Characterization of denatured metallothioneins by reversed phase coupled with on-line chemical vapour generation and atomic fluorescence spectrometric detection

A new analytical hyphenated technique is proposed for determination and characterization of thiolic proteins, based on reverse phase chromatography (RPC) coupled on-line with cold vapour generation atomic fluorescence spectrometry (CVGAFS). Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol l(-1) urea and p-hydroxymercurybenzoate (PHMB). The derivatized proteins are separated on a C4 Vydac Reverse Phase column. Post-column on-line reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of both the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury, also in the presence of methanol in the RPC eluent phase. Hg(II) is selectively detected by AFS in a Ar/H-2 miniaturized flame after sodium borohydride reduction to Hgdegrees. Under optimized conditions, on-line bromine treatment gives a 98 +/- 2% recovery of both free and protein-complexed PHMB. The effect of methanol on the sensitivity of Hg(II) detection was studied and controlled. RPC-CVGAFS system has been applied to the analysis of metallothioneins from rabbit liver (MTRL) standard solutions, and their commercial isoforms MT-1 and MT-2. The analysis of denatured, PHMB-complexed MTs allowed the determination of the number of thiolic groups complexed by PHMB. It was found that MTs from rabbit liver have 10.0 +/- 0.3 (MT-1) and 6.7 +/- 0.3 (MT-2 and MTRL)-SH groups complexed by PHMB. The detection limit (LODc) for PHMB in 95% methanol in the optimized conditions was about 9.3 x 10(-9) mol l(-1) and for the denatured MTs LODc was about 8.6 x 10(-10) mol l(-1), taking into account an approximate complexating ratio PHMB:MTs of 7:1. (C) 2004 Elsevier B.V. All rights reserved.