A NEW METHOD FOR QUANTITATIVE-DETERMINATION OF POLYSACCHARIDES IN THEYEAST-CELL WALL - APPLICATION TO THE CELL-WALL DEFECTIVE-MUTANTS OF SACCHAROMYCES-CEREVISIAE

A reliable acid hydrolysis method for quantitative determination of th e proportion of P-glucan, mannan and chitin in Saccharomyces cerevisia e cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of th e wall per gram of cell dry mass. This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex pol ysaccharides. It resulted in an almost complete release of glucose, ma nnose and glucosamine residues from cell wall polysaccharides. After c omplete removal of sulfate ions by precipitation with barium hydroxide , the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric det ection. The superiority of this method over the hydrolysis in either t rifluoroacetic or hydrochloric acid resides in its higher efficiency r egarding the release of glucose from beta 1,6-glucan and of glucosamin e from chitin. The sulfuric acid method was successfully applied to de termine the beta-glucan, mannan and chitin contents in cell walls of g enetically well-characterized yeast mutants defective in cell wall bio synthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for th e characterization of cell walls from S. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and bio technological applications. (C) 1998 John Wiley & Sons, Ltd.