IIIB

Quantitation of Tryptophan in Proteins

Alastair Aitken and Mich~le Learmonth
1. Introduction

1.1. Hydrolysis Followed by Amino Acid Analysis

Accurate measurement of the amount oftryptophan 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 N 2 at 110~ 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 HC1, containing 0.5-6% (v/v) thioglycolic, acid at 110~ 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.
Alkaline hydrolysis followed by amino acid analysis is also used for the estimation
of tryptophan. The complete hydrolysis of proteins is achieved with 2-4M sodium
hydroxide at 100~ 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 other amino 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 76). A further complication of this
method is possible contamination resulting from autodigestion of the enzymes.

1.2. Measurement of Tryptophan Content by og

The absorption of protein solutions in the UV is the result oftryptophan and tyrosine
(and to a very minor, and negligible, extent phenylalanine and cysteine). The absorpFrom: The Protein Protocols Handbook
Edited by: J. M. Walker Humana Press Inc., Totowa, NJ

29

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Aitken and Learmonth

tion maximum will depend on the pH of the solution, and spectrophotometric measurements are usually made in alkaline solutions. Absorption curves for tryptophan and
tyrosine show that at the points of intersection, 257 and 294 nm, the extinction values
are proportional to the total tryptophan + tyrosine content. Measurements are normally
made at 294.4 nm, since this is close to the maximum in the tyrosine curve (where Ae/A~,
the change in extinction with wavelength, is minimal), and in conjunction with the extinction at 280 nm (where Ae/AX, is minimal for tryptophan), the concentrations of each of
the two amino acids may be calculated. This is the method of Goodwin and Morton (2).

2. Materials
1.
2.
3.
4.

3Mp-toluenesulfonic acid.
0.2% tryptamine 3-[2-Aminoethyl] indole (Pierce, Chester, UK).
3Mmercaptoethanesulfonic acid (Pierce).
1MNaOH.

3. Methods

3.1. Quantitation of Tryptophan by Acid Hydrolysis

1. To the protein dried in a Pyrex glass tube (1.2 x 6 cm or similar, in which a constriction
has been made by heating in an oxygen/gas flame) is added 1 mL of 3Mp-toluenesulphonic
acid, containing 0.2% tryptamine (0.2% 3-[2-aminoethyl] indole) (3).
2. The solution is sealed under vacuum and heated in an oven for 24-72 h at 110~ in vacuo.
3. Altematively, the acid used may be 3M mercaptoethanesulfonic acid, The sample is
hydrolyzed for a similar time and temperature (4).
4. The tube is allowed to cool and cracked open with a heated glass rod held against a horizontal scratch made in the side of the tube.
5. The acid is taken to near neutrality by carefully adding 2 mL of 1MNaOH. An aliquot of
the solution (which is still acid) is mixed with the amino acid analyzer loading buffer.
6. Following this hydrolysis, quantitative analysis is carried out for each of the amino acids
on a suitable automated instrument.

3.2. Alkaline Hydrolysis

1. To the protein dried in a Pyrex glass tube (as above, Section 3.1. step 1) 0.5 mL of 3M
sodium hydroxide is added.
2. The solution is sealed under vacuum and heated in an oven for 4-8 h at 100~ in vacuo.
3. After cooling and cracking open, the alkali is neutralized carefully with an equivalent
amount of 1M HC1. An aliquot of the solution is mixed with the amino acid analyzer
loading buffer and analyzed (as above, Section 3.1. step 6).

3.3. Measurement of Tryptophan Content by UV

1. The protein is made 0.1M in NaOH.
2. Measure the absorbance at 294.4 and 280 nm in cuvets (transparent to this wavelength,
i.e., quartz) in a spectrometer.
3. The amount of tryptophan (w) is estimated from the relative absorbances at these wavelengths by the method of Goodwin and Morton (2) shown in Eq. (1), where x = total
mol/L, w = tryptophan mol/L, and ( x - w) = tyrosine mol/L, lgy = Molar extinction of
tyrosine in 0.1M alkali at 280 n m = 1576. e,w = Molar extinction of tryptophan in 0.1M
alkali at 280 nm = 5225.

Quantitation of Tryptophan

31

Also, x is measured from E294.4 (the molar extinction at this wavelength). This is 2375
for both Tyr and Trp (since their absorption curves intersect at this wavelength). An accurate reading of absorbance at one other wavelength is then sufficient to determine the
relative amounts of these amino acids.
E280 = w ew + (x-w)~:y

(1)

W -" (E280 - x ~,y)/(F, w - ~,y)

(2)

Therefore:
4. An alternative method of obtaining the ratios of Tyr and Trp is to use the formulae derived
by Beaven and Holiday (5).
MTyr = (0.592 K294- 0.263 K280) 10-3
MTrp = (0.263 K280- 0.170 K294) x 10-3

(3)
(4)

where MTyr and MTrp are the moles of tyrosine and tryptophan in 1 g of protein, and K294
and K280 are the extinction coefficients of the protein in 0.1M alkali at 294 and 280 nm.
Extinction values can be substituted for the K values to give the molar ratio of tyrosine
to tryptophan according to the formula:MTyr/MTrp = (0.592 E294- 0.263 E280/0.263 E280- 0.170 E294)

(5)

4. Notes
1. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of
protein at the same wavelength, and hence a few percent of nucleic acid can greatly influence the absorption.
2. In this analysis, the tyrosine estimate may be high and that of tryptophan low. If amino
acid analysis indicates absence of tyrosine, tryptophan is more accurately determined at
its maximum, 280.5 nm.
3. Absorption by most proteins in 0.1MNaOH solution decreases at longer wavelengths into
the region 330--450 nm, where tyrosine and tryptophan do not absorb. Suitable blanks for
294 and 280 nm are therefore obtained by measuring extinctions at 320 and 360 nm and
extrapolating back to 294 and 280 nm.
4. In proteins, in a peptide bond, the maximum of the free amino acids is shifted by 1-3 nm
to a longer wavelength, and pure peptides containing tyrosine and tryptophan residues are
better standards than the free amino acids. A source of error may be owing to turbidity in
the solution, and if a protein shows a tendency to denature, it is advisable to treat with a
low amount of proteolytic enzyme to obtain a clear solution.

References
1. Matsubara, H. and Sasaki, R. M. (1969) High recovery of tryptophan from acid hydrolysates of proteins. Biochem. Biophys. Res. Commun. 35, 175-181.
2. Goodwin, T. W. and Morton, R. A. (1946) The spectrophotometric determination of
tyrosine and tryptophan in proteins. Biochem. J. 40, 628-632.
3. Liu, T.-Y. and Chang, Y. H. (1971) Hydrolysis of proteins with p-toluenesulphonic acid. J.
Biol. Chem. 246, 2842-2848.
4. Penke, B., Ferenczi, R., and Kovacs, K. (1974) A new acid hydrolyisis method for determining tryptophan in peptides and proteins. Anal. Biochem. 60, 45-50.
5. Beaven, G. H. and Holiday, E. R. (1952) Utraviolet absorption spectra of proteins and
amino acids. Adv. Protein Chem. 7, 319.