A mixed standard of metallothionein containing isoforms 1 and 2 was obtained from Sigma, as well as dithiothreitol (DTT) and Tris buffers.  Three mg of lyophilized Cd, Zn, and Cu MT was dissolved in 1 ml of ultrapure 20 mM tris buffer pH 8.6.  To this mixture 20 mg of DTT was added to prevent oxidation of the cysteine thiolate groups and incubated overnight.  The pH was then adjusted to 1 via rapid addition of 0.1M HCl.  The mixture was mixed thoroughly and left to incubate for 50 minutes.  It was then dialyzed in an Amicon pressure dialysis unit with a Millipore YM 100 membrane (1000 MW cutoff) under nitrogen gas.  This procedure was repeated four times to insure that all metals and DTT had been washed through the membrane and into the effluent.  The apo-MT was then washed with aliquots of 20 mM tris buffer until the pH reached 8.6.  Two mls of apo-MT in 20 mM tris buffer was recovered from the Amicon and analyzed for both protein and metal content.

The apo-MT was fractioned using size exclusion chromatography on a Phenomenex BioSep 2000 column.  The protein was eluted using 20 mM tris (pH 7.2) at a flow rate of 1 ml/min.  The eluent was monitored automatically for absorbing species using a Beckman 168 diode array detector and analyzed for metal content by a directly coupled Hewlett Packard ICP-MS.