The development of an amperometric biosensor for the reduced glutathione determination in serum is described, The biosensor is based on glutathione peroxidase (GSH-Px, EC 1.11.1.9) immobilized onto a pyrolytic graphite-working electrode using carbodiimide as enzymatic condensing reagent. This resulted in an amperometric biosensor with good sensitivity and stability. The reduced glutathione (GSH) was enzymatically converted to glutathione disulfide (GSSG) in the presence of hydroperoxide. which was monitored amperometrically by its electrooxidation at +0.65 V vs. SCE (saturated calomel electrode). Glutathione measurement was carried out by maintaining the ratio between GSH and hydrogen peroxide at 2:1 (25 degreesC), The amperometric response of the biosensor was linearly proportional to the GSH concentration between 1.9 X 10(-5) and 1.4 X 10(-4) mol/l, in 0.1 mol/l phosphate buffer (pH = 7.8), containing 0.1 mol/l KCI and 0.5 mmol/l Na(2)H(2)EDTA, as the supporting electrolyte, In presence of interfering compounds, the recoveries ranged between 97.2% and 110.7%. The biosensor useful lifetime was at least 2 months when it was evaluated after continuous use. Serum samples analyzed by this biosensor showed a good correlation with the results from the spectrophotometric method (Ellman's reagent) used as reference, presenting relative deviations lower than 7.0%. The low apparent Michaelis-Menten constant value. K-M(app) = 1.6 mmol/l. demonstrated that GSH-Px immobilized on pyrolytic graphite exhibited a high affinity to GSH, without loss of enzymatic activity. (C) 2001 Elsevier Science B.V. All rights reserved.308416715567