Some hints to avoid pitfalls in quantitative determination of glutathione peroxidase (EC 1.11.1.9).

Methods of quantitative determination of glutathione peroxidase were reinvestigated with respect to reliability and specificity. The determination of product (GSSG) by a coupled enzymatic oxidation of NADPH, as described by PAGLIA and VALENTINE, had to be modified as follows: 1. The concentration of GSH was lowered to a maximum of 1 mM in the incubation mixture to avoid product inhibition of GSSG reductase. 2. The concentration of H2O2 was increased to guarantee a sufficient amount of substrate to survive the lag phase of the coupled enzymatic test. The pretreatment of the samples proposed by PAGLIA and VALENTINE (13) was föund'to be insufficient for obtaining exact data on GSH peroxidase activity as all hemoglobin derivatives tested, including cyanmethemoglobin, caused a significant unspecific oxidation of GSH by H2O2. The determination of GSH peroxidase activity described by SCHNEIDER and FLOHE (3) as well as their procedure to purify the enzyme prior to measurement was found to be reliable and adequately reproducible. In view of the lower apparative requirements, however, the procedure of PAGLIA and VALENTINE offers a more suitable clinical screening test if the possible errors mentioned below are considered. The influence of the medium on GSH peroxidase activity was investigated. The definitions of the enzyme unit are discussed with respect to the kinetic behaviour of GSH peroxidase. Our unit of activity is defined as the amount of enzyme dissolved in 1 m/ which effects a difference of 1 in the logarithms of the GSH concentrations per minute.