title: Binding Specificity and Possible Analytical Applications of the Cytokinin-binding Antibody, Anti-N-Benzyladenosine.
abstract: Antibodies elicited in rabbits by immunization with an N(6)-benzyladenosine-bovine serum albumin conjugate bound N(6)-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 to 330 nanomoles of cytokinin binding sites per gram protein. Affinity constants were 8.8 x 10(8) molar(-1) for 6-benzylaminopurine, 8.4 x 10(7) molar(-1) for kinetin, 9.1 x 10(7) molar(-1) for 6-(3-methyl-2-butenylamino)purine, 6.6 x 10(6) molar(-1) for 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine, and 2.0 x 10(4) molar(-1) for 6-methylaminopurine. Affinity constants were below the limit of detectability (<10(4) molar(-1)) for benzylamine, adenine, and other adenine derivatives without an N(6)-side chain. The N(6)-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 [(3)H]6-(3-methyl-2-butenylam-ino)purine from 2 nanomolar solutions and readily trapped [(14)C]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.
authors: Department of Forestry, University of Wisconsin, Madison, Wisconsin 53706.
authors: H A
authors: HA
authors: Constantinidou