Protein Protocols Protein Protocols

Quantitation of Tryptophan in Proteins 41
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Quantitation of Tryptophan in Proteins
Alastair Aitken and Michèle Learmonth
1. Introduction
1.1 Hydrolysis Followed by Amino Acid Analysis
Accurate measurement of the amount of tryptophan in a sample is problematic, as 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 6 M hydrochloric acid is most frequently used. The reaction is usually carried
out in evacuated sealed tubes or under nitrogen 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 valine, leucine, and isoleucine). As well
as complete destruction of tryptophan, small losses of serine and threonine occur, for
which corrections are made. 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 6 N 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
Subheading 3.1. may result in almost quantitative recovery of tryptophan.
Alkaline hydrolysis followed by amino acid analysis is also used for the estimation
of tryptophan. The complete hydrolysis of proteins is achieved with 2–4 M sodium
hydroxide at 100°C for 4–8 h. This is of limited application for routine analysis,
because cysteine, serine, threonine, and arginine are destroyed in the process and
partial destruction by deamination of asparagine and glutamine to aspartic and
glutamic acids occurs.
The complete enzymatic hydrolysis of proteins (where tryptophan would be quantitatively
recovered) is difficult, because most enzymes attack only specific peptide
bonds rapidly. Often a combination of enzymes is employed (such as Pronase), and
extended time periods are required (see Chapter 82). A further complication of this
method is possible contamination resulting from autodigestion of the enzymes.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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