Amino Acid Composition Determination
The pioneering studies of Emil Fischer [301] at the turn of the century established the role of amino acids and the peptide bond in the composition and chemistry of proteins. In the light of contemporary techniques, his methods of protein degradation and
- PDF / 4,185,344 Bytes
- 33 Pages / 504.567 x 720 pts Page_size
- 11 Downloads / 276 Views
Amino Acid Composition Determination JOHN W. EVELEIGH and GEORGE D. WINTER
I. General A. Early Methods The pioneering studies of EMIL FISCHER [301] at the turn of the century established the role of amino acids and the peptide bond in the composition and chemistry of proteins. In the light of contemporary techniques, his methods of protein degradation and subsequent fractionation of the products appear cumbersome and of historical interest only. Yet it is significant that for nearly 50 years these original publications provided the major methods available to the analytical protein chemist. The basic amino acids were precipitated with phosphotungstic acid and subsequently fractionated as their silver salts [575]. Neutral and acidic amino acids were esterified with ethanol and hydrochloric acid and the esters separated by fractional distillation [298]. These and allied methods were fraught with practical difficulties and the variable yields made them far from quantitative. The availability of isotopes in the late 1940's enabled the early separation methods to be quantitated using the isotope dilution principle [828]. The isotopes 0 4, N15, S35, and deuterium were successfully employed. However, the low specific activity of the radioactive isotopes available at that time coupled with poor efficiencies of detection systems necessitated the use of relatively large quantities of protein sample. An alternative to the purely chemical approach was offered by the development of the microbiological determination technique [977]. This method utilized cultures of Neurospora and Lactobacilli strains whose metabolic characteristics enabled the determination of specific amino acids at the milligram level. By turbidimetric assay of the growth rate directly, or titration of the lactic acid or carbon dioxide produced, an estimate of the amino acid concentration could be obtained. Non-specificity of the mutant strains and the purely relative nature of the method contributed largely to its replacement by chromatographic methods. In 1944, CONSDEN, GORDON and MARTIN [212] introduced the technique of paper chromatography. Its application to complex mixtures of amino acids allowed hitherto impossible separations to be readily achieved with a minimum of expense and experience. The inherent sensitivity and the ease of adaptation to specific applications made the method one of the major analytical aids to protein research.
S. B. Needleman (ed.), Protein Sequence Determination © Springer-Verlag Berlin Heidelberg 1970
92
Amino Acid Composition Determination
Concurrent with the development of the paper chromatographic method was the application pf column separation techniques. Early attempts used differential absorption of the amino acids on solids such as aluminum oxide, silica gel and activated charcoal. The use of starch columns eluted with butanol, propanol and dilute hydrochloric acid, was described by MOORE and STEIN [700]. The colorimetric reaction of the collected fractions with ninhydrin allowed the first truly quantitative separa
Data Loading...
