Zhejiang Provincial Key Lab of Mariculture & Enhancement, Zhejiang Marine Fisheries Research Institute, Tiyu Road 28, Zhoushan 316021, China.
College of Food and Pharmaceutical Sciences, Ningbo University, Qixing South Road 169, Ningbo 315800, China.
Molecules. 2022 Apr 3;27(7):2320. doi: 10.3390/molecules27072320.
We report here the purification of a novel metal-binding protein from ( MT-1) by gel and ion-exchange chromatography. SDS-PAGE and MALDI-TOF analyses demonstrated that isolated MT-1 was of high purity with a molecular weight of 12.4 kDa. The fluorescence response to SBD-F derivatives revealed that MT-1 contained a large number of sulfhydryl groups, which is a general property of metallothioneins. Zn and Cu metal stoichiometries for MT-1 were 3.97:1 and 0.55:1, respectively. The proportion of cysteine (Cys) residues in the amino acid composition was 32.69%, and aromatic amino acids were absent. The peptide sequence coverage with calmodulin (accession AOA3S8FSK5) was 60%. Infrared spectroscopy of MT-1 revealed two obvious peaks at absorption frequencies for the amide I band and the amide II band. CD spectra revealed that the secondary structure was mainly composed of random coil (57.6%) and β-sheet (39.9%). An evaluation of in vitro antioxidant activity revealed that isolated MT-1 has strong reducing activities, exhibiting scavenging rates for DPPH and OH of 77.8% and 75.8%, respectively (IC values 0.57 mg/mL and 1.1 mg/mL). MT-1 may be used as a functional additive in cosmetics, health foods, and medical products, as well as a reference material for quantitative analysis of metallothionein in such products.
我们通过凝胶和离子交换层析从 （MT-1）中纯化了一种新型的金属结合蛋白。SDS-PAGE 和 MALDI-TOF 分析表明，分离得到的 MT-1 具有高纯度，分子量为 12.4 kDa。对 SBD-F 衍生物的荧光响应表明，MT-1 含有大量的巯基，这是金属硫蛋白的一般特性。MT-1 的 Zn 和 Cu 金属化学计量比分别为 3.97:1 和 0.55:1。氨基酸组成中半胱氨酸（Cys）残基的比例为 32.69%，不存在芳香族氨基酸。钙调蛋白（登录号 AOA3S8FSK5）的肽序列覆盖率为 60%。MT-1 的红外光谱在酰胺 I 带和酰胺 II 带的吸收频率处显示出两个明显的峰。CD 光谱表明，二级结构主要由无规卷曲（57.6%）和β-折叠（39.9%）组成。体外抗氧化活性评价表明，分离得到的 MT-1 具有较强的还原活性，对 DPPH 和 OH 的清除率分别为 77.8%和 75.8%（IC 值分别为 0.57 mg/mL 和 1.1 mg/mL）。MT-1 可作为化妆品、保健品和医药产品中的功能性添加剂，以及这些产品中金属硫蛋白定量分析的参考物质。
