A previously unknown protein has been isolated in your...

A previously unknown protein has been isolated in your laboratory. Others in your lab have determined that the protein sequence contains 172 amino acids. They have also determined that this protein has no tryptophan and no phenylalanine. You have been asked to determine the possible tyrosine content of this protein. You know from your study of this chapter that there is a relatively easy way to do this. You prepare a pure $50 \mathrm{mM}$ solution of the protein, and you place it in a sample cell with a $1-\mathrm{cm}$ path length, and you measure the absorbance of this sample at $280 \mathrm{nm}$ in a UV-visible spectrophotometer. The absorbance of the solution is $0.372 .$ Are there tyrosines in this protein? How many? (Hint: You will need to use Beer's law, which is described in any good general chemistry or physical chemistry textbook. You will also find it useful to know that the units of molar absorptivity are $\left.M^{-1} \mathrm{cm}^{-1} .\right)$

Key Concepts

Protein Quantification by Spectroscopy

Using UV–visible spectroscopy to quantify protein composition involves measuring the absorbance at specific wavelengths that correspond to the absorption of aromatic amino acids. This analytical method capitalizes on the known relationships provided by the Beer–Lambert Law and the specific molar absorptivity values to determine the concentration of the protein and, in cases where only certain residues are present, to deduce the number of these residues in the protein.

UV Absorption of Aromatic Amino Acids

Proteins absorb ultraviolet light primarily due to the presence of aromatic amino acids like tyrosine, tryptophan, and phenylalanine. When certain aromatic residues are absent, any observed absorbance at around 280 nm can be attributed to the remaining types. This concept allows for the estimation of the content of a specific aromatic residue within the protein by applying quantitative methods using their known extinction coefficients.

Molar Absorptivity (Extinction Coefficient)

Molar absorptivity is a measure of how strongly a substance absorbs light at a given wavelength, expressed in M?¹cm?¹. In the context of proteins, each aromatic amino acid contributes a characteristic molar absorptivity. Knowing these values allows one to calculate the number of specific residues (such as tyrosine) present in a protein by comparing the observed absorbance with the expected contribution of each residue.

Beer?Lambert Law

The Beer?Lambert Law relates the absorbance of a solution to its concentration, path length, and the molar absorptivity (extinction coefficient). This concept is fundamental in spectroscopy for quantifying the concentration of absorbing species, as it states that absorbance is directly proportional to both the concentration of the molecule in solution and the length of the light path through the sample.

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