Väänänen Antti J, Kankuri Esko, Rauhala Pekka
Institute of Biomedicine (Pharmacology), Biomedicum Helsinki, P.O. Box 63, University of Helsinki 00014, Finland.
Free Radic Biol Med. 2005 Apr 15;38(8):1102-11. doi: 10.1016/j.freeradbiomed.2005.01.007.
Protein oxidation, irreversible modification, and inactivation may play key roles in various neurodegenerative disorders. Therefore, we studied the effects of the potentially in vivo occurring nitric oxide-related species on two different markers of protein oxidation: protein carbonyl generation on bovine serum albumine (BSA) and loss of activity of a cysteine-dependent protease, papain, in vitro by using Angeli's salt, papanonoate, SIN-1, and S-nitrosoglutathione (GSNO) as donors of nitroxyl, nitric oxide, peroxynitrite, and nitrosonium ions, respectively. Angeli's salt, SIN-1, and papanonoate (0-1000 microM) all generated a concentration-dependent increase in carbonyl formation on BSA (107, 60, and 45%, respectively). GSNO did not affect carbonyl formation. Papain was inhibited by Angeli's salt, SIN-1, papanonoate, and GSNO with IC50 values of 0.62, 2.3, 54, and 80 microM, respectively. Angeli's salt (3.16 microM)-induced papain inactivation was only partially reversible, while the effects of GSNO (316 microM) and papanonoate (316 microM) were reversible upon addition of excess DTT. The Angeli's salt-mediated DTT-irreversible inhibition of papain was prevented by GSNO or papanonoate pretreatment, hypothetically through mixed disulfide formation or S-nitrosylation of the catalytically critical thiol group of papain. These results, for the first time, compare the generation of carbonyls in proteins by Angeli's salt, papanonoate, and SIN-1. Furthermore, these results suggest that S-nitrosothiols may have a novel function in protecting critical thiols from irreversible oxidative damage.
蛋白质氧化、不可逆修饰及失活可能在多种神经退行性疾病中起关键作用。因此，我们通过使用安吉利盐、对氨基苯磺酸、SIN-1和S-亚硝基谷胱甘肽（GSNO）分别作为硝酰基、一氧化氮、过氧亚硝酸盐和亚硝基离子的供体，研究了体内可能存在的一氧化氮相关物质对两种不同蛋白质氧化标志物的影响：牛血清白蛋白（BSA）上蛋白质羰基的生成以及半胱氨酸依赖性蛋白酶木瓜蛋白酶在体外的活性丧失。安吉利盐、SIN-1和对氨基苯磺酸（0 - 1000 microM）均使BSA上的羰基形成呈浓度依赖性增加（分别为107%、60%和45%）。GSNO不影响羰基形成。木瓜蛋白酶受到安吉利盐、SIN-1、对氨基苯磺酸和GSNO的抑制，其IC50值分别为0.62 microM、2.3 microM、54 microM和80 microM。安吉利盐（3.16 microM）诱导的木瓜蛋白酶失活仅部分可逆，而加入过量二硫苏糖醇（DTT）后，GSNO（316 microM）和对氨基苯磺酸（316 microM）的作用是可逆的。GSNO或对氨基苯磺酸预处理可防止安吉利盐介导的木瓜蛋白酶对DTT不可逆的抑制，推测是通过形成混合二硫键或木瓜蛋白酶催化关键巯基的S-亚硝基化。这些结果首次比较了安吉利盐、对氨基苯磺酸和SIN-1在蛋白质中羰基的生成情况。此外，这些结果表明S-亚硝基硫醇可能在保护关键巯基免受不可逆氧化损伤方面具有新功能。
