Below is an excellent article regarding the implementation of L-Tryptophan in the test, and calibration of UV optical instrumentation.
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Contribution of L-Tryptophan to the Ultraviolet Absorpation Spectra of Proteins
The ultravoilet (UV) absorption spectrum of proteins is the most commonly used method for determining the concentration of a protein in solution. A typical UV spectrum of a protein is shown below.
Figure 1: Typical Protein UV Absorption Spectrum
Protein spectra have a large peak in the far UV between 190 and 205 nm. Only the tail of this peak is shown in Figure 1 above. The near UV protein spectrum typically has a minimum around 250 nm, and a peak around 280 nm that tails off to baseline around 330 nm. In addition to the concentration of the protein, a UV spectrum can tell one a lot about the protein sample. The minimum at 250 nm is normally half the height of the peak at 280 nm. If DNA is present, as is often the case during the early stages of purification, then the 250 nm to 280 nm ratio will increase. If the protein sample contains protein aggregates, the tail on the 280 nm peak will fail to reach the baseline by 330nm, due to the light scattering of the aggregates.
Protein Concentration Determination by Ultraviolet Spectroscopy
The concentration of a pure protein is proportional to the absorbed light measured in the near UV, as stated in the Beer's Law (1854).
A=ε l c
Where A is the absorption, negative log of the transmitted light intensity divided by the incident light intensity; ε is the molar absorption coefficient, sometimes referred to as the extinction coefficient; l is the path length the light passes through; and c is the concentration. The standard units for ε, l and c are L mol-1 cm-1, cm and mol L-1 (Molar or M), respectively.
Solving for concentration, we get the concentration of the protein is equal to the absorbance (A) divided by the molar absorption coefficient of the protein (ε) and the path length (l).
c=A / ε l
Most proteins have a molar absorption coefficient in L mol-1 cm-1 within a factor of two of their molecular weight in Daltons, and with the linear response of most UV spectrophotometers best below absorbances of 1, it's recommended that protein UV spectra be collected at concentrations around or just below 1 g/L (mg/ml) using a standard 1 cm path-length cell at 280 nm.
Amino Acid Contributions to Ultraviolet Spectra of Proteins
The shape of the peak at 280 nm depends on the protein sequence and the environment of the amino acids that contribute to the 280 nm peak. There are only three amino acids that significantly contribute to the shape of the 280 nm peak: Tryptophan (Trp or W), Tyrosine (Tyr or Y) and Cysteine (Cys or C), when it is in a disulfide bond with another cysteine residue; see Figure 2.
Figure 2: UV Absorption Spectra of Amino Acids
Tryptophan, Tyrosine, Phenylalanine, and Histidine spectra were obtained from Oregon Medical Laser Center's PhotochemCAD by Jonathan Lindsey. The Cystine sectrum was obtained from Fred W. Ward, (1923). All of the spectra were scaled to their molar absorption values.
L-Tryptophan has the largest contribution to the absorption spectrum of a protein on a by-amino acid basis, thus it is commonly used as a standard to calibrate UV spectrophotometers for protein concentration determinations. In solution, the amino acid cysteine forms a dimer, cystine, through the formation of a disulfide bond between the thiol groups. It is this disulfide bond in cystine that contributes to the 280 nm peak in a protein's UV spectrum. Under reducing conditions, such as in a cell, or in the presence of dithiothreitol, the disulfide bonds between the cysteine residues are broken.
Molar Absorption Coefficient Prediction
There a a number of web-based protein analysis services available for prediction of the molar absorption coefficient of a protein (ε[protein]) around the 280 nm peak, including ExPASy ProtParam, and Protein Calculator by Chris Putnam at The Scripps Research Instutute. Both use the Edelhoch et al. (1967) method for calculating ε(protein), in which the number of tryptophan residues in the protein sequence (N(Trp)) multiplied by the tryptophan molar absorption coefficient (ε(Trp)) is added to the number of tyrosine residues in the protein sequence (N(Tyr)) multiplied by the tyrosine molar absorption coefficient (ε(Tyr)) plus the number of cysteine residue pairs in the protein sequence (N(Cystine)) multiplied by the cystine molar absorption coefficient (ε(Cystine)).ε(protein)=N(Trp) x ε(Trp) + N(Tyr) x ε(Tyr) + N(Cystine) x ε(Cystine) ExPASy ProtParam uses the molar absorption coefficients from Pace et al. (1995) from 80 proteins in water. ProteinCalculator uses the Gill and von Hippel (1989) coefficients, based on data from 18 denatured proteins in 6 M guanidine hydrochloride; see Table 1 below.
Table 1: Molar Absorption Coefficients Used in
Protein Molar Absorption Predictions at 280 nM
Both of these methods return mean percent deivations of less than 4%. But there are are cases where the percent deviation is greater than 10%, such as with Insulin where the perdicted values range from 1.9% to 13.66% higher than the observed values at 280nm depending on the insulin sample (Pace et al. (1995); Gill and von Hippel (1989)). In the ExPASy ProtParam documentation states “Generally, the calculated values deviated much more often (>10%) from the measured for proteins that do not contain Trp residues. This is due to the fact that Trp contributes much more to the overall extinction coefficient than does Tyr and cystines, and that the Trp extinction coefficient is less sensitive to the environment than the one for Tyr.”
Beer, A. (1854) Einleitung in die höhere Optik.
Fred W. Ward (1923) Biochem J. 17(6): 898–902; The Absorption Spectra of Some Amino Acids.
Edelhoch, H. (1967) Biochemistry 6, 1948-1954. Spectroscopic determination of tryptophan and tyrosine in proteins.
Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) Protein Sci. 11, 2411-2423. How to measure and predict the molar absorption coefficient of a protein.
Gill, S.C. and von Hippel, P.H. (1989) Analytical Biochemistry, 182: 319-326. Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data.
ExPASy ProtParam documentation
Protein Calculator by Chris Putnam at The Scripps Research Instutute
Chemical Nature of the Amino Acids by Michael W. KingI'm a paragraph.
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This image was created by John Altman at the Emory Vaccine Center.
Reference Solution ε(Trp) ε(Tyr) ε(Cystine)
Pace et al. (1995) Water 5500 1490 125
Gill and von Hippel (1989) GuHCl 5600 1280 120
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