Study of the disulfide reduction of denatured proteins by liquid chromatography coupled with on-line cold-vapor-generation atomic-fluorescence spectrometry (LC-CVGAFS).

Abstract

Hydrophobic-interaction chromatography coupled on-line with chemical-vapor-generation atomic-fluorescence spectrometry (HIC-CVGAFS), optimized recently for the analysis of thiol-containing proteins under denaturing conditions, has been used to study the chemical reduction of denatured proteins. Four proteins chosen as models (human serum albumin (HSA), bovine serum albumin (BSA), alpha-lactalbumin (alpha-Lac) from bovine milk, and lysozyme from chicken egg (Lys)) were denatured with urea and reduced with dithiothreitol (DTT), with selenol as catalyst. The method is based on derivatization of the -SH groups of proteins with p-hydroxymercurybenzoate (PHMB), followed by HIC separation and post-column on-line reaction of the derivatized reduced, denatured proteins with bromine generated in situ. HgII, derived from rapid conversion of uncomplexed and protein-complexed PHMB, is selectively detected by AFS in an Ar/H2 miniaturized flame after sodium borohydride (NaBH4) reduction to Hg degrees . The yield of the reduction was studied as a function of reductant concentration, reduction time (tred), and urea concentration. Results showed that the optimum values for DTT and selenol concentrations and for tred were between 1 and 100 mmol L(-1) and between 1 and 20 min, respectively, depending on the protein studied. The percentage disulfide bond reduction increases as the urea concentration used for protein denaturation increases, giving a single-step sigmoid increment for single-domain, low-MW proteins (alpha-Lac and Lys), and a two-step sigmoid increment for multi-domain, high MW proteins (HSA and BSA). The shapes of plots of percentage reduced disulfide against urea concentration are characteristic of each protein and are correlated with the location of S-S in the protein. Under the adopted conditions complete protein denaturation is the conditio sine qua non for obtaining 100% S-S reduction. The detection limit for denatured, reduced proteins examined under the optimized conditions was found to be in the range 1-5 x 10(-12) mol L(-1) (10-30 pg), depending on the protein considered.