a heterodimer in which the two subunits with masses of 32 and 27 kDa, are covalently connected by disulfide bonds. TCA-I and TCA-II are glycoproteins with 6.2% and 6.8% covalently bound neutral sugar, respectively. CD spectroscopic studies indicate that the two isolectins are very similar in secondary structure and contain about 8 to 10% alpha-helix, 37-38% beta-sheet, 20% beta-turns, and 32-33% unordered structures. These isolectins have similar carbohydrate specificities as revealed by hemagglutination-inhibition assays. Carbohydrate specificity, subunit size and composition, and secondary structure of TCA isolectins suggest close similarity to type-II ribosome inactivating proteins. The agglutination activity of TCA-I was found to be highest in the pH range 7.0-8.0. The lectin activity was unaffected between 0 and 40 degrees C, but decreased dramatically above 40 degrees C. Association constant for the interaction of TCA-I with lactose was determined by monitoring ligand-induced changes in the protein intrinsic fluorescence characteristics as 7.42 x 10(3) M(-1) at 25 degrees C. The exposure and accessibility of the tryptophan residues of TCA-I and the effect of ligand binding on them have been probed by quenching studies employing neutral and ionic quenchers.
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad, India.
authors.forename: Nabil Ali Mohammed
authors.initials: NA
authors.lastname: Sultan
authors.forename: M
authors.initials: M
authors.lastname: Kavitha
authors.forename: Musti J
authors.initials: MJ
authors.lastname: Swamy