Study of the competitive inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine using capillary zone electrophoresis

摘要

This article describes how capillary zone electrophoresis was used to evaluate the inhibitory behavior of EHNA or erythro-9-(2-hydroxy-3-nonyi)adenine on the enzymatic deamination of adenosine by adenosine deaminase. The technique used for carrying out the assay was electrophoretically mediated micro analysis (EMMA). A fused-silica capillary was filled with phosphate buffer containing the inhibitor at a concentration that was adequate to inhibit the enzyme. After subsequent injection of the enzyme adenosine deaminase and the substrate adenosine, enzymatic reaction, electrophoretic separation and quantification of substrate and reaction product all took place inside the capillary. Reaction velocities were estimated from the peak area of the reactionproduct inosine. Michaelis-Menten and Lineweaver-Burk plots were constructed and the inhibitory constant K_i of EHNA was determined by comparison of the K_m of adenosine deaminase in the presence of inhibitor with the K_m in a solution without inhibitor.