CHROMATOGRAPHIC CHARACTERIZATION OF A HEXAPEPTIDE CONTAINING 3 THIOL-GROUPS, INTRINSIC TO THE METALLOTHIONEIN STRUCTURE

The hexapeptide Lys-Cys-Thr-Cys-Cys-Ala [56-61] MT I is a molecule int rinsic to the metallothionein structure. Its chromatographic behaviour on a reversed-phase column, using electrochemical detection has been studied in order to get a better understanding of its chemical stabili ty under various conditions. The chromatogram of the peptide consists of two peaks. The optimization procedure for their separation is prese nted and a hydrodynamic voltammogram has been generated. The relative proportion of the two peaks can vary with experimental conditions and is clearly dependent of the peptide concentration. Following the vario us experiments to which the peptide was submitted, namely elution afte r air oxidation, under inert atmosphere, in basic pH, after addition o f an oxidizing agent (H2O2) and after addition of a reducing agent (2- mercaptoethanol), a reaction scheme was suggested: transformations of the reduced hexapeptide (three free thiol groups) into - as a first st ep - its intramolecular oxidized form (one SS bond, one free SH) and t hen into - as a second step - the fully oxidized dimeric form (no more free SH). Electrochemical detection allows a quantitative follow-up o f the thiol oxidation and hence, of the disappearance of the reduced p eptide.