Protein UV absorption - starring tryptophan, Beer's Law, & more

Like all molecules, Trp is made up of atoms (individual carbons, hydrogens, etc.). Atoms link up by sharing pairs of electrons - you need 2 for a single bond & 4 for one of the shorter, stronger, double bonds. But what if you don’t quite have enough? You might want to join an electron commune! (otherwise know as resonance/electron delocalization/conjugation). Tryptophan’s a fan of this. Tryptophan is AROMATIC. That doesn’t mean it smells nice, it just means that it has rings and, in the rings, after they’ve “spent” 1 electron each on the bonds to their neighbors they donate their “extra” into a communal shared stock. Those atoms that opt into this commune get to share, and this leads to electron delocalization above and below aromatic rings, kinda like a donut.
more here: bit.ly/tryptoph...
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And the aromatic-ness makes it “visible” in the “invisible” range. Spectroscopy uses light to measure things by taking advantage of different molecules’ tendency to absorb certain wavelengths of light to different extents (e.g proteins absorb strongly at one wavelength and DNA absorbs strongly at another). So, basically, if you shine light through a solution and then look to see what light makes it through you can infer things about what was in that solution. The more light that’s “missing,” the more that was absorbed and thus the more of that molecule there was. ⠀
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This comes in really handy. For example, we have a UV detector that monitors what comes off protein-purification columns so you can tell where your protein comes off. And we often use a NanoDrop spectrophotometer to measure concentration of molecules like nucleic acids.⠀
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In addition to concentration, a lot of information about purity can be gained by looking at where they shouldn’t be absorbing much light. The height of the peak you usually focus on for a particular molecule is where it absorbs best - this corresponds to how much stuff is there (concentration) whereas ratios between peaks can tell you about how pure that stuff is.⠀
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But how does all this actually work? And how does Trp fit in?⠀
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Light (electromagnetic radiation) is little packets of energy traveling in waves. Different colors have different wavelengths of light with different energies. 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⠀
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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. ⠀
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Electron delocalization through resonance involves the merging of some neighboring molecules’s orbits (in the case of aromatic rings, they merge into that shared donut). And this lowers the cost to move to promote an electron. ⠀
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All proteins absorb some UV. Proteins peak at 280 & 230. The 230nm absorbance is from the generic backbone part - corresponds to absorbance by the peptide bonds linking the letters (those have some resonance too remember). The parts of proteins that absorb at 280 are aromatic rings (sound familiar?) Only 3 of the 20 common amino acids have these, and different proteins have different numbers of these 3 amino acids. So different proteins absorb 280nm light differently, which is reflected by different extinction coefficients. ⠀
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Since Trp is the main absorber at 280, the the UV280 absorbance per protein is gonna depend mainly 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 “estimated extinction coefficient” for your exact protein, using free online software tools like Expasy ProtParam, which you can stick into Beer’s law. I say estimated because context matters - the local environment around the absorbing part can influence how eager it is to absorb a photon. much more here: bit.ly/bradforduv

[@sofiaherrera8506]

I’ve been looking for so long but no video could explain this as perfectly as you did! Thank you very much

[@thebumblingbiochemist]

Glad it helped!