 Y east 14, 1297–1306 (1998) A New Method for Quantitative Determination of Polysaccharides in the Yeast Cell Wall. Application to the Cell Wall Defective Mutants of Saccharomyces cerevisiae NATHALIE DALLIES, JEAN FRANC q OIS* AND VERONIQUE PAQUET Centre de Bioingenierie Gilbert Durand, UM R-CNRS 5504, LA. INRA, Departement de Genie Biochimique et Alimentaire, Institut National des Sciences Appliquees, Complexe Scientifique de Rangueil, 31077 T oulouse Cedex 04, France A reliable acid hydrolysis method for quantitative determination of the proportion of -glucan, mannan and chitin in S accharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimized version of Saeman’s procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After complete 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 detection. The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from 1,6-glucan and of glucosamine from chitin. The sulfuric acid method was successfully applied to determine the -glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S . cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications.  1998 John Wiley & Sons, Ltd.   — cell wall; chitin; -glucan; S accharomyces cerevisiae INTRODUCTION The cell wall of S accharomyces cerevisiae may account for between 20 and 30% of the cell dry mass. It is mainly composed of mannoproteins and -glucans (85–90% of cell wall dry mass) and a smaller amount of chitin (1–3%) and lipids (2–5%) (Fleet, 1991; Klis, 1994). The proportion of these components may vary according to the strains and the culture conditions (McMurrough and Rose, 1967; Catley, 1988). Methods available for cell wall architecture analysis are based on the separation of structural components by either chemical or enzymatic methods. The chemical fractionation of the cell wall polysaccharides results in three main frac- tions: an alkali-soluble fraction consisting mainly of 1,3-glucan and mannan and some 1,6-glucan; an alkali-insoluble acid-insoluble fraction, con- taining 1,3-glucan linked to chitin; and an acid-soluble alkali-insoluble fraction composed of *Correspondence to: J. M. Franc ¸ois, Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, LA. INRA, Departement de Genie Biochimique et Alimentaire, Institut National des Sciences Appliquees, Complexe Scientifique de Rangueil, 31077 Toulouse Cedex 04, France. Tel: (+ 33) 561559492; fax: (+ 33) 561559400; e-mail: fran_jm@insa-tlse.fr CCC 0749–503X/98/141297–10 $17.50  1998 John Wiley & Sons, Ltd. Received 10 February 1998 Accepted 9 May 1998