Purification and physicochemical characterization of two galactose-specific isolectins from the seeds of Trichosanthes cordata

✍ Scribed by Nabil Ali Mohammed Sultan; M. Kavitha; Musti J. Swamy

• Publisher: John Wiley and Sons
• Year: 2009
• Tongue: English
• Weight: 360 KB
• Volume: 61
• Category: Article
• ISSN: 1521-6543
• DOI: 10.1002/iub.174

✦ Synopsis

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% α‐helix, 37–38% β‐sheet, 20% β‐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°C, but decreased dramatically above 40°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 × 10^3^ M^−1^ at 25°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. © 2009 IUBMB IUBMB Life, 61(4):457–469, 2009