Analytical Biochemistry 1996-09-05

A novel method for chemical modification of functional groups other than a carboxyl group in proteins by N-ethyl-5-phenylisooxazolium-3'-sulfonate (Woodward's reagent-K): inhibition of ADP-induced platelet responses involves covalent modification of aggregin, an ADP receptor.

R N Puri, R W Colman

Index: Anal. Biochem. 240(2) , 251-61, (1996)

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Abstract

The chemical reaction of N-ethyl-5-phenylisooxazolium-3'-sulfonate (Woodward's Reagent-K, WR-K) with a carboxyl group yields an enol ester that cannot be reduced by sodium borohydride in an aqueous solution, while other nucleophiles such as sulfhydryl, hydroxyl, amino, and imidazole groups, react with WR-K to yield unsaturated ketones that are capable of being reduced by sodium borohydride in an aqueous medium. Aggregin, a 100-kDa protein on the surface of human blood platelets has been identified as an ADP receptor. Autoradiography of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the samples of solubilized human blood platelets modified by WR-K and then reduced by tritiated sodium borohydride (NaB[3H]4) showed the presence of a prominent band corresponding to a 100-kDa radiolabeled protein. Labeling of platelets by WR-K and NaB[3H]4 was inhibited by ADP, ATP, and thiol group modifying reagents. WR-K blocked completely labeling of platelets by [beta-32P]-8-(4-bromo-2, 3-dioxo-butylthio)adenosine-5'-diphosphate, an ADP-affinity analog that selectively and covalently labels aggregin (Puri, R. N., Kumar, A., Chen, H., Colman, R. F., and Colman, R. W. (1995) J. Biol. Chem. 256, 24482-24488). WR-K also inhibited ADP-induced platelet shape change, aggregation, and mobilization of intracellular Ca2+ and blocked ADP-induced inhibition of stimulated adenylate cyclase activity. The results show conclusively that WR-K inhibited ADP-induced platelet responses by preventing binding of ADP to aggregin and suggest that ADP binding domain of aggregin contains an essential thiol group. The method of labeling proteins by WR-K and NaB[3H]4, hitherto not used to distinguish among functional groups modified by WR-K, offers a useful and convenient alternative to previously used ultraviolet spectral methods which cannot be used to investigate the modified proteins in intact cellular systems.