Kepinska Marta, Krizkova Sona, Guszpit Ewelina, Rodrigo Miguel A Merlos, Milnerowicz Halina
Department of Biomedical and Environmental Analysis, Faculty of Pharmacy with Division of Laboratory Medicine, Wroclaw Medical University, Poland.
Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Czech Republic.
Adv Clin Exp Med. 2018 Nov;27(11):1601-1608. doi: 10.17219/acem/98916.
Metallothioneins (MTs) constitute a family of evolutionary conserved low molecular weight proteins with small variations in their amino acid sequences. They play a role in the regulation of trace metals metabolism, in the detoxification of heavy metal ions and in mechanisms controlling growth, differentiation and proliferation of cells.
The aim of this study was to evaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due to the common use of MT from rabbit liver as a model protein, the properties of the rabbit and human MTs were compared.
Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization.
In chip CE analysis, changes in the range of 5-8 kDa corresponding to the MT monomer, as well as some peaks of 13-14 kDa corresponding to dimers in both species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. In the Brdicka reaction analysis, a lower concentration of MTs from both organisms coincided with a decrease in the signal corresponding to MT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025 mg/mL) of rabbit MT.
The applied methods allowed for the characterization of MTs and gave complementary information about MT isoforms. Altered electrochemical activity of human and rabbit MTs, despite the same number of -sulfhydryl (-SH) groups, was observed, which may be due to different availability of MT cysteinyl groups.
金属硫蛋白（MTs）是一类进化保守的低分子量蛋白质家族，其氨基酸序列略有差异。它们在痕量金属代谢调节、重金属离子解毒以及控制细胞生长、分化和增殖的机制中发挥作用。
本研究旨在使用先进的分析方法评估人和兔MTs的纯度及特性。由于兔肝MT常被用作模型蛋白，因此对兔和人MTs的特性进行了比较。
采用毛细管电泳（CE）、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和布氏反应对人和兔MTs进行表征。
在芯片CE分析中，观察到对应于MT单体的5-8 kDa范围内的变化，以及两种物种中对应于二聚体的13-14 kDa的一些峰。使用MALDI-MS鉴定出兔（MT-2D）和人（MT-1A、MT-1G、MT-1G+Cd和MT-2A）MTs。在布氏反应分析中，两种生物体中较低浓度的MTs与对应于MT水平（Cat2）的信号降低相吻合。然而，人MT在相同浓度（0.025 mg/mL）下比兔MT给出更高的Cat2峰。
所应用的方法能够对MTs进行表征，并提供有关MT同工型的补充信息。尽管巯基（-SH）基团数量相同，但观察到人和兔MTs的电化学活性发生了改变，这可能是由于MT半胱氨酰基团的可及性不同所致。
