Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.
The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.
D001120:Arginine; D001665:Binding Sites; D002241:Carbohydrates; D002942:Circular Dichroism; D006386:Hemagglutination Tests; D006639:Histidine; D005779:Immunodiffusion; D007785:Lactose; D037102:Lectins; D008239:Lysine; D037121:Plant Lectins; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014443:Tyrosine
D002241:Carbohydrates; D037102:Lectins; D037121:Plant Lectins; C016940:abrus agglutinin; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D001120:Arginine; D007785:Lactose; D008239:Lysine