I I IB

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 condi- tions, 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 measure- ment 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 ana- lyzer is not readily available. Acid hydrolysis in the presence of 6N HC1, contain- ing 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 quanti- tatively 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 absorp-

From: The Protein Protocols Handbook Edited by: J. M. Walker Humana Press Inc., Totowa, NJ

29

30 Aitken and Learmonth

tion maximum will depend on the pH of the solution, and spectrophotometric measure- ments 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 extinc- tion 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. 3Mp-toluenesulfonic acid. 2. 0.2% tryptamine 3-[2-Aminoethyl] indole (Pierce, Chester, UK). 3. 3Mmercaptoethanesulfonic acid (Pierce). 4. 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 hori-

zontal 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 wave-

lengths 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 nm= 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 accu- rate 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)

Therefore: W -" (E280 - x ~ ,y ) / (F , w - ~,y) ( 2 )

4. An alternative method of obtaining the ratios of Tyr and Trp is to use the formulae derived by Beaven and Holiday (5).

MTy r = (0.592 K294- 0.263 K280) • 10 -3 (3) MTrp = (0.263 K280 - 0.170 K294) x 10 -3 (4)

where MTy r 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/MTr p = (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 influ- ence 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 hydroly-

sates 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 deter-

mining 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.