UV absorbance of proteins: practical guide to A280 measurements for finding protein concentration

UV-Vis spectroscopy is commonly used to measure protein concentration based on absorbance of 280 nm light…
blog form: bit.ly/bradforduv
note: if you want to learn about other methods including BCA & Lowry, check out this past post bit.ly/proteina...
Light (electromagnetic radiation) (EMR) is little packets of energy traveling in waves. Different colors have different wavelengths of light with different energies (this is also true for “invisible” colors - wavelengths outside of the visible spectrum - like radio waves, which are lower frequency and ultraviolet (UV) waves which are higher frequency. Different molecules absorb different wavelengths of light to different extents, and this can be quantified by a number called the extinction coefficient (ε), which tells you how well a molecule absorbs light of a particular wavelength. More on why here: bit.ly/2CfaXbJ You can then use this equation called Beer’s law to figure out how much of a protein or DNA or anything that absorbs based on it
But TLDR, it has to do with how much energy the outer electrons of the molecule have and how much more energy they need to get to the next level - electrons can be thought of as living in “houses” called molecular orbitals - it’s not that they “always” live in one place - they’re constantly zipping around, but these orbitals are where you have the greatest chance of finding them. Orbitals farther from the nuclei of the molecules (where the positive protons and neutral neutrons are held) require electrons to have higher energy to live there. If a molecule gets hit by a photon of the optimal energy, the photon can get absorbed and its energy used to promote a lower energy electron to a higher energy, further from the nucleus, orbital.
Electron delocalization through resonance involves the merging of some neighboring molecules’s orbits into that shared donut. And this lowers the cost to move to promote an electron - so the rings in aromatic amino acids can absorb UV light - Tryptophan (Trp, W), Phenylalanine (Phe, F), and Tyrosine (Tyr, Y) can all contribute, but Trp is the main contributor, so the UV280 absorbance per protein is gonna depend on how many Trps that protein has. “Abnormal” Trp numbers can trip you up! “Too many” and you can underestimate and “too few” and you can overestimate protein concentration if you go by the “average,” but if you know the protein’s sequence you can calculate the extinction coefficient for your exact protein
Extinction coefficients can be given in terms of molarity if you know the sequence of the protein. but when you don’t (and even sometimes when you do) they’re given in mg/mL terms. They tell you the absorbance value (A) that corresponds to 10 mg/mL (1%) or 1 mg/mL (0.1%). Those percentages come from the weight/volume percentage convention that a 1% solution corresponds to 1 g/100 mL - more on why here: bit.ly/37LsTrq
You can calculate the estimated extinction coefficient using free online software tools like Expasy ProtParam. I say estimated because context matters - the local environment around the absorbing part can influence how eager it is to absorb a photon
When I do this for BSA I see this:
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 42925
Abs 0.1% (=1 g/l) 0.638, assuming all pairs of Cys residues form cystines
Ext. coefficient 40800
Abs 0.1% (=1 g/l) 0.607, assuming all Cys residues are reduced
First it tells me the values under oxidizing conditions and below that it tells me the values under reducing conditions (the intracellular environment is reducing and we usually add reducing agents like DTT or β-mercaptoenthanol) to protein solutions to keep them happy outside the cell). It tells me this because cysteine crosslinks can also absorb, where applicable.
Then I can plug this into Beer’s law (or have the computer do it for me) if I measure the absorbance.
You measure this absorbance using something called a spectrophotometer. Basically it shines light through a solution and measures to what extent different wavelengths make it through (are transmitted) versus don’t make it through (are absorbed). We looked at how this can be converted into concentration of solute (dissolved molecules) using Beer’s Law bit.ly/2N4nzXE
The equation is: A = εcl
A = absorbance
ε = extinction coefficient (aka molar absorptivity coefficient) - specific for particular molecule & particular wavelength; units of L mol-1cm-1
c = concentration (in mol/L) - this is molarity - a mole is just a chemist’s “baker’s dozen” - it’s Avogadro’s number (6.022 x 10^23) of something - solute molecules or donuts, it’s just a number bit.ly/2r4RnrX
l = path length (in cm)
Finished in comments

[@nill5217]

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[@thebumblingbiochemist]

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[@Ani-v7k]

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[@thebumblingbiochemist]

Happy it helped! Maybe sometime in the future, but I'd need to do more research first in order to adequately cover it. In the meantime, you might enjoy: bit.ly/insulinsequencing

[@Ani-v7k]

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[@ByakuyaKuchiki-y7w]

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[@thebumblingbiochemist]

Hope this can help: bit.ly/biochemistrystartersguide Best of luck!