Kinetic enzyme assay for determining the CO2 content of body fluids using PEP carboxylase with inhibitor

摘要

An improved kinetic assay is provided for spectrophotometrically determining the dissolved carbon dioxide (CO2) content of a body fluid (e.g., blood, plasma or serum), wherein at about pH 8.0 the CO2 is present substantially as bicarbonate ion (HCO3-). The assay sample is first subjected to a coupled reaction mixture containing phosphoenolpyruvate (PEP) and phospho-enolpyruvate carboxylase (PEPC) which enzymatically catalyzes conversion of HCO3- to oxaloacetate (OA). The OA produced is subjected to a second coupled reaction mixture containing nicotinamide adenine dinucleotide (NADH) and malate dehydrogenase (MDH) which thereby reduces the OA to malate and oxidizes the NADH to AND+. The CO2 level in the body fluid sample may indirectly be determined by spectrophotometrically measuring the change in NADH concentration. According to the invention, an amount of an inhibitor is added to the reaction mixture which renders the reaction kinetics of the system essentially first order over the entire bicarbonate concentration range of interest. A useful inhibitor substantially satisfies a model represented by the monoexponential equation At=(Ao-A INFINITY )e-kt+A INFINITY [I] A sufficient quantity of the inhibitor is present to substantially cause the coupled reactions to follow the model equation [I] and yield a number which is within about +/-200% of the noise level of the spectrophotometer used for the assay when each absorbance value is applied to the equation <IMAGE> [II] Best results have been obtained through use of an inhibitor which provides an anion selected from the group consisting of ClO3-, ClO4-, SCN-, or I-.