All proteins absorb light. The peptide bond has a weak absorption at 230 nm, for
ID: 193520 • Letter: A
Question
All proteins absorb light. The peptide bond has a weak absorption at 230 nm, for example, associated with excitation in the orbital of the O-C-N-H system and its resonance structures. The strongest absorption of light (in the absence of a prosthetic group like a heme or ligand) comes from absorbance by the aromatic amino acid side chains between 240 and 300 nm. Below is a picture of absorption spectra for these side chains. Use it to calculate: 1. 2. 3. E091 for the side chain excitation transition. (20 points) The absorption cross section for each amino acid. (25 points) The magnitude of the transition dipole for each amino acid. (25 points) 40,000 20,000 10,000 5,000 Trp molar extinction coefficient 2.000 1,000 500 200 100 50 Phe 20 10 200 220 24 320 (Alt + A) UV absorbance spectra of the three aromatic amino acids phenylalanine, tryptophan, and tyrosine When you have completed your calculations, answer the following questions (10 points each): 1. 2. Why do Phe and Tyr absorb light at different wavelengths? Why do the extinction coefficients of these amino acids differ so dramatically? (in other words, what does the extinction coefficient mean)Explanation / Answer
Ans 1)
In the near-UV the molar absorbance of phenylalanine is much smaller than that of tyrosine and tryptophan, and the spectrum of a protein (such as Figure) between 240 and 300nm is therefore dominated by the contributions from the Tyr and Trp side-chains. Phe residues contribute fine structure (‘wiggles’) to the spectrum between 250 and 260 nm. The aromatic amino acids do not absorb above 310 nm, and therefore protein absorbance should be zero at wavelengths greater than 310 nm. Proteins without Trp residues do not absorb above 300 nm.
Ans 2)
Estimation of protein concentration by UV-light absorption is not accurate for complex protein solutions (e.g., cell lysates) because of the composition of proteins with different absorption coefficients.
Beer’s Law states that molar absorptivity is constant (and the absorbance is proportional to concentration) for a given substance dissolved in a given solute and measured at a given wavelength. For this reason, molar absorptivities are called molar absorption coefficients or molar extinction coefficients.
The extinction coefficient refers to “a measure of the rate of transmitted light via scattering and absorption for a medium.”
Most protein extinction coefficients (percent) range from 4.0 to 24.0.
W, Trp, Tryptophan=5500 M-1cm-1
Y, Tyr, Tyrosine=1490 M-1cm-1
F, Phe, Phenylalanine=200 M-1cm-1
Each protein has a distinct UV spectrum as well as an extinction coefficient at 280 nm (280). The specific UV spectrum is based on its amino acid composition. Major contributions to the spectra stem from aromatic tryptophan (W) and tyrosine (Y) residues with high extinction coefficients of 5500 and 1490 M-1cm-1. Phenylalanine (F) absorbs maximally at 260 nm but little at 280 nm.