Stability of metallothionein isoforms by capillary zone electrophoresis

Stability of isoforms on storage of metallothionein (MT) was investigated by capillary zone electrophoresis (CZE) at neutral pH. The mouse liver was removed 1 day after injection of zinc, and homogenized in 0.25 M sucrose with 100 mM TRIS-HCl buffer, pH 7.4. The specimens for MT analysis were obtained by two partial purification methods, i.e., heat treatment (HT) and ethanol/chloroform precipitation (ECP) methods. The specimens were stored at either 4 degrees C or -80 degrees C for 1 and 3 days, and peak areas of MT-1 and MT-2 isoforms obtained by CZE were compared. When specimens were obtained by HT method, both MT isoforms were stable at -80 degrees C within 3 days. However, the peak area of MT-1 stored at 4 degrees C was gradually decreased to one third on the 3rd day. The MT-2 peak did not decrease the next day, but decreased to 57% on the 3rd day. In contrast, peak areas of both MT isoforms decreased remarkably the 1st day, when specimens obtained from ECP method were stored at 4 degrees C and -80 degrees C. From these results, we conclude that stability of MT isoforms is different depending on the purification methods, and MT isoforms are not stable after partial purification, especially by ECP method, even if stored at -80 degrees C.