ISSN print edition: 0366-6352 ISSN electronic edition: 1336-9075 Registr. No.: MK SR 9/7 Published monthly

A validated method to assess glutathione peroxidase enzyme activity

Ahmed Yasser Ahmed, Saadon Abdulla Aowda, and Mahmoud Hussein Hadwan

Chemistry Dept, College of Science, University of Babylon, Hilla City, Iraq

E-mail: ahmedyassr51@gmail.com

Received: 11 June 2021  Accepted: 16 August 2021

Abstract:

Abstract

This article presents a reliable, effective and easy procedure for measuring glutathione peroxidase (Gpx) activity. Enzyme samples were incubated with phosphate buffer, which included the appropriate concentrations of glutathione (GSH) and peroxide as substrates, to determine Gpx activity. In the CUPRAC method, the CUPRAC reagent Cu(Nc)₂²⁺ was added to stop the enzymatic reaction after a sufficient period of incubation. The unreacted substrates acted to reduce the Cu(II)–neocuproine complex into the strongly coloured Cu(I)–neocuproine complex that could be measured spectrophotometrically at 450 nm. GPx activity was linked to a decrease in the absorbance of the coloured Cu(I)–neocuproine complex. The Box–Behnken design was used to optimise the formation of the Cu(I)–neocuproine complex. Response surface methodology was applied to determine the accuracy of the method. This new protocol was confirmed by performing the Bland–Altman plot analysis of Gpx activity in matched samples through the Gpx–DTNB assay. The correlation coefficient between the two protocols was 0.9967. This result indicated that the new protocol was very accurate and on par with the comparison method.

Graphic abstract

Keywords: Bland–Altman plot; Box–Behnken design; CUPRAC method; Glutathione; Response surface methodology

Full paper is available at www.springerlink.com.

DOI: 10.1007/s11696-021-01826-1

Chemical Papers 75 (12) 6625–6637 (2021)