Isoform separation of metallothioneins by capillary zone electrophoresis with Tris–tricine buffer in the presence or absence of methanol

Abstract

A simple capillary electrophoretic method for analysis of metallothionein (MT) isoforms has been developed. Rabbit liver metallothionein (MT) isoforms are readily separated by capillary zone electrophoresis (CZE) with 150 mM Tris(hydroxymethyl)aminomethane (Tris)–150 mM N-Tris(hydroxymethyl)methylglycine (Tricine) buffer (pH 7.50) using an uncoated polyimide cladded fused-silica capillary column thus offering an alternative method for MT separations under original conditions. The effects of buffer concentration, buffer pH and buffer composition on the separation of MT isoforms were investigated. The effect of the addition of organic solvent, MeOH, as buffer modifier on the separation was also studied. Addition of MeOH to the buffer system enhanced the separation and additional information of the heterogeneity within each isoform group was gained. Also the effect of pH on the separation is enhanced when using organic solvent modified buffer. Enhanced information is gained by using photodiode array detection allowing recording of UV spectra of the putative isoform peaks thus offering the possibility of characterisation of the peaks obtained by comparing them to the unique characteristics of metallothionein metal–thiol bonds. The Tris–tricine method presented in this paper allows the separation of peaks that were found to need two separate runs under different conditions for separation to occur when using Tris–borate buffer. Rabbit liver MT, theoretically a mixture of MT-1 and MT-2, exhibited two major peaks, one medium sized peak and several smaller peaks. The experiments indicate that rabbit liver MT exhibited peaks which were not found in either MT-1 or MT-2 isoforms or they were found only as small residues. Both MT-1 and MT-2 samples exhibited one major peak and several smaller peaks. Residues of MT-2 were found in the MT-1 sample and vice versa.