Solubilization of yeast cell-wall β-(1→3)- d-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction

• Published in: Carbohydrate research 1999-03, Vol.316 (1), p.161-172
• Main Authors: Ohno, Naohito, Uchiyama, Michiharu, Tsuzuki, Aiko, Tokunaka, Kazuhiro, Miura, Noriko N., Adachi, Yoshiyuki, Aizawa, Maki W., Tamura, Hiroshi, Tanaka, Shigenori, Yadomae, Toshiro
• Format: Article
• Language: English
• Subjects: beta-Glucans; Candida; Candida - chemistry; Candida spp; Cell wall; Cell Wall - chemistry; cell wall components; Chromatography, Gel; Dimethyl Sulfoxide; Enzyme-Linked Immunosorbent Assay; Glucans - analysis; Glucans - isolation & purification; Limulus Test; Magnetic Resonance Spectroscopy; MALDIMS; Mannans - chemistry; Oxidation-Reduction; Sodium Hydroxide; Sodium Hypochlorite; Solubility; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; β-Glucan
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/S0008-6215(99)00049-X

The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a β-(1→3)- d-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. β-(1→3)- d-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me 2SO) extraction (yield of 9.6±4.1%) of acetone-dried whole-cell preparations. The β-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of β-(1→3)- and β-(1→6)- d-glucosidic linkages. The specific activity of the β-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10 −11 g/mL.