Cysteine and Phosphoserine containing Peptide Sequences of Ovalbumin

Abstract

TO investigate whether certain amino-acid sequences, for example, cysteinylglycine¹, occur with greater than chance frequency in different proteins^2,3, a method has been devised for the isolation and identification of cysteine containing sequences, and for peptide sequences containing O-phosphoserine, a compound with which at least one of the two phosphorus residues in ovalbumin⁴ has now been identified. 2-gm. portions of four times crystallized, performic acid oxidized² ovalbumin were precipitated and washed with trichloroacetic acid, hydrolysed with 40 ml. of 11 N hydrochloric acid for 3–7 days at 37°, and hydrochloric acid removed in vacuo below 25°. A strongly acid peptide fraction was separated from the hydrolysate by washing through a 4 cm. × 10 cm. ‘Dowex 50’ (H⁺ form) column with water, and the effluent was dried in vacuo, neutralized in small volume, and applied to a 1 cm. × 50 cm. ‘Dowex 2’ column (pH 3, Cl⁻ form). Slow elution with 400 ml. hydrochloric acid rising continuously from 0.001 to 0.05 N provided fractions containing only one to three peptides, separable by one-dimensional paper chromatography. Composition and N-terminal amino-acid of each peptide were determined by paper chromatography of sealed-capillary hydrolysates (11 N hydrochloric acid, at 110°) of aliquots, with and without treatment with dinitrofluorobenzene⁵.