Perrin Caroline, Vergely Catherine, Zeller Marianne, Rochette Luc
Laboratoire de Physiopathologie et Pharmacologie Cardiovasculaires Expérimentales, Facultés de Médecine et Pharmacie, Dijon, France.
Cell Mol Biol (Noisy-le-grand). 2002;48 Online Pub:OL267-70.
The aim of this study was 1/ to assess the efficacy of calpain inhibitors: leupeptin and calpain inhibitor-1, to inhibit calpain activity in vitro, 2/ to measure the scavenging abilities of these compounds against free radicals. The efficacy of calpain inhibitors to block calpain activity was tested with azocasein as a substrate for calpain. Leupeptin and calpain inhibitor-1 inhibited calpain activity in the same range of concentrations, the IC50 being 0.14 microM and 0.09 microM, respectively. We measured the antioxidant properties of leupeptin and calpain inhibitor-1 using the allophycocyans assay after identification offree radical species produced by the complex H2O2 + Cu(++). Electron paramagnetic resonance (EPR) spin-trapping studies performed by using DMPO showed that a quartet signal (hyperfine couplings aN = aH = 14.9 G) arisen from DMPO-OH was formed. We found a correlation between leupeptin concentration and oxygen radical absorbance capacity (ORAC) values (r2 = 0.975) indicating an in vivo antioxidant capacity. In contrast, calpain inhibitor-1 showed no protection. In conclusion, our findings indicate that leupeptin and calpain inhibitor-1 are equipotent inhibitors on calpain activity but exhibit diffrent antioxidant efficacy.
1/评估钙蛋白酶抑制剂（亮抑蛋白酶肽和钙蛋白酶抑制剂-1）在体外抑制钙蛋白酶活性的功效；2/测定这些化合物清除自由基的能力。以偶氮酪蛋白作为钙蛋白酶的底物，测试钙蛋白酶抑制剂阻断钙蛋白酶活性的功效。亮抑蛋白酶肽和钙蛋白酶抑制剂-1在相同浓度范围内抑制钙蛋白酶活性，其半数抑制浓度（IC50）分别为0.14微摩尔和0.09微摩尔。在鉴定出由H2O2 + Cu(++)复合物产生的自由基种类后，我们使用别藻蓝蛋白测定法测量了亮抑蛋白酶肽和钙蛋白酶抑制剂-1的抗氧化特性。使用二甲基吡啶氧化物（DMPO）进行的电子顺磁共振（EPR）自旋捕获研究表明，形成了源自DMPO-OH的四重信号（超精细偶合aN = aH = 14.9 G）。我们发现亮抑蛋白酶肽浓度与氧自由基吸收能力（ORAC）值之间存在相关性（r2 = 0.975），表明其具有体内抗氧化能力。相比之下，钙蛋白酶抑制剂-1没有显示出保护作用。总之，我们的研究结果表明，亮抑蛋白酶肽和钙蛋白酶抑制剂-1对钙蛋白酶活性具有同等效力的抑制作用，但表现出不同的抗氧化功效。
