Abstract
BackgroundMetallothionein (MT) characterized by their low molecular weight and high cysteine content.ResultsTwo recombinant proteins of MT-I and MT-II overproduced in E. coli (M15) was purified by Ni2+-chelated affinity chromatography. The molecular mass of MT-I and MT-II are ca. 6,600 and 8,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Total antioxidant status, DPPH radical scavenging activity, reducing power method, Fe2+-chelating ability, ferric thiocyanate (FTC) method, and protecting calf thymus DNA against hydroxyl radical-induced damage were studied. The MT-I and MT-II proteins with a concentration of 100 μg/mL exhibited the highest activity (expressed respectively as 61.72 ± 0.13 and 74.28 ± 1.15 μM Trolox equivalent antioxidative capacity, TEAC) in total antioxidant status test. Like total antioxidant status, DPPH radical scavenging activity, reducing power, Fe2+-chelating ability, FTC activity, and protecting calf thymus DNA against hydroxyl radical-induced damage all showed that MT-1 and MT-II proteins have antioxidant activities. In this study, we also found that antioxidant activities of MT-I and MT-II increased from 17% and 16% (0 h) to about 26% and 28% (24 h) after 24 h hydrolysis by trypsin. Smaller peptides increased the antioxidant activities. Four and three peptides, respectively, from MT-I and MT-II protein sequences for testing antioxidative activity were synthesized according to tryptic hydrolysis simulation. The obtained MSSGCK, CGSDCK, LTLEGSSEK, ATEGGHACK, CGNGCGGCK, and CDPCNCK showed IC50 values of 309.87, 1423.37, 3925.54, 561.32, 300.76, and 610.12 μM, respectively, when scavenging activity of DPPH radicals (%) was measured.ConclusionsThese findings mean that a cysteine residue is most important in antiradical activities. It was suggested that MT-I and MT-II might contribute their antioxidant activities against hydroxyl and peroxyl radicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0064-4) contains supplementary material, which is available to authorized users.
Highlights
Metallothionein (MT) characterized by their low molecular weight and high cysteine content
Cultures of the transformed E. coli (M15) each overexpressed a protein of the expected molecular mass, which was purified by affinity chromatography in Ni-nitrilotriacetic acid (NTA) columns (Qiagen), according to the manufacturer’s instructions
MT-I or MT-II was found as a soluble protein in the supernatant (Figure 1, lane 2), and was absent in protein extracts obtained from E. coli transformed with pQE-31 vector (Figure 1, lane 1)
Summary
Metallothionein (MT) characterized by their low molecular weight and high cysteine content. DPPH radical scavenging activity, reducing power method, Fe2+-chelating ability, ferric thiocyanate (FTC) method, and protecting calf thymus DNA against hydroxyl radical-induced damage were studied. DPPH radical scavenging activity, reducing power, Fe2+-chelating ability, FTC activity, and protecting calf thymus DNA against hydroxyl radical-induced damage all showed that MT-1 and MT-II proteins have antioxidant activities. From MT-I and MT-II protein sequences for testing antioxidative activity were synthesized according to tryptic hydrolysis simulation. MT is a member of the low molecular weight protein family rich in cysteine and with the ability to coordinate heavy metal atoms (such as zinc, copper, and selenium). During the past few years, natural antioxidants and compounds with radical scavenging activity have been found, such as phenolic compounds (Chang et al 2007), Bowman-Birk Inhibitor (Huang et al 2010), and trypsin inhibitor from sweet potato root (Huang et al 2012). The objectives of this work were to investigate the antioxidant property of MT-I and MT-II from sweet potato tuberous root in comparison with chemical compounds such as butylated hydroxytoluene (BHT), reduced glutathione or ascorbate in a series of in vitro tests
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