UV-INDUCED CROSS-LINKING OF ABSCISIC-ACID TO BINDING-PROTEINS

Conditions for UV-induced cross-linking of abscisic acid (ABA) through its enone chromophore to binding proteins were evaluated. The effects of a UV-light band between 260 and 530 nm on both unconjugated and pr otein-conjugated ABA, as well as on anti-ABA antibodies as models of A BA-binding proteins were determined. UV irradiation caused both isomer ization and photolysis of ABA, but increasing the lower irradiation bo undary to 345 nm strongly reduced photolysis and largely prevented iso merization. When conjugated to alkaline phosphatase (AP), ABA remained stable when using either a 320 or a 345 nm filter. At these wavelengt hs both binding of ABA to antibodies as well as AP enzymatic activity were maintained. UV-induced cross-linking of monoclonal anti-ABA antib odies to immobilized ABA was analysed by immunoassays. Optimal cross-l inking was achieved after a 5 min irradiation period at 0 degrees, usi ng a long pass, cut-on filter to quench wavelengths below 290 nm. This cross-linking faithfully reflected cognate binding activity.