I would like to compare the change in the absorption spectrum of a protein with the change in pH. However the protein concentration in one pH is not necessarily equal to the concentration at a different pH. Am I supposed to divide each spectrum by its maximum absorption? Or am I supposed to divide each spectrum to its absorbance at a specific wavelength (e.g. divide both spectrums with their respective absorbances at 210nm)? I also found this formula using (A-Amin)/(Amax-Amin). Because the increase and decrease of the peak can also indicate something. However depending on what I use as my divisor, these increase and decrease will also change. Can someone please enlighten me?
You can get a good estimate of protein concentration if you have a pure protein in solution and you know its primary structure. Using Beer's-Lambert Law,
A(280 nm) = ε(280 nm)bc, you can determine concentration, c, by estimating the extinction coefficient at 280 nm, ε, and measuring the absorption at 280 nm, A.
ε(280 nm) = (# of tryptophanyl residues)x(5600 M^-1cm^-1) +
(#of tyrosines)x(1400 M^-1cm^-1)
If there are any disulfides in your protein, then add to the above equation the (# of disulfides)x(120M^-1cm^-1).
You can also use the abosrption of the peptide bond near 210 nm, then the extinction coefficient, ε(214 nm) = (# of peptide bonds)x(923 M^-1cm^-1), however you will have major contribution of absorption from tryptophan at this wavelength.
The ratio (A-Amin)/(Amax-Amin), usually (A at 280 nm)/(A at 254 nm) is a unique value for each protein and can be used as a criterion for protein purity.
Thank you for the answers! I was actually doing the protein concentration determination at 280nm, but the thing is something's up and it seems like the molar absorbance at 280nm of the protein I'm using suddenly decreased just because I used a sample in a basic buffer. I can't explain though why this is so.
Dear Christine,
If you have independent solubility data vs. pH, you can normalize the spectra to identical concentration. If not, it is not easy.
In the pH dependent ansorption spectra usually there is a so-called isosbestic point, where the absoprtion of all pH-s are identical. (It is supposed that the buffer does not change the spectrum).
http://www.rose-hulman.edu/~brandt/Fluorescence/Absorbance_Spectroscopy.pdf
http://www.clinchem.org/content/25/1/143.full.pdf
http://cdn.intechopen.com/pdfs-wm/30981.pdf
http://www.yorku.ca/skrylov/Teaching/BioanalyticalChemistry/Part_3_Using_Light_for_detection.pdf
I think this link is useful,
http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/
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