ISOFORM SEPARATION OF METALLOTHIONEINS BY CAPILLARY-ZONE-ELECTROPHORESIS WITH TRIS-TRICINE BUFFER IN THE PRESENCE OR ABSENCE OF METHANOL

A simple capillary electrophoretic method for analysis of metallothion ein (MT) isoforms has been developed. Rabbit liver metallothionein (MT ) isoforms are readily separated by capillary zone electrophoresis (CZ E) with 150 mM Tris(hydroxymethyl)aminomethane (Tris)-150 mM N-Tris(hy droxymethyl)methylglycine (Tricine) buffer (pH 7.50) using an uncoated polyimide cladded fused-silica capillary column thus offering an alte rnative method for MT separations under original conditions. The effec ts of buffer concentration, buffer pH and buffer composition on the se paration of MT isoforms were investigated. The effect of the addition of organic solvent, MeOH, as buffer modifier on the separation was als o studied. Addition of MeOH to the buffer system enhanced the separati on 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 g ained by using photodiode array detection allowing recording of UV spe ctra of the putative isoform peaks thus offering the possibility of ch aracterisation of the peaks obtained by comparing them to the unique c haracteristics of metallothionein metal-thiol bonds. The Tris-tricine method presented in this paper allows the separation of peaks that wer e found to need two separate runs under different conditions for separ ation to occur when using Tris-borate buffer. Rabbit liver MT, theoret ically a mixture of MT-1 and MT-2, exhibited two major peaks, one medi um sized peak and several smaller peaks. The experiments indicate that rabbit liver MT exhibited peaks which were not found in either MT-1 o r MT-2 isoforms or they were found only as small residues. Both MT-1 a nd MT-2 samples exhibited one major peak and several smaller peaks. Re sidues of MT-2 were found in the MT-1 sample and vice versa. (C) 1998 Elsevier Science B.V. All rights reserved.