Houston M, Chumley P, Radi R, Rubbo H, Freeman B A
Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL 35233-6810, USA.
Arch Biochem Biophys. 1998 Jul 1;355(1):1-8. doi: 10.1006/abbi.1998.0675.
Nitric oxide (.NO) and peroxynitrite (ONOO-) inhibit enzymes that depend on metal cofactors or oxidizable amino acids for activity. Since xanthine oxidase (XO) is a 2(2Fe2S) enzyme having essential sulfhydryl groups linked with Mo-pterin cofactor function, the influence of .NO and ONOO- on purified bovine XO was determined. Physiological (</=1 microM) and supraphysiological (</=100 microM) concentrations of dissolved .NO gas did not inhibit the catalytic activity or alter the spectral characteristics of XO at 25 degreesC and pH 7.0, differing from reports showing XO inhibition by .NO. The apparent decrease in XO activity observed previously was the result of depressed rates of uric acid accumulation in XO assay systems, due to ONOO--mediated oxidation of uric acid upon reaction of residual .NO with XO-derived superoxide (O*-2). Nitric oxide derived from S-nitrosoglutathione also did not inhibit cultured vascular endothelial cell XO activity. In contrast, purified and vascular endothelial cell catalase, a heme enzyme reversibly inhibited by .NO, was inhibited by similar concentrations and rates of production of . NO. In contrast to .NO, ONOO- inhibited XO (0.2 microM, 50 mU/ml) with an IC50 of 57 microM (for 3 microM/min infusion of ONOO-) or 120 microM (for bolus addition of ONOO-). Addition of 1% bovine serum albumin, 50 microM xanthine, or 10 microM uric acid protected XO from inactivation by ONOO-. Thus, in the presence of purine substrates and other more readily oxidized components of the biological milieu, XO should not be inhibited by either .NO or ONOO-. These observations reveal that .NO will not serve as an indirect antioxidant by inhibiting XO-derived production of reactive species and that the XO-derived products O*-2 and uric acid readily modify the reactivities of .NO and ONOO-.
一氧化氮（·NO）和过氧亚硝酸盐（ONOO⁻）会抑制那些依赖金属辅因子或可氧化氨基酸来发挥活性的酶。由于黄嘌呤氧化酶（XO）是一种具有与钼蝶呤辅因子功能相连的必需巯基的双（2Fe₂S）酶，因此测定了·NO和ONOO⁻对纯化的牛XO的影响。在25℃和pH 7.0条件下，生理浓度（≤1μM）和超生理浓度（≤100μM）的溶解·NO气体均未抑制XO的催化活性，也未改变其光谱特征，这与显示·NO抑制XO的报道不同。先前观察到的XO活性明显下降是由于XO检测系统中尿酸积累速率降低所致，这是由于残余的·NO与XO衍生的超氧阴离子（O₂⁻*）反应时，ONOO⁻介导了尿酸的氧化。源自S - 亚硝基谷胱甘肽的一氧化氮也未抑制培养的血管内皮细胞的XO活性。相比之下，纯化的和血管内皮细胞过氧化氢酶是一种可被·NO可逆抑制的血红素酶，受到类似浓度和产生速率的·NO的抑制。与·NO不同，ONOO⁻以57μM的IC₅₀（对于以3μM/分钟的速度注入ONOO⁻）或120μM（对于一次性添加ONOO⁻）抑制XO（0.2μM，50 mU/ml）。添加1%的牛血清白蛋白、50μM黄嘌呤或10μM尿酸可保护XO不被ONOO⁻灭活。因此，在存在嘌呤底物和生物环境中其他更容易氧化的成分的情况下，XO不应被·NO或ONOO⁻抑制。这些观察结果表明，·NO不会通过抑制XO衍生的活性物种的产生而作为间接抗氧化剂，并且XO衍生的产物O₂⁻*和尿酸很容易改变·NO和ONOO⁻的反应活性。
