Kruszyna H, Kruszyna R, Rochelle L G, Smith R P, Wilcox D E
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755-3835.
Biochem Pharmacol. 1993 Jul 6;46(1):95-102. doi: 10.1016/0006-2952(93)90352-w.
Nitrovasodilators react with hemoglobin (Hb) to form heme(III) and nitric oxide (NO)Hb. These reactions can be exploited as models for events that take place at the cellular level leading to the biological effects of the prodrugs. Sodium nitroprusside (SNP) is known to undergo a one-electron reduction in its reaction with heme(II), resulting in the labilization of the cyanide ligand trans to the NO ligand. This reduced form is here called "penta." Upon dissociation of the trans-cyanide, the resulting species is here called "tetra." Dissociation of the trans-cyanide is obligatory for transfer of the NO to a heme(II) group. NO release from penta is blocked by excess free cyanide in solution, which prevents the formation of tetra. As reported here, both penta and tetra had unique EPR signals when frozen at -196 degrees, but only tetra gave an EPR signal at 22 degrees. NOHb also has a unique EPR signal, but it could not be detected when SNP was incubated with Hb in air or 10 or 5% oxygen. NOHb was detected in similar incubations under 1% oxygen, but the levels were 3- to 10-fold lower than those found under 100% nitrogen. The concentration of tetra was also much lower under 1% oxygen and penta was not detectable, suggesting that oxygen may either shift the penta-tetra equilibrium towards tetra or that penta may be susceptible to oxidation by molecular oxygen. Nitroglycerin (GTN) also generated much less NOHb but more heme(III) under 1% oxygen than under nitrogen. Carbon monoxide (CO), which binds to heme(II), completely blocked the reactions of SNP and GTN with Hb, whereas N-ethylmaleimide (NEM) alkylation of globin sulfhydryl groups increased both NOHb and heme(III) formation. 13C NMR studies on uniformly 13C-labeled SNP suggested that oxygen had little effect on the concentrations of the NMR-detectable species in the reaction. In summary, the most oxygen-sensitive step in the nitrosylation of Hb by SNP was probably the transfer of NO to heme(II). However, the penta-tetra equilibrium was affected by oxygen, temperature and cyanide. No evidence was found for the involvement of the globin sulfhydryl groups in either the GTN or the SNP reaction with Hb.
硝基血管扩张剂与血红蛋白（Hb）反应形成血红素（III）和一氧化氮（NO）血红蛋白。这些反应可被用作细胞水平上发生的事件的模型，这些事件导致前药产生生物学效应。已知硝普钠（SNP）在与血红素（II）反应时会发生单电子还原，导致与NO配体反位的氰化物配体不稳定。这种还原形式在这里称为“五价体”。反位氰化物解离后，产生的物种在这里称为“四价体”。反位氰化物的解离是将NO转移到血红素（II）基团所必需的。溶液中过量的游离氰化物会阻止五价体释放NO，从而防止四价体的形成。如本文报道，五价体和四价体在-196℃冷冻时都有独特的电子顺磁共振（EPR）信号，但只有四价体在22℃时给出EPR信号。NO血红蛋白也有独特的EPR信号，但当SNP在空气或10%或5%氧气中与Hb孵育时无法检测到。在1%氧气的类似孵育中检测到了NO血红蛋白，但水平比在100%氮气下低3至10倍。在1%氧气下四价体的浓度也低得多，且未检测到五价体，这表明氧气可能会使五价体-四价体平衡向四价体移动，或者五价体可能易被分子氧氧化。与氮气相比，在1%氧气下硝酸甘油（GTN）产生的NO血红蛋白也少得多，但产生的血红素（III）更多。与血红素（II）结合的一氧化碳（CO）完全阻断了SNP和GTN与Hb的反应，而球蛋白巯基的N-乙基马来酰亚胺（NEM）烷基化增加了NO血红蛋白和血红素（III）的形成。对均匀13C标记的SNP进行的13C核磁共振研究表明，氧气对反应中核磁共振可检测物种的浓度影响很小。总之，SNP使Hb亚硝基化过程中对氧气最敏感的步骤可能是NO转移到血红素（II）。然而，五价体-四价体平衡受氧气、温度和氰化物的影响。未发现有证据表明球蛋白巯基参与了GTN或SNP与Hb的反应。
