Simultaneous determination of denatured proteins by hydrophobic interaction chromatography

The quantitation of denatured proteins by hydrophobic interaction chromatography (HIC) with a mobile phase containing 8.0 mol l(-1) urea is proposed. The following couples of enzymes, taken as model molecules, have been examined: glyceraldehyde 3-phosphate dehydrogenase and aldolase from rabbit skeletal muscle, alcohol dehydrogenase and phosphoglucose isomerase from baker's yeast. For each denatured protein, reproducibility, linearity range, recovery (> 97%) and determination limit are reported. The feasibility of the simultaneous determination of such protein couples in synthetic mixtures has been tested, and HIC proved a useful tool for separation of denatured proteins, in particular those having similar relative molecular mass and/ or charge.