An efficient strategy for quickly determining adenosine deaminase and purine nucleoside phosphorylase kinetics and screening their inhibitors.

Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) are of great significance in the study of the fine regulation of purine metabolism. It will provide an important basis for the study of the pathogenesis of immunodeficiency diseases, leukemia and other diseases related to purine metabolism abnormality, as well as for the target of drug discovery and development. Here, a new online capillary electrophoresis and post-calculation strategy was proposed for the quick determination of adenosine deaminase and purine nucleoside phosphorylase kinetic constant and their inhibitors screening. With unique injection procedure, special separation method and innovative calculation strategy, the enzyme kinetics could be obtained through combing the product curves containing enzyme reaction kinetics in a CE run. Furthermore, the weak product signal and the difficult separation issues for inosine, adenosine and hypoxanthine under the neutral online enzyme reaction conditions were effectively solved for the first time by the adjusted gravity mediated-capillary electrophoresis (AGM-CE). Compared with the traditional offline method, the proposed strategy was reliable and efficient. It also was successfully used in inhibitor screening of adenosine deaminase and purine nucleoside phosphorylase. Results proved that the new strategy had great potential in enzyme analysis and inhibitor screening.