Polarographic study of the O-chain lipopolysaccharide of Brucella melitensis after labelling as the N-malylamide derivative

Abstract

A method is described for the electrochemical labelling of the antigenic O-chain polysaccharide of Brucella melitensis based on the conversion of N-formylated groups into the N-maleyl derivative. After alkaline hydrolysis of native lipopolysaccharide the O-chain polysaccharide containing free amino groups in the glycosyl units was obtained. The reaction of this biopolymer with maleic anhydride in borax buffer yielded the N-maleylamide derivative. Dialysis was used to purify the labelled polysaccharide and IR spectra used for characterization. The differential pulse polarographic behaviour of the N-maleylamide polysaccharide derivative was studied, and two polarographic peaks were obtained. The first was in the 1–10 pH range, peak intensity and peak potential depending on pH ( E P = −0.53−0.063 pH); the second appears in the 10–12 pH range and the E P potential remains practically constant at −1.51 V (vs. SCE). The analytical possibilities of the peak appearing at −1.00 V (vs. SCE) in Britton—Robinson buffer (pH = 7.4) were useful for N-maleylamide polysaccharide derivative determination in the 30–4 30 μg/ml range, with a 2.6% relative standard deviation. The polarographic process is irreversible and shows adsorption of the biopolymer onto the electrode surface. The polarographic signal remained stable during both experiments and after 3 months of cool storage of an N-maleyl-polysacch solution.