Harper's Illustrated Biochemistry, Volume 1 - Murray R. 1993

Structure and Functions of Proteins and Enzymes
Peptides
Determination of the Amino Acid Composition of Peptides

First of all, peptide bonds are cleaved via Hydrolysis. Because peptide bonds are stable at neutral pH, acid or base catalysis is employed. Enzymatic Catalysis is less suitable for complete hydrolysis. Complete Protein Hydrolysis into its constituent Amino Acids is inevitably accompanied by a partial loss of Certain amino acid residues. Hydrolysis is best performed in 6 N HCl at 110 °C in an evacuated sealed tube. Under these conditions, Tryptophan and Cysteine are completely destroyed, and cystine is partially destroyed. In the presence of metals, a partial loss of Methionine and Tyrosine is observed. Glutamine and asparagine are quantitatively deamidated to glutamate and aspartate. The content of Serine and Threonine is also underestimated, to an extent that increases with the duration of hydrolysis. Finally, some bonds between neutral residues (Val-Val, Ile-Ile, Val-Ile, Ile-Val) are only 50% hydrolyzed even after 20 h. Typically, parallel samples are hydrolyzed for 24, 48, 72, and 96 h. Then, the data for serine and threonine are plotted on a semi-logarithmic scale and extrapolated to zero time. For valine and isoleucine, the results obtained from 96-h hydrolysis are used. Dicarboxylic acids and their amides are determined as a sum and reported as "Glx" or "Asx". Prior to hydrolysis, cysteine and cystine are converted into acid-stable derivatives (e.g., cysteic acid). Alkaline hydrolysis is used for the analysis of tryptophan; however, this causes the destruction of serine, threonine, Arginine, and cysteine, as well as the racemization of all amino acids. Upon completion of hydrolysis, the Amino Acid Composition can be determined using automated Ion-exchange Chromatography (see Fig. 3.12) or high-pressure liquid chromatography.

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Fig. 4.7. Gel filtration of Cyanogen bromide (CB) fragments of human fetal globin. Chromatography was performed on a Sephadex G-50 Column equilibrated with 1% HCOOH, followed by elution with the same solution. The numbering of the α- and γ-chain fragments is arbitrary. (Courtesy of J. D. Pearson et al., Department of Biochemistry, Purdue University.)

Fig. 4.8. High-performance reverse-phase liquid chromatography: elution profile of cyanogen bromide (CB) fragments of human fetal globin. The numbering of the α- and γ-chain fragments is arbitrary. (Courtesy of J. D. Pearson et al., Department of Biochemistry, Purdue University.)