Tolmacheva Anna S, Blinova Elena A, Ermakov Evgeny A, Buneva Valentina N, Vasilenko Nataliya L, Nevinsky Georgy A
Siberian Division of Russian Academy of Sciences, Institute of Cytology and Genetics, Lavrentiev Ave., 10, Novosibirsk, Russia.
Siberian Division of Russian Academy of Sciences, Institute of Chemical Biology and Fundamental Medicine, Lavrentiev Ave., 8, Novosibirsk, 630090, Russia.
J Mol Recognit. 2015 Sep;28(9):565-80. doi: 10.1002/jmr.2474. Epub 2015 May 6.
We present the evidence showing that small fractions of electrophoretically homogeneous immunoglobulin G (IgGs) from the sera of healthy humans and their Fab and F(ab)2 fragments oxidize 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2 O2 and through an oxidoreductase activity in the absence of H2 O2 . During purification on protein G-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me(2+) ions. After extensive dialysis of purified Abs against agents chelating metal ions, the relative peroxidase activity decreased dependently of IgG analyzed from 100 to 10-85%, while oxidoreductase activity from 100 to 14-83%. Addition of external metal ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. Chromatography of the IgGs on Chelex non-charged with Cu(2+) ions results in the adsorption of a small IgG fraction bound with metal ions (5%), while Chelex charged with Cu(2+) ions bind additionally ~38% of the total IgGs. Separation of Abs on both sorbents results in IgG separation to many different subfractions demonstrating various affinities to the chelating resin and different levels of the specific oxidoreductase and peroxidase activities. In the presence of external Cu(2+) ions, the specific peroxidase activity of several IgG subfractions achieves 20-27 % as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of these fractions is ~4-6-fold higher than that for HRP. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activities of human IgGs could also play an important role in the protection of organisms from oxidative stress and toxic compounds.
我们提供的证据表明，来自健康人血清的电泳纯免疫球蛋白G（IgG）及其Fab和F(ab)2片段的小部分，在有H2O2存在时通过过氧化物酶活性氧化3,3'-二氨基联苯胺，在无H2O2时通过氧化还原酶活性氧化3,3'-二氨基联苯胺。在蛋白G-琼脂糖凝胶上纯化和凝胶过滤过程中，多克隆IgG会部分失去Me(2+)离子。用螯合金属离子的试剂对纯化后的抗体进行广泛透析后，相对过氧化物酶活性根据所分析的IgG从100%下降到约10 - 85%，而氧化还原酶活性从100%下降到14 - 83%。向透析和未透析的IgG中添加外部金属离子会导致其活性显著增加。IgG在未负载Cu(2+)离子的Chelex上进行色谱分析，会导致一小部分与金属离子结合的IgG被吸附（约5%），而负载Cu(2+)离子的Chelex会额外结合约38%的总IgG。在两种吸附剂上分离抗体导致IgG分离为许多不同的亚组分，这些亚组分对螯合树脂表现出不同的亲和力以及不同水平的特异性氧化还原酶和过氧化物酶活性。在存在外部Cu(2+)离子的情况下，与辣根过氧化物酶（HRP，设定为100%）相比，几个IgG亚组分的特异性过氧化物酶活性达到20 - 27%。这些组分的氧化还原酶活性比HRP高约4 - 6倍。抗氧化酶如超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶已知是防止DNA、蛋白质和脂质氧化修饰的关键防御机制。人IgG的过氧化物酶和氧化还原酶活性也可能在保护生物体免受氧化应激和有毒化合物的影响中发挥重要作用。
