Determination of free hemoglobin in plasma based on its peroxidatic characteristic

Based on peroxdatic characteristic of hemoglobin, enzyme kinetic behavior was studied with eriochrome black T as hydrogen donor substrate. Reaction velocity depends upon hemoglobin and substrate concentrations, and a Michaelis-Menten K-m value and a V-m value were measured at pH 7.5. The mechanism that eriochrome black T was cleaved into aromicdiazonium ions by catalytic effect of hemoglobin was discussed. A catalytic-kinetics spectrophotometric method was proposed for the determination of hemoglobin. The initial rate of fad reaction of eriochrome black T at the wavelength of 615 nm was monitored, permitting a detection limit of 3.6 x 10(-9) mol/L hemoglobin. A linear calibration graph was obtained over the hemoglobin concentration range of 2 x 10(-8) similar to 2 x 10(-6) mol/L, and the relative standard deviation at a hemoglobin concentration of 7 x 10(-7) mol/L was 2.1% (n = 7). Satisfied results were obtained in the determination of hemoglobin in plasma samples by this method.