Product/Service

BIOXYTECH GPx-340 Colorimetric Assay for Cellular Glutathione Peroxidase

Source: OXIS Research a division of OXIS Health Products, Inc.
Immediately upon receipt, theNADPH Reagent should be stored at less than or equal to -10°C
Component Storage:

Immediately upon receipt, theNADPH Reagent should be stored at less than or equal to -10°C. Unopened vials are stable until expiration if kept at -10°C or below. The remainder of the reagents should be stored at 2-8°C and are stable unopened until expiration date.

Introduction:

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 (Ursini, 1995). 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-O-H) 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 (Ursini, 1995). The GPx enzymes accept a wide variety of organic peroxides as substrates. However, with the exception of phospholipid hydroperoxide GPx and perhaps pl·GPx, 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.

4. Thaw GPx samples and place on ice.

5. Bring all reagents to assay temperature (recommended 23-25°C).

6. Turn on spectrophotometer, set to measure absorbance at 340 nm and set the assay temperature (recommended 23-25°C).

7. Zero the spectrophotometer at 340 nm using deionized water.

For Research Use Only