Chemical Modification and Denaturation Effects on the Hemagglutinating Activity of Two Pterocarpus Species Seeds Lectins

Abstract

Aims: Pterocarpus osun and Pterocarpus soyauxii seeds lectins were subjected to various chemical modifications in order to detect the amino acid residues involved in their hemagglutinating and sugar-binding activities. Methodology: The lectins were purified using salt precipitation and size exclusion chromatography. Hemagglutinating activity and sugar specificity of the lectins were also established. Chemical modification of arginine was done using phenylglyoxal hydrate, and 5,5- dithiobis-(2-nitrobenzoic acid) (DTNB) was used to modify cysteine. Phenylmethylsulfonyl fluoride (PMSF) was employed for serine modification and tryptophan residue was modified with N-bromosuccinimide (NBS). Denaturants effects on the hemagglutinating activity were carried out with chaotropic agents, acid, disulphide bridge reducer and cross-linker agent. Results: Pterocarpus osun seeds lectin is mannose specific while Pterocarpus soyauxii seeds lectin is galactose/lactose-binding lectin. Hemagglutinating activities of the two lectins were completely lost when tryptophan residue was modified with NBS and the loss was reversed by dialysis. Modifications of Cysteine, Arginine and Serine have no effect on the hemagglutinating activity of P. osun lectin. Nevertheless, the modifications of same amino acids slightly reduced the activity of P. soyauxii lectin, which dialysis and prolonged incubation were able to overturn. Mannose was found to bind and inhibit hemagglutinating activity of P. osun lectin in the presence of various modifiers but galactose and lactose could not inhibit the hemagglutinating activity of P. soyauxii lectin in the presence of modifiers. All denaturants employed significantly affected the hemagglutinating activity of the two lectins. However, the effects were reversible except for P. osun lectin denatured with 8M urea. Conclusion: The results revealed that tryptophan residue is essential for hemagglutinating activity of the Pterocarpus species seeds lectins studied in this research. Cysteine, Arginine and Serine are also needed for sugar binding by P. soyauxii lectin but not so important in P. osun sugar binding ability.