Analytical separations by highvoltage paper electrophoresis. Amino acids in protein hydrolysates.

The determination of the amino acid composition of peptides and proteins is usually carried out on acid hydrolysates by ion-exchange chromatography, by following the methods developed by Moore & Stein and their collaborators (Moore, Spackman & Stein, 1958). This method is precise but time-consuming, requiring up to 600 individual determinations for a single protein analysis. Automatic scanning of the column effluent (Spackman, Stein & Moore, 1958) obviates this objection, but increases complication and cost. An alternative group of methods for amino acid analysis are based on paper-chromatographic separation of the individual amino acids, followed by quantitative determination by means of some modification of the ninhydrin reaction. A considerable literature (reviewed by Lederer & Lederer, 1957; Hanes, 1961) describes the many variants of this method, but most are subject to considerable errors due mainly to the limitations of the ninhydrin reaction, and to losses on the paper during the chromatographic separation and the subsequent manipulations. An exhaustive study of these sources of error has been made by Hanes and his associates (Connell, Dixon & Hanes, 1955; Hanes, 1961; Wade, Matheson & Hanes, 1961; Hanes, Harris, Moscarello & Tigane, 1961; Matheson, Tigane & Hanes, 1961; Tigane, Wade, Tze-Fei Wong & Hanes, 1961), who have developed two new paper-chromatographic systems for the separation of 16 of the 18 common amino acids, and also a modified ninhydrin reagent. These improvements constitute a considerable advance over previous methods, but at the cost of a relatively complex technique. Recoveries of individual amino acids were within ± 10 %. No applications to protein hydrolysates were reported. Levy (1954) has described a method of amino acid analysis based on the paper-chromatographic separation of the dinitrophenyl derivatives, followed by spectrophotometric determination at 360 mp. This method appears to be markedly inferior to ion-exchange chromatography (see Hedbom, 1961, for a direct comparison). Whitehead (1958) has described an isotope-dilution method based on acetylation of the amino acids with [14C]and [3H]-acetic anhydride, and a combined chromatographic separation of the acetyl compounds. This method does not appear to have been widely used, although it is applicable to microgram amounts of protein. Recent advances in the technique of high-voltage paper electrophoresis (reviewed comprehensively by Michl, 1958) have provided an analytical method of extremely high resolving power for small molecules. The present paper describes systems for the complete separation of all the amino acids commonly occurring in protein hydrolysates. Combined with the improved cadmium-ninhydrin method ofHeilmann, Barollier & Watzke (1957) this provides a new method of amino acid analysis, which reduces the estimations required to one for each amino acid present in the hydrolysate, while the precision is comparable with that obtained with the ion-exchange method; 250 ,ug. of protein suffices for a complete amino acid. analysis. Rowe, Ferber & Fischer (1958) have described an analogous method, which was, however, applicable to only 10 of the 18 commonly occurring amino acids. Preliminary accounts of the present work have already been published (Atfield & Morris, 1960a, b).