A systematic approach to the characterization of selenized yeast supplements in terms of the speciation of selenium was developed. The optimized fractionation procedures included the sequential leaching of water soluble, cell-wall bound, and membrane-protein selenium followed by a further fractionation of each extract by high-resolution size-exclusion chromatography. The stability of fractions collected as chromatographic peaks was investigated in the presence of a proteolytic enzyme (pronase XIV) and trypsin in order to discriminate between selenium-containing peptides and other selenocompounds. Reversed-phase HPLC of tryptic digests of size-exclusion chromatographic fractions allowed the identification of selenopeptides by MALDI and electrospray MS. The complexity of the speciation of the water-soluble selenium in yeast was confirmed. Surprisingly, selenomethionine in the water insoluble fraction was found to be bound physically to cell wall constituents rather than being incorporated chemically into the protein structure, in contrast to former studies.

An analytical approach allowing the identification of unknown seleniummetabolites in selenium-rich yeast was described. Anion-exchange HPLC of the Se-