STUDY OF MAMMALIAN METALLOTHIONEIN POLYMORPHISM BY REVERSE PHASE HPLC WITH ON-LINE DIODE ARRAY AND ELECTROCHEMICAL DETECTION

Abstract

The polymorphism of rabbit liver metallothionein RL and of its two isoforms RL-1 and RL-2 has been studied by means of reverse phase HPLC with on-line diode array (DAD) and electrochemical (EC) detection. Separations have been carried out in an acidic mobile phase (TFA). Using the typical UV absorption characteristics of MT, as well as the electrochemical response of the various peaks and comparing the morphology of the chromatograms obtained with a gradient at pH 3 and at pH 2, we reached the following conclusions: RL-1 has three subisoMTs which are apoforms at pH 3 and four other Cu, Cd metalloforms; RL-2 has a less complex polymorphism with one major and one minor subisoMTs under apoform at pH 3; RL has four main subisoMTs which are apoforms at pH 3, coming either from RL-1 or from RL-2 and two main other metalloforms including a pure Cu form not found in the isoforms. In all three MTs, an extra peak has been identified by its non MT-type UV spectrum, its time-dependence increase, its total lack of electrochemical response, and its larger variation of retention time with increasing temperature. This could be a degradation product of MT.

ACKNOLEDGEMENT

The authors wish to thank Josephine McCourt of our laboratory for the determination of the metal concentrations in the metallothionein samples.