Quantitation of Tryptophan in Proteins
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Accurate measurement of the amount of tryptophan in a sample is problematic, since it is completely destroyed under normal conditions employed for the complete hydrolysis of proteins. Strong acid is ordinarily the method of choice, and constant boiling hydrochloric acid, 6M, is most frequently used. The reaction is usually carried out in evacuated sealed tubes or under N2 at 110°C for 18–96 h. Under these conditions, peptide bonds are quantitatively hydrolyzed (although relatively long periods are required for the complete hydrolysis of bonds to valine, leucine, and isoleucine). As well as complete destruction of tryptophan, small losses of serine and threonine occur, which are corrected for. The advantages of amino acid analysis include the measurement of absolute amounts of protein, provided that the sample is not contaminated by other proteins. However, it may be a disadvantage if an automated amino acid analyzer is not readily available. Acid hydrolysis in the presence of 6N HCl, containing 0.5–6% (v/v) thioglycolic, acid at 110°C for 24–72 h, in vacuo will result in greatly improved tryptophan yields (1), although most commonly, hydrolysis in the presence of the acids described in Section 3.1. may result in almost quantitative recovery of tryptophan.
KeywordsFree Amino Acid Peptide Bond Amino Acid Analysis Seal Tube Complete Hydrolysis
- 2.Goodwin, T. W. and Morton, R. A. (1946) The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem. J. 40, 628–632.Google Scholar