Mammalian metallothionein sub-isoform separation by RP-HPLC with online UV and electrochemical detection

The present article reviews the most significant results obtained over the last five years on the use of HPLC to study mammalian MT polymorphism. Firstly, it has been shown that reverse phase HPLC with electrochemical (EC, coulometric) detection allowed us to follow the chemical evolution of a trithiolic hexapeptide intrinsic to the MT structure. The EC response is proportional to the number of free thiols, which permits the identification of the reduced and oxidised forms of the peptide which are well separated. This EC detection was then applied to MT characterisation. Four MTs from rabbit Liver and horse kidney were subjected to RP-HPLC (TFA/acetonitile) with on-line UV and EC detection. They were found to exhibit a different polymorphism. Actually, two types of peaks were observed, those equally detected by UV and by EC being attributed to original thiol containing sub-isoforms and those less hydrophobic, detected by UV but hardly at ail by EC, being attributed to oxidised forms containing disulphides instead of thiols, hence not detectable in our EC mode. The elutions were then carried out at various temperatures between 25 and 60 degrees C. This appears to have two main effects: small retention time decrease of all peaks with increasing temperature and large effect on peak 'detectability": when the temperature rises, a drastic alteration of the oxidised peaks is observed. As a result, between 20 and 40 degrees C all peaks are detected and resolved, while at 60 degrees C only those peaks assumed to be the original sub-isoforms, hence heat-stable, are present. Previous to that, separation of MT by size exclusion chromatography had shown that they were all partially dimerised (5 to 15% of molar rates of dimers). Although small, these levels of non-monomer MT are not negligible.