Enkephalin amino acid analysis

Figure 13. Tryptic peptides derived from adrenal opioid peptides F and I (see Fig. 7) (from Kimura et a/., 1980). Chromatography was carried out on an Ultrasphere octyl column using a gradient of n-propanol at pH 4.0. (A) Standard a, [Met ]enkephalin-Lys b, [Met ] enkephalin[Arg ] c, [Met ]enkephalin d, [Leu ]enkephalin e, [Met ]enkephalin-Arg -[Phe ]. (B) Tryptic digest of peptide F. (C) Tryptic digest of peptide I. Aliquots of the collected fractions were tested for opioid activity and hydrolyzed for amino acid analysis.

One of the first applications of HPLC in the clinical field was the quantitation of theophylline in asthmatic infants. This highly accurate measurement was an important test because of the very low amount of sample required and the accuracy of the determination (see Fig. 1-10). More recent clinically related HPLC separations include drugs and drug metabolites, neurochemicals and their metabolites, histamines, thyroid hormones, and enkephalins. The earliest bioresearch applications of HPLC included the determination of peptides, proteins, and amino acids. Application of HPLC to the analysis of these compounds remains important, as indicated by the rapid growth in references (Fig. 1-11). Bioresearch remains one of the most rapidly expanding growth areas of LC. 

In some cases, redundant amino acids may render analysis more complex. In the case of the opioid pentapeptide enkephalin, Tyr-Gly-Gly-Phe-Met, analysis has been aided by the incorporation of stereochemically deuterated glycine residues. Such studies have shown that conformational averaging in the peptide backbone must occur in order to account for the observed spin-spin couplings (Fishman et a/., 1978). 

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