Characterisation of Metallothionein Isoforms by Reverse Phase High Performance Liquid Chromatography with On-Line UV and Electrochemical Detection

Abstract

Four metellothioneins (MTs), rabbit liver MT (RL), MT-1 (RL-1) and MT-2 (RL-2) and horse kidney MT (HK), were subjected to reversed-phase HPLC with on-line UV and electrochemical (EC) detection, the latter comprising of a graphite electrode. The MTs are found to exhibit a different polymorphism and the varioius peaks differ in their detectability depending on the detection mode. RL-2 (1 major peak) and HK (3 peaks) have peak patterns which are stable with time and varying pH, all peaks being detected by EC and UV modes. The 2 other MTs also exhibit peaks which are eluted within the same retention time range as the previous mentioned MTs and which giveEC and UV signals (3 peaks for RL-1 and 3 for RL), but which also shows some other peaks - eluted earlier - which are perfectily detected in UV but not or very little in EC. Stable with time at neutral and basic pH, the RL-1 and RL chromatograms are highly evolutional in acid pH. 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.JRC.D-Institute for Reference Materials and Measurements (Geel