Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening

Anal Biochem. 2016 Aug 1;506:31-44. doi: 10.1016/j.ab.2016.04.021. Epub 2016 May 9.

Abstract

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.

Keywords: Adenosine deaminase; Capillary electrophoresis; Inhibitor screening; Purine nucleoside phosphorylase; Red blood cells; Traditional Chinese medicines.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Deaminase / analysis*
  • Adenosine Deaminase / metabolism
  • Dose-Response Relationship, Drug
  • Electrophoresis, Capillary / methods*
  • Enzyme Inhibitors / analysis*
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Medicine, Chinese Traditional
  • Purine-Nucleoside Phosphorylase / analysis*
  • Purine-Nucleoside Phosphorylase / antagonists & inhibitors*
  • Purine-Nucleoside Phosphorylase / metabolism
  • Structure-Activity Relationship

Substances

  • Enzyme Inhibitors
  • Purine-Nucleoside Phosphorylase
  • Adenosine Deaminase