Effect of amino acid residue and oligosaccharide chain chemical modifications on spectral and hemagglutinating activity of Millettia dielsiana Harms. ex Diels. lectin

• Published in: Acta biochimica et biophysica Sinica 2005-01, Vol.37 (1), p.47-54
• Main Authors: Gao, Shun, An, Jie, Wu, Chuan-Fang, Gu, Ying, Chen, Fang, Yu, Yuan, Wu, Qia-Qing, Bao, Jin-Ku
• Format: Article
• Language: English
• Subjects: Acrylamide - chemistry; Amino Acids - chemistry; Arginine - chemistry; Binding Sites; Carbohydrates - chemistry; Circular Dichroism; Hemagglutination; Mannose - chemistry; Millettia - metabolism; Oligosaccharides - chemistry; Oxygen - metabolism; Plant Lectins - chemistry; Protein Conformation; Spectrometry, Fluorescence; Spectrophotometry - methods; Tryptophan - chemistry; Ultraviolet Rays
• ISSN: 1672-9145
• DOI: 10.1093/abbs/37.1.47

The effects of modifying the carbohydrate chain and amino acids on the conformation and activity of Millettia dielsiana Harms. ex Diels. lectin (MDL) were studied by hemagglutination, fluorescence and circular dichroism analysis. The modification of tryptophan residues led to a compete loss of hemagglutinating activity; however, the addition of mannose was able to prevent this loss of activity. The results indicate that two tryptophan residues are involved in the carbohydrate-binding site. Modifications of the carboxyl group residues produced an 80% loss of activity, but the presence of mannose protected against the modification. The results suggest that the carboxyl groups of aspartic and glutamic acids are involved in the carbohydrate-binding site of the lectin. However, oxidation of the carbohydrate chain and modification of the histidine and arginine residues did not affect the hemagglutinating activity of MDL. Fluorescence studies of MDL indicate that tryptophan residues are present in a relatively hydrophobic region, and the binding of mannose to MDL could quench tryptophan fluorescence without any change in lambda(max). The circular dichroism spectrum showed that all of these modifications affected the conformation of the MDL molecule to different extents, except the modification of arginine residues. Fluorescence quenching showed that acrylamide and iodoacetic acids are able to quench 77% and 98% of the fluorescence of tryptophan in MDL, respectively. However, KI produced a barely perceptible effect on the fluorescence of MDL, even when the concentration of I(-) was 0.15 M. This demonstrates that most of tryptophan residues are located in relatively hydrophobic or negatively charged areas near the surface of the MDL molecule.