Caccia S, Denisov I, Perrella M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Biophys Chem. 1999 Jan 11;76(1):63-72. doi: 10.1016/s0301-4622(98)00219-1.
The kinetics of the reaction between NO and O2 was determined by measuring the time course of the decrease in the concentration of NO with a quench-flow technique. NO and O2 were mixed rapidly and reacted for periods of time varying from 10 to 50 s. A second rapid mixing with a solution containing an excess of deoxyhemoglobin and sodium hydrosulfite trapped free NO as nitrosylhemoglobin and reduced O2. The spectrum of the mixture of deoxy- and nitrosylhemoglobin was recorded within 30 s from the second mixing, before any appreciable dissociation of NO from the protein, by means of a flow-cell mounted on-line with the quench-flow apparatus. The amount of NO not consumed in the auto-oxidation reaction was calculated from the proportion of nitrosylhemoglobin in the mixture. As NO and O2 bind deoxyhemoglobin at comparable rates and NO is oxidized to nitrate by oxyhemoglobin, the ratio of hemoglobin/(NO + O2) had to be optimized to avoid the interference of this oxidation reaction. The kinetics was first and second order with respect to O2 and NO, respectively and third order overall with a rate constant k = 4 x kaq = 4 x 2.23 (+/- 0.26) x 10(6) M-2 s-1 at 20 degrees C, invariant in the pH range 7-9, in agreement with published values obtained by different methodologies.
通过采用猝灭流动技术测量一氧化氮（NO）浓度降低的时间进程，确定了NO与氧气（O₂）之间反应的动力学。将NO和O₂快速混合，并使其反应10至50秒不等的时间。与含有过量脱氧血红蛋白和亚硫酸氢钠的溶液进行第二次快速混合，将游离的NO捕获为亚硝基血红蛋白，并还原O₂。在第二次混合后的30秒内，在NO从蛋白质上发生任何明显解离之前，借助与猝灭流动装置在线连接的流通池记录脱氧血红蛋白和亚硝基血红蛋白混合物的光谱。根据混合物中亚硝基血红蛋白的比例计算出自氧化反应中未消耗的NO量。由于NO和O₂以相当的速率与脱氧血红蛋白结合，且NO会被氧合血红蛋白氧化为硝酸盐，因此必须优化血红蛋白/（NO + O₂）的比例，以避免这种氧化反应的干扰。该动力学分别对O₂和NO为一级和二级，总体为三级，在20℃时速率常数k = 4×kaq = 4×2.23（±0.26）×10⁶ M⁻² s⁻¹，在pH值7至9的范围内不变，这与通过不同方法获得的已发表值一致。
