Differential determination of the 3-Deoxy-D-mannooctulosonic acid residues in lipopolysaccharides of Salmonella minnesota rough mutants.

Abstract

Different applications of the thiobarbituric acid assay were used to determine C4/5‐unsubstituted, heptosyl‐substituted and total 3‐deoxy‐d‐mannooctulosonic acid (dOclA) in lipopolysaccharides of Salmonella minnesota rough mutants. C4/5‐unsubstituted dOclA is determined without prior hydrolysis of the lipopolysaccharides by periodate oxidation near neutral pH at 4°C using the methyl ketoside of dOclA as a standard. C4/5‐unsubstituted dOclA, and dOclA which is substituted by other dOclA residues, is quantified after hydrolysis in 0.1 M acetate buffer pH 4.4 at 100°C/1 h. Total dOclA, including heptosyl‐substituted dOclA, is measured after hydrolysis in 1 M HCl at 100°C for 1–4 h. For S. minnesota R595, 760–790 nmol/mg lipopolysaccharide are determined independently of the hydrolysis conditions employed. In R7, one half, and in R345, two thirds of the total dOclA content are detected after mild acid hydrolysis. Hydrolysis in 1 M HCl at 100°C for 1 h (R7) and 4 h (R345) are required to liberate the heptosyl‐substituted dOclA residues. For the three lipopolysaccharides tested, determination of C4/5‐unsubstituted dOclA revealed that about one third of the total dOclA is reactive in this application of the thiobarbituric acid assay. By strong acid hydrolysis, a 3‐deoxy‐2‐ketoaldonic acid is identified in the lipopolysaccharide of Vibrio cholerae. Upon mild and strong acid hydrolysis, a 3‐deoxy‐2‐ketoaldonic acid is identified in the lipopolysaccharide of Vibrio cholerae. Upon mild and strong acid hydrolysis, dOclA undergoes degradation which is recognized by (a) decreased reactivity in the thiobarbituric acid assay (b) changed ultraviolet spectra and (c) development of compounds which are reactive in the thiobarbituric acid assay after reduction with sodium borohydride. The rate of degradation depends on the strength of the acid but is the same for reference and lipopolysaccharide‐liberated dOclA under a given hydrolysis condition.