O₂ solubility in aqueous media determined by a kinetic method

A kinetic method for the determination of O₂ solubility in air-saturated aqueous solutions of widely varying composition and temperature is described. It is based on the precise molar stoichiometry between the rates of uptake of H⁺ and O₂, measured with response-matched electrodes, in the reaction NADH + H⁺ + 1 2O₂ → NAD⁺ + H₂O, catalyzed by an NADH oxidase preparation. To the initially anaerobic test system, which contains an excess of NADH and NADH oxidase in a buffered medium, an aliquot of the O₂-containing solution to be tested is added and the rates of both O₂ uptake and H⁺ uptake are recorded; the H⁺ electrode is calibrated against standard HCl. From these data the amount of O₂ in the aliquot is calculated. Some representative values for O₂ solubility at 25°C and 760 mm in air-saturated systems are (i) distilled H₂O, 516 nmol O/ml, (ii) 0.15 m KCl, 480 nmol O/ml, and (iii) 0.25 m sucrose, 458 nmol O/ml. Data and equations are also given for the solubility of O₂ at 760 mm in air-saturated and lightly buffered 0.15 m KCl and 0.25 m sucrose over the range 5 to 40°C. In the method described the rates of O₂ and H⁺ uptake are precisely linear and stoichiometric when NADH is present in large excess over O₂. However, when O₂ is in excess and small additions of 340-nm-standardized NADH are made, as in earlier methods based on NADH oxidation, the endpoint is approached very gradually and tends to overestimate O₂ solubility, owing to (i) the higher K_m for NADH than for O₂, (ii) the relatively slow response of the Clark O₂ electrode, and (iii) the incomplete oxidation of NADH in the presence of 340-nm-absorbing inhibitory substances.