Solubilization of yeast cell-wall beta-(1-->3)-D-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction.
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 beta-(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. beta-(1-->3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The beta-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 beta-(1-->3)- and beta-(1-->6)-D-glucosidic linkages. The specific activity of the beta-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.
Tokyo University of Pharmacy and Life Science, School of Pharmacy, Japan.
N
N
Ohno
M
M
Uchiyama
A
A
Tsuzuki
K
K
Tokunaka
N N
NN
Miura
Y
Y
Adachi
M W
MW
Aizawa
H
H
Tamura
S
S
Tanaka
T
T
Yadomae
D002175:Candida / Q000737:chemistry*; D002473:Cell Wall / Q000737:chemistry*; D002850:Chromatography, Gel; D004121:Dimethyl Sulfoxide; D004797:Enzyme-Linked Immunosorbent Assay; D005936:Glucans / Q000032:analysis / Q000302:isolation & purification*; D008033:Limulus Test; D009682:Magnetic Resonance Spectroscopy; D008351:Mannans / Q000737:chemistry; D010084:Oxidation-Reduction; D012972:Sodium Hydroxide; D012973:Sodium Hypochlorite; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D047071:beta-Glucans
D005936:Glucans; D008351:Mannans; D047071:beta-Glucans; C064197:beta-1,6-glucan; D012972:Sodium Hydroxide; C033363:beta-1,3-glucan; D012973:Sodium Hypochlorite; D004121:Dimethyl Sulfoxide