4-amino-5-hydrazino-1-2-4-triazole-3-thiol and metaperiodate

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.

Topics: Bacteria; Chromogenic Compounds; Formaldehyde; Glycols; Periodic Acid; Polysaccharides; Sulfhydryl Compounds

We have adapted the purpald assay (M. S. Quesenberry and Y. C. Lee, Anal. Biochem. 234, 50-55, 1996) to quantify lipopolysaccharide (LPS) content in solution in 96-well microtiter plates at room temperature. This method employs the oxidation of unsubstituted terminal vicinal glycol groups in 2-keto-3-deoxyoctonate (Kdo) and l-(or d-)glycero-d-manno-heptose of LPS molecules by periodate to release formaldehyde. The formaldehyde is quantified at 550 nm (or 530-570 nm) by reacting with purpald reagent followed by oxidation with NaIO4. The sensitivity of the purpald assay is comparable to that of the Kdo assay for LPS determination. However, the purpald assay is superior to the Kdo assay because: (i) No acid hydrolysis of the samples and no boiling in the assay process are required; thus, it can be directly carried out with microtiter plates for a large number of samples at room temperature. (ii) The purpald assay can detect many types of LPS from various bacteria since LPS contains Kdo and heptose which possess unsubstituted terminal vicinal glycol in its structure, while the Kdo assay cannot detect LPS from certain bacteria (e.g., Haemophilus influenzae, Bordetella pertussis, and Vibrio cholerae) due to substitution at the C-4 and C-5 positions of Kdo.

Topics: Bordetella pertussis; Chemistry Techniques, Analytical; Evaluation Studies as Topic; Formaldehyde; Haemophilus influenzae; Heptoses; Lipopolysaccharides; Oxidation-Reduction; Periodic Acid; Sugar Acids; Sulfhydryl Compounds; Vibrio cholerae

A simple, rapid, and sensitive spectrophotometric assay procedure for the determination of as low as 2 microM solutions of formaldehyde and acetaldehyde using an alkaline 4-amino-5-hydrazino-3-mercapto-1,2,4-triazole reagent is described. The method is particularly useful for determination of these aldehydes (0.5-50 nmol) when produced by the periodate oxidation of various glycols and can be applied to the assay of dilute solutions of sugars or polyols.

Topics: Acetaldehyde; Chemical Phenomena; Chemistry; Formaldehyde; Glycols; Oxidation-Reduction; Periodic Acid; Spectrophotometry; Sulfhydryl Compounds