Effect of temperature variation on metallothionein sub-isoform separation by reverse phase high performance liquid chromatography

Sub-isoforms of rabbit liver metallothionein (MT-RL) and of its two main isoforms (RL-1 and RL-2) have been readily separated at room temperature on a reverse phase column using a gradient of TFA and acetonitrile. MT samples were prepared at neutral and acidic pH. The detection was double, consisting of two UV and electrochemical (EC) detectors on line, the EC (coulometric) detection being based on the oxidation of the thiol groups into disulphides at the graphite electrode. 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 all by EC, being attributed to oxidised species containing disulphides instead of thiols, hence not detectable in our EC mode. All peaks in RL could be attributed to either RL-1 or RL-2 contribution, except for one of the oxidised forms. The elutions were then carried out at various temperatures between 25 and 60°C. The temperature appears to have two main effects on MT subisoform separation: moderate enthalpy changes (-1 to -2 kcal.mol) represented on the chromatograms by small retention time decreases with increasing temperature; important effect on peak 'detectability': when the temperature rises, a clear alteration of the oxidised peaks is observed, especially above 50°C. As a result, between 20 and 40°C all peaks are perfectly detected and resolved, while at 60°C only those peaks assumed to be the original sub-isoforms, hence heat-stable, are present.