KINETIC ENZYME ASSAY FOR DETERMINING THE C02 CONTENT OF BODY FLUIDS USING PEP CARBOXYLASE WITH INHIBITOR

摘要

An improved kinetic assay is provided for spectrophotometrically determining dissolved carbon dioxide (CO2) in bodyfluid wherein at pH 8.0 the CO2 is present substantially as bicarbonate ion (HCO3). The sample is subjected to a reactionmixture containing phosphoenolpyruvate (PEP) and phosphoenolpyruvate carboxylase (PEPC) which enzymatically catalyzesconversion of HCO3 to oxaloacetate (OA). The OA produced is subject to a second reaction mixture containingNADH and malate dehydrogenase (MDH) which reduces the OA to malate and oxidizes the NADH to NAD +. The CO2level in the sample may be indirectly determined by measuring the change in NADH concentration. An inhibitor is added tothe reaction mixture which renders the reaction kinetics of the system essentially first order over the entire bicarbonateconcentration range of interest. Best results have been obtained through use of an inhibitor which provides an anion selectedfrom the group consisting of C10 3, C10 4, SCN, or I.