Quantification of bacterial polysaccharides by the purpald assay: measurement of periodate-generated formaldehyde from glycol in the repeating unit.
We have adapted the purpald assay for measurement of bacterial polysaccharides (PS) containing substituted and/or unsubstituted glycol (SG or UG) in residues such as glycerol, ribitol, arabinitol, furanosyl galactose, and sialic acid. For the purpald assay of UG-containing PS, 50 microL of PS samples was consecutively reacted with 50 microL of 16 mM NaIO4 for 20 min, 50 microL of 136 mM purpald reagent in 2 N NaOH for 20 min, and 50 microL of 64 mM NaIO4 for 20 min in a 96-well tissue culture plate followed by a measurement of absorbance at 550 nm with a plate reader. For SG-containing PS, conversion of SG to UG with 25 micro;L of 0.3 N NaOH, 1 h at room temperature for de-O-acetylation followed by 25 microL of 0.6 M H2SO4, 1 h at 80 degrees C for acid hydrolysis of PS precedes the periodate treatment in the purpald assay. The concentration of the samples can be calculated from the sample absorbance and the reference standard curve constructed from the reference concentrations of the same PS (well-characterized) and their corresponding absorbance values assayed in the same plate. The purpald assay provides a tool in addition to the existing ones for the measurement of glycol-containing PS. Among the usefulness of this method are the determinations of the glycerol content in the phospho-glycerol-containing PS and the SG and UG contents and structural integrity in PS and conjugate vaccines.
Lee
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Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic Products, OVRR, CBER, FDA, 8800 Rockville Pike, Bethesda, Maryland 20892, USA.
Frasch
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D001419:Bacteria / Q000737:chemistry*; D002863:Chromogenic Compounds / Q000032:analysis; D005557:Formaldehyde / Q000032:analysis* / Q000737:chemistry; D006018:Glycols / Q000737:chemistry*; D010504:Periodic Acid / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis*; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry
D002863:Chromogenic Compounds; D006018:Glycols; D011134:Polysaccharides; D013438:Sulfhydryl Compounds; D010504:Periodic Acid; C006537:4-amino-5-hydrazino-1,2,4-triazole-3-thiol; D005557:Formaldehyde; C009288:metaperiodate