Paper Electrophoresis of Polyglutamyl Peptide

AFTER capsulated B. anthracis had grown in a modified synthetic medium of Brewer et al. 1 , a mixture of growth products was isolated from the glass-filtered medium which gave a precipitate with copper sulphate. Crude polyglutamyl peptide was obtained from this precipitate 2 . An attempt was made to separate the components of the original mixture of growth products by paper electrophoresis using a tank as described by Flynn and Mayo 3 , and it was found that the peptide could not be detected using naphthalene black, bromo-phenol blue, ninhydrin or ultra-violet light 4 . We found it could be detected by taking advantage of the acidic nature of the peptide, using the following technique : (1) paper strips with the peptide applied are run in a suitable buffer, for example, veronal/veronal sodium at p H. 8.6 or acetic acid/sodium acetate at p H 4.5, and dried at 100°; (2) the buffer is washed out with two changes of 80 per cent ethanol; (3) the strips are treated with N /50 hydrochloric acid in 90 per cent ethanol; (4) the acid is thoroughly removed with ethanol.; (5) after drying, the strips are dipped into N /300 alcoholic sodium hydroxide containing 0.04 per cent bromo-cresol purple, which is repeated if necessary until colour differentiation is obtained, and the strips are blotted.