MEASUREMENT OF PROTEIN-CONTENT IN YEAST-CELLS WITH THE AID OF FLUORESCAMINE

We studied the following main steps of the yeast protein assay using reaction with fluorescamine, a fluorescent reagent specific for the amine nitrogen: 1) pretreatment of cells; 2) interaction with fluorescamine; and 3) determination of protein concentration from the fluorescence intensity. We have validated the applicability of the extraction-free protein determination in permeabilized cells after removal of amino acids. We also optimized cell permeabilization and pelleting, as well as the concentration of yeast biomass and fluorescamine in the reaction mixture, pH, and volume of the reaction. We reviewed the algorithms for defining of the range of measurable protein concentrations on a laboratory fluorimeter and for the algorithms for defining of the range of measurable protein concentrations on a laboratory fluorimeter and for the calculation of the true yeast protein content from the determined concentration and the data obtained for a standard protein using specificity coefficient. The optimal procedure for the quantifying of yeast protein-by fluorescence analysis-and the advantages of this technique over conventional methods are described.