Characterization of Metallothionein Isoforms by Reverse Phase High Performance Liquid Chromatography with On-Line UV and Electrochemical Detection
作者:
G. Bordin,
F.Cordeiro Raposo,
A.R. Rodriguez,
期刊:
Journal of Liquid Chromatography & Related Technologies
(Taylor Available online 1996)
卷期:
Volume 19,
issue 19
页码: 3085-3104
ISSN:1082-6076
年代: 1996
DOI:10.1080/10826079608015809
出版商: Taylor & Francis Group
数据来源: Taylor
摘要:
Four metallothioneins (MTs), rabbit liver MT (RL), MT-1 (RL 1) and MT-2 (RL 2) and horse kidney MT (HK), were subjected to reverse phase HPLC with on-line UV and electrochemical (EC) detection, the latter comprising of a graphite electrode. The EC detection is based on the direct oxidation of the thiols contained in the MT chains, into disulphides. MT samples were prepared at different pH values and eluted with a gradient of TFA and acetonitrile. The MTs are found to exhibit a different polymorphism and the various peaks differ in their detectability depending on the detection mode. RL 2 (one major peak) and HK (three peaks) have peak patterns which are stable with time and varying pH, all peaks being detected by both EC and UV modes. The two other MTs also exhibit peaks which are eluted within the same retention time range as the previously mentioned MTs and which give EC and UV signals (three peaks for RL 1 and four for RL), but which also show some other peaks - eluted earlier - which are perfectly detected in UV but not or very little in EC. This latter information means that these species contain none, or very few, thiol groups. Stable with time at neutral and basic pH, the RL 1 and RL chromatograms are highly evolutional in acid pH with the formation of more of the non-EC detected species and a decrease in the doubly detected ones. We can therefore assume that the peaks detected by both EC and UV modes correspond to the original thiol containing isoforms, while those less hydrophobic of RL 1 and RL correspond to modified species, probably containing disulphide bonds which explains the lack of EC response at the graphite electrode. Since many MTs are highly reactive, this UV/EC combination appears to be a very good tool for a quick identification of reduced (thiolic) isoforms from oxidised (with disulphides) species.
点击下载:
PDF (783KB)
返 回