Antioxidant activities of two metallothionein-like proteins from sweet potato (Ipomoea batatas [L.] Lam. `Tainong 57’) storage roots and their synthesized peptides

Huang et al. Botanical Studies 2014, 55:64 http://www.as-botanicalstudies.com/content/55/1/64 RESEARCH Open Access Antioxidant activities of two metallothionein-like proteins from sweet potato (Ipomoea batatas [L.] Lam. ‘Tainong 57’) storage roots and their synthesized peptides Shyh-Shyun Huang1†, Jeng-Shyan Deng2†, Hsien-Jung Chen3, Yaw-Huei Lin4* and Guan-Jhong Huang5* Abstract Background: Metallothionein (MT) characterized by their low molecular weight and high cysteine content. Results: Two 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. Conclusions: These 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. Keywords: Sweet potato; Metallothionein; Recombinant protein; Antioxidant activity Background Recently, attention has been focused on a role for metallothionein (MT) as free radical scavenger. 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). Plant MTs generally contain * Correspondence: ; † Equal contributors 4 Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan 5 School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan Full list of author information is available at the end of the article two smaller cysteine-rich domains (4–8 cysteines each) and a large spacer region (30–50 residues) devoid of this amino acid. The distribution of cysteine residues, as well as the length of the spacer region served to further classify plant MTs into four types (Huang et al. 2013). Expression of MT genes has been reported in a variety of plant tissues and has been proposed to function in metal homeostasis/ detoxification, oxidative stress defense, salinity and carbonate stress, and development regulation (Sato and Bremner 1993). MT genes from sweet potato enhanced tolerance to heavy metals and abiotic stresses, which can be used for phytoremediation or tolerance to environmental stresses © 2014 Huang et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Huang et al. Botanical Studies 2014, 55:64 http://www.as-botanicalstudies.com/content/55/1/64 Chen et al. 2003a, 2003b). In addition, the antioxidant function of MT is attributed to the presence of a large number of cysteine residues, which besides metal binding are also capable of reactive oxygen species (ROS) scavenging (Kumar et al. 2012). Plants are equipped with multiple enzymes and nonenzymatic molecules that are involved in the decomposition of ROS (Huang et al. 2007). Under normal conditions, the physiologically important intracellular levels of ROS are kept at low levels by various enzyme systems participating in the redox homeostasis. Therefore, oxidative stress can also be viewed as an imbalance between the prooxidants and antioxidants in the cell (Kehrer 1993). The ROS play an important role in the degenerative or pathological processes of various serious diseases, such as aging, cancer, coronary heart disease, Alzheimer’s disease, neurodegenerative disorders, atherosclerosis, and inflammation (Huang et al. 2008). 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. Methods Materials Ascorbic acid, glutathione, DPPH (2, 2-diphenyl-1picrylhydrazyl), EDTA (ethylenediaminetetraacetic acid), FTC (ferric thiocyanate), sodium bicarbonate, and Tris (hydroxylmethyl) aminomethane were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO USA). Total antioxidant status assay kit was purchased from CalbiochemNovabiochem Corp (San Diego, California, USA). 95% ethanol was purchased from Riedel-de Haen Chemical Co. (Germany). Expression of MT-I and MT-II in E. coli. MT-I (AAD10220) and MT-II (FJ418632) with its prosequence were expressed in E. coli. The coding sequence was amplified from cDNA MT-I and MT-II using an oligonucleotide (MT-I, 5′-GGA TCC AGA GAT GTC TTC CGG TTG C −3′; MT-II, 5′-GGA TCC AAA AAT GTC TTG CTG TG-3′), with a Bam HI site (underlined) at the putative initial Met residue, and an oligonucleotide (MT-I, 5′- GAC CCT TGC AAC TGT AAG CTT CAA −3′; MT-II, 5′- GCA ATT GCA AGT GAG ATG CGAA G CTT −3′), with a Hind III site at the 3′ end. The PCR fragment was subcloned in pGEM T-easy vector. The plasmid was then digested with Bam HI and Hind III and the excised fragments were subcloned in Page 2 of 9 pQE31 expression vector (QIAexpress expression system, Qiagen). The resulting plasmid, termed pQE-MT-1 and pQE-MT-II, was introduced into E. coli (M15). Cultures of t (...truncated)