Approaches to the determination of metallothionein(s) by high-performance liquid chromatography–quartz tube atomic absorption spectrometry

Abstract

The operation of a quartz T-tube interface for coupling liquid flowing streams with AAS, when optimized for Ag detection, provided low to sub-nanogram responses for flow injections into aqueous carrier streams. Limits of detection were not greatly different to those for Cd, Cu or Zn but were dependent on the mobile phase composition. The interface was used to monitor the Ag, Cd, Cu or Zn bound to individual metallothionein (MT) isoforms which had been partially resolved by size-exclusion or ion-exchange HPLC. Commercial MT isolates from rabbit liver were readily resolved into 4–5 Cd-, Cu- and/or Zn-containing fractions within 10–15 min using a solvent programme which increased the tris(hydroxymethyl)methylamine content over time. In addition to the major Cd–MT-I and Cd–MT-II isoforms [retention time (t_R)= 5.84 and 7.58 min], two rapidly eluting fractions (t_R= 2.22 and 3.21 min) collectively accounted for 21–24% of the Cd-response to the MT mixture and the MT-I enriched standards. By contrast, the MT-II standard contained only trace amounts of Cd bound to the t_R= 2.22 min fraction and virtually none bound to the t_R= 3.21 min fraction. Zn was associated predominantly with the MT-II isoform and with a more retained fraction which did not contain the other metals. Cu, which represented only a small fraction (<1%) of the metal content of these polypeptides, was present in greater proportion in the MT-I isoform than in the MT-II isoform and was prevalent in the early-eluting fraction with t_R= 3.25 min. An Ag-saturation procedure rapidly and efficiently replaced the bound Cd, Cu and Zn in these polypeptides but modified the chromatographic behaviour of the products appreciably.