Protein Protocols Protein Protocols

Quantitation of Tryptophan in Proteins 43
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 mole/L; w = tryptophan mole/L; (x – w) = tyrosine mole/L.
e y = Molar extinction of tyrosine in 0.1 M alkali at 280 nm = 1576.
e w = Molar extinction of tryptophan in 0.1 M alkali at 280 nm = 5225.
Also, x is measured from E 294.4 (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.
Therefore,
E 280 = w ε w + (x – w)ε y
w = (E 280 – x ε y )/( ε w - ε y)
4. An alternative method of obtaining the ratios of Tyr and Trp is to use
the formulae (Eq. 2) derived by Beaven and Holiday (5).
M Tyr = (0.592 K 294 – 0.263 K 280) × 10 –3
= (0.263 K 280 – 0.170 K 294) × 10 –3
where M Tyr and M Trp are the moles of tyrosine and tryptophan in 1/ g of protein, and K 294 and
K 280 are the extinction coefficients of the protein in 0.1 N 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:
4. Notes
M Tyr / M Trp = (0.592 E 294 – 0.263 E 280)/( 0.263 E 280 – 0.170 E 294) (2)
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 0.1 M NaOH solution decreases at longer wavelengths
into the region 330–450nm 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 due 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.
(1)