Binding Specificity and Possible Analytical Applications of the Cytokinin-binding Antibody, Anti-N6-Benzyladenosine

Publisher Website
Google Scholar
Abstract
Antibodies elicited in rabbits by immunization with an N6-benzyladenosine-bovine serum albumin conjugate bound N6-benzyladenosine specifically. The affinity constants and specific binding site concentrations for a number of cytokinins and related compounds were estimated by nonlinear least squares analysis of direct or competitive ultrafiltration data. The antisera contained 230-330 nmole of cytokinin binding sites/g protein. Affinity constants were 8.8 .times. 108 M-1 for 6-benzylaminopurine, 8.4 .times. 107 M-1 for kinetin, 9.1 .times. 107 M-1 for 6(3-methyl-2-butenylamino)purine, 6.6 .times. 106 M-1 for 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine and 2.0 .times. 104 M-1 for 6-methylaminopurine. Affinity constants were below the limit of detectability (< 104 M-1) for benzylamine, adenine and other adenine derivatives without an N6-side chain. The N6-substituent was thus immunodominant, but the purine moiety was also necessary for binding affinity. The antibodies were immobilized on cyanogen bromide-activated Sepharose with 95% retention of binding capacity and without apparent change in affinity constants. Columns of the immobilized antibody retained 64% of the [3H]6-(3-methyl-2-butenylamine)purine from 2 nM solutions and readily trapped [14C]6-benzylaminopurine that had been added to crude extracts of cabbage. Aqueous 10% pyridine adjusted to pH 7.3 with formic acid effectively eluted bound cytokinins from gel columns without loss of binding capacity of the immobilized antibody.