Description
Introduction: The Analyte
Cellular glutathione peroxidase (c-GPx, EC 1.11.1.9) is a member of a family of GPx enzymes whose function is to detoxify peroxides in the cell.1 Because peroxides can decompose to form highly reactive radicals, the GPx enzymes play a critical role in protecting the cell from free radical damage, particularly lipid peroxidation. The GPx enzymes catalyze the reduction of H2O2 to water and organic peroxides (R-O-O-H) to the corresponding stable alcohols (R-OH) using glutathione (GSH) as a source of reducing equivalents:
With the exception of phospholipid-hydroperoxide GPx, a monomer, all of the GPx enzymes are comprised of 4 identical subunits (monomer Mr 22-23 kDa). Each subunit contains a molecule of selenocysteine in the enzyme active site. The selenocysteine is thought to participate directly in electron donation to the peroxide substrate and become oxidized in the process. The enzyme then uses glutathione as an electron donor to regenerate the reduced form of the selenocysteine.1 The GPx enzymes accept a wide variety of organic peroxides as substrates. However, with the exception of phospholipid hydroperoxide GPx and perhaps plGPx, the enzymes exhibit a strong preference for glutathione as a source of reducing equivalents. Phospholipid-hydroperoxide GPx (Mr 19 kDa) is the only enzyme with significant activity on esterified phospholipids and cholesterol in membranes.
Principles Of The Procedure
This assay is an indirect measure of the activity of c-GPx.7 Oxidized glutathione (GSSG), produced upon reduction of an organic peroxide by c-GPx, is recycled to its reduced state by the enzyme glutathione reductase (GR):
The oxidation of NADPH to NADP + is accompanied by a decrease in absorbance at 340 nm (A340) providing a spectrophotometric means for monitoring GPx enzyme activity. The molar extinction coefficient for NADPH is 6220 M-1 cm-1 at 340 nm. To assay c-GPx, a cell or tissue homogenate is added to a solution containing glutathione, glutathione reductase, and NADPH. The enzyme reaction is initiated by adding the substrate, tert-butyl hydroperoxide and the A340 is recorded. The rate of decrease in the A340 is directly proportional to the GPx activity in the sample