Binding Mechanism Of Horseradish Peroxidase To Nitric Oxide And Its Application To Nitric Oxide Determination

In this paper, binding of nitric oxide (NO) to horseradish peroxidase (HRP) has been investigated by UV absorption spectrometry, and based on the reaction of HRP with NO, a novel detection method of NO has been developed with second-order derivative spectrometry.Binding of nitric oxide to HRP has been investigated by UV absorption spectrometry in 0.2mol/L anaerobic phosphate buffer solution (pH 7.4). Based on this binding equilibrium, a model equation for evaluating the binding constant of NO to HRP is developed and the binding constant is calculated to be (1.55±0.06)×10⁴L/mol, indicating that HRP can form a stable complex with nitric oxide. The type of inhibition by NO is validated on the basis of studying initial reaction rates of HRP-catalyzed oxidation of guaiacol in the presence of hydrogen peroxide and nitric oxide. The inhibition mechanism is found to follow an apparent non-competitive inhibition by Lineweaver–Burk method. Based on this kinetic mechanism, the binding constant is also calculated to be (5.22±0.06)×104L/mol. The values of the binding constant determined by the two methods are almost identical. This further demonstrated that the non-competitive inhibition mechanism suggested in the study was reasonable. The non-competitive inhibition model is also applicable to studying the effect of NO on other metalloenzymes, which catalyze the two-substrate reaction with the"ping-pong"mechanism.Based on the reaction of HRP with nitric oxide, a novel detection method of NO has been developed with second-order derivative spectrometry in an anaerobic phosphate buffer solution. The effects of pH and HRP concentration on the determination of NO in HRP system were investigated, and the conditions for the measurements were optimized. Some possible coexisting substances, such as nitrite, nitrate and hydrogen peroxide, were tested. The linear regression equation of standard curve was found to be h=8.89×10^-2CNO–1.56×10^-3 with the relevant coefficient of 0.9966 (n=5) in the NO concentration range of 0.085-1.3μM. The relative standard deviations were less than 3%. Based on the standard deviation of 5 bank measurements and a signal-to-noise ratio of 3, the detection limit for NO was 0.032μM. The proposed method was successfully applied to the determination of nitric oxide levels in serum samples.