Protein Protocols Protein Protocols

42 Aitken and Learmonth
1.2. Measurement of tryptophan content by UV
The method of Goodwin and Morton (2) is described in Subheading 3.3.
The absorption of protein solutions in the UV is due to tryptophan and tyrosine (and
to a very minor, and negligible, extent phenylalanine and cysteine). The absorption
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, as this is close to the maximum in the tyrosine curve (where ∆ε/∆λ, the
change in extinction with wavelength, is minimal), and in conjunction with the extinction
at 280 nm (where ∆ε/∆λ is minimal for tryptophan) the concentrations of each of
the two amino acids may be calculated (see Notes 1 and 2).
2. Materials
1. 3 M p-toluenesulfonic acid.
2. 0.2% 3-(2-Aminoethyl) indole.
3. 3 M Mercaptoethanesulfonic acid (Pierce-Warriner).
4. 1 M NaOH.
3. Methods
3.1. Quantitation of Tryptophan by Acid Hydrolysis
1. To the protein dried in a Pyrex glass tube (1.2 × 6 cm or similar, in which a constriction
has been made by heating in an oxygen/gas flame) is added 1 mL of 3 Mp-toluenesulfonic
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 24–72h at 110°C, in vacuo.
3. Alternatively, the acid used may be 3 M 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 1 M NaOH. 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 described in Subheading 3.1., step 1),
0.5mL of 3 M sodium hydroxide is added (See Notes 3).
2. The solution is sealed under vacuum and heated in an oven for 4–8 h at 100°C, in vacuo.
3. After cooling and cracking open, while one is wearing safety goggles, the alkali is neutralized
carefully with an equivalent amount of 1 M HCl. An aliquot of the solution is
mixed with the amino acid analyzer loading buffer and analyzed (as described in
Subheading 3.1., step 6).
3.3. Measurement of Tryptophan Content by UV
1. The protein is made 0.1 M in NaOH.
2. Measure the absorbance at 294.4 nm and 280 nm in cuvettes transparent to this
wavelength (i.e. quartz) in a spectrometer (see Note 4).