APPROACHES TO THE DETERMINATION OF METALLOTHIONEIN(S) BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY QUARTZ TUBE ATOMIC-ABSORPTION SPECTROMETRY

The operation of a quartz T-tube interface for coupling liquid flowing streams with AAS, when optimized for Ag detection, provided low to su b-nanogram responses for how injections into aqueous carrier streams, Limits of detection were not greatly different to those for Cd, Cn or Zn but were dependent on the mobile phase composition, The interface w as used to monitor the Ag, Cd, Cu or Zn bound to individual metallothi onein (MT) isoforms which had been partially resolved by size-exclusio n or ion-exchange HPLC, Commercial MT isolates from rabbit liver were readily resolved into 4-5 Cd-, Cu- and/or Zn-containing fractions with in 10-15 min using a solvent programme which increased the tris(hydrox ymethyl)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 accou nted for 21-24% of the Cd-response to the MT mixture and the MT-I enri ched standards, By contrast, the MT-II standard contained only trace a mounts of Cd bound to the t(R)=2.22 min fraction and virtually none bo und to the t(R)=3.21 min fraction, Zn was associated predominantly wit h the MT-II isoform and with a more retained fraction which did not co ntain 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 prev alent in the early-eluting fraction with t(R)=3.25 min. An Ag-saturati on procedure rapidly and efficiently replaced the bound Cd, Cu and Zn in these polypeptides but modified the chromatographic behaviour of th e products appreciably.