Chemical and biological properties of bacterial flagellin following iodination and oxidation by chloramine-T

Abstract

(1) Flagellin (mol. wt. 40,000), isolated from the flagella of Salmonella adelaide, was iodinated by the use of the oxidant, chloramine-T. (2) It was found that at pH 7·0, chloramine-T oxidized two of the three methionine residues of flagellin to methionine sulphoxide. An attempt was made to establish conditions for the iodination of flagellin with chloramine-T where minimal methionine oxidation occurred. However, methionine oxidation was an almost unavoidable by-product of the iodination reaction. (3) A technique was developed to monitor the extent of oxidation of the individual methionine residues in proteins, based on the observation that methionine in its sulphoxide form is resistant to cleavage by cyanogen bromide. By using polyacrylamide gel electrophoresis to identify and quantitate the partial breakdown products resulting from incomplete cyanogen bromide cleavage, an estimate of methionine oxidation was obtained. (4) Using this technique, at pH 7·0, the methionines at positions 111 and 331 were, respectively, 40 and 70 times more susceptible to chloramine-T oxidation than the methionine at position 287. It was also found that substantial quantities of methionine sulphoxide existed in native flagellin. (5) Iodination and methionine oxidation were pH dependent. Iodination was more efficient under alkaline conditions, whereas methionine oxidation was favoured at acid pH. From these results it is proposed that under acidic conditions, the methionines of flagellin are presented at the surface of the molecule, whereas under alkaline conditions the methionines are less exposed. (6) Complete oxidation of methionines 111 and 331 did not affect the antigenicity or immunogenicity of flagellin. In contrast, such oxidation modified the polymerizing properties of flagellin, salt-induced and seed-induced polymerization revealing different changes in polymerizing behaviour. It was concluded that methionines 111 and 331 are not associated with the antigenic determinants of flagellin, but may be associated with the polymerizing sites of the protein. Show more