Artefacts in the Chromatography of Mixtures of Amino-acids containing Cysteine

Abstract

IN the course of an investigation of the amino-acid metabolism of Staphylococcus aureus¹, using paper partition chromatography, I used the bromine water oxidation method, devised by Consden and Gordon², to avoid trouble during the separation of mixtures containing cysteine. The following procedure was used: to a neutralized solution of cysteine and glutamic acid (10 mgm. per ml. of each acid) enough bromine water is added to oxidize all the cysteine to cysteic acid (about 0.1 ml. of bromine water saturated at 20° C. per mgm. of cysteine hydrochloride); the solution is then evaporated to dryness in vacuo at room temperature to remove the slight excess of bromine and the hydrobromic acid formed during the reaction. The residue is dissolved in a small amount of distilled water and dried three times, then made up to the original volume and neutralized. Chromatographic separation on paper (Whatman No. 1 or No. 3), with butanol/acetic acid/water as solvent³, shows, as illustrated on the diagram, four unexpected spots giving slowly a reddish-purple colour with ninhydrin. The two top ones (α₁ very weak, and α₂, stronger) give, after elution and acid hydrolysis (in 6N hydrochloric acid, 3 hr. at 105° C), strong cysteic acid spots on a new ehromatogram. The two others (β, strong, and γ, weak) run between the two initial amino-acids and are hydrolysed into cysteic and glutamic acids; they move also as single spots in a propanol/water mixture⁴ (80 : 20, v : v). These spots are not due to incomplete oxidation, since excess of bromine water does not prevent their appearance; working at 0° C. does not affect the result.