Glutaraldehyde cross-linking of lectins to marker enzymes: protection of binding site by specific sugars.
The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site of jacalin, which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti alpha-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins.
Appukuttan
P S
PS
Division of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India.
Chacko
B K
BK
Geetha
M
M
Annamma
K I
KI
Mathai
J
J
D000818:Animals; D001665:Binding Sites; D002241:Carbohydrates; D002417:Cattle; D003432:Cross-Linking Reagents; D005976:Glutaral; D006735:Horseradish Peroxidase; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D037121:Plant Lectins
D002241:Carbohydrates; D003432:Cross-Linking Reagents; D037102:Lectins; D037121:Plant Lectins; C043696:jacalin; D006735:Horseradish Peroxidase; D005976:Glutaral