Amino Acid Analysis

Amino acid analysis in the Core has been discontinued due to lack of samples. However, we try to help investigators to get their samples analyzed from an outside core facility.

All samples for amino acid analysis should be salt-free. Contamination of samples with non-volatile primary amines (e.g., Tris, glycine), salts, or detergents affects the accuracy analysis. These materials may be removed by ethanol-precipitation, trichloroacetic acid precipitation, reverse-phase extraction, gel filtration, ion-exchange separation, or dialysis.

The most accurate determination of the amino acid composition of a protein is obtained by "timed analyses," in which a series of analyses are carried out on sample aliquots hydrolyzed for varying periods of time, e.g., 24, 48, and 72 hours. Some amino acids, such as Ser, Thr and Tyr, are partially degraded under hydrolysis conditions. These amino acids are estimated by extrapolating experimental values to time zero. Amino acids, such as Ile, Leu and Val, are released slowly during hydrolysis because peptide bonds involving these residues are more resistant to hydrolysis. The amounts of these amino acids are estimated by the values obtained after 72-hr of hydrolysis. Cystine and cysteine decompose under normal hydrolysis conditions. These amino acids may be converted to stable derivatives, such as carboxymethyl cysteine, pyridylethyl cysteine, and cysteic acid, before hydrolysis. Alternatively, 1/2Cys is converted to a mixed-disulfide derivative during the hydrolysis reaction.

Tryptophan decomposes under normal hydrolysis conditions. Therefore, tryptophan analysis can be carried out after hydrolysis in 3 M mercaptoethanesulfonic acid. Because tryptophan residues are scarce in protein, tryptophan analysis requires a larger amount of sample material than for other amino acids. Tryptophan analysis is carried out only when requested. Alternatively, the tryptophan content can also be determined by UV-spectroscopy. Spectroscopic determination is advantageous in that the material can be recovered for other studies.