Seeing UV-VIS in a Different Light: How to Measure Anything with Chemical Biology's Easiest Multi-Tool

UV-Vis is the most underappreciated tool in all of protein science. If you asked me what the quickest to use, conceptually accessible, and versatile tool in my lab is then I would easily tell you it is my UV-Vis spectrometer. I have spent a plurality of all of my time during my PhD thesis at or around my beloved spectrometer and it is the place where my undergraduate mentees spend about 90% of their time. It has been -by far- the most informative tool at my disposal. In admiration, here is my love letter to my UV-Vis: a list of everything you can easily measure about a protein using only a UV Vis spectrometer (with as many open-access citations as I could find). Like a fancy new pair of earbuds, this article's coming to you in two parts. Part one will describe the intrinsic protein absorbances and chemical functionalizations that can be performed to quantify protein concentration and chemical functionalities. Part two will describe the online resources and chromogenic probes everyone should use in their lab to quickly troubleshoot a challenging assay with UV-Vis.

A quick note about what this article is. It will not dive into the theory of how Beer's Law works which has been elegantly summarized elsewhere. (1) It also won't be a how-to guide for measuring a protein sample using a UV-Vis spectrometer or how to analyze data. I also won't summarize the numerous techniques that are really just UV-Vis with added accoutrements including -but not limited to- fluorescence, circular dichroism, and FPLC/HPLC. Those would be great articles and if you comment which one you want on the post I can write that too. Finally, it will not be a database of molar absorptivity values for every chromophore in the world. I don't have time and other people are working on it. (2, 3) Hopefully it will inspire you to find more uses for the most versatile instrument available in a biochemistry lab.

Part I - Absorbances for Protein Concentration Determination and Functionality Identification

1 - Protein Concentration / Protein Folding

Ah protein concentration, every biochemist's favorite use of the UV-Vis. Nano-drops were specifically developed just to do this: a single-use instrument rivaled in popularity only by the household bagel guillotine. However, there are a few things that you may not know about the classical intrinsic protein chromophores (Trp and Tyr at 280 nm, Phe at 260 nm, and misc amide bonds around 210-220 nm). I would like to offer a friendly reminder that the original papers measuring the molar absorptivity of tyrosine and tryptophan were conducted in 6M guanidinium hydrochloride. (4) As such, the molar absorptivity of a canonical sidechain chromophore on a protein is highly dependent on its local environment. This allows UV-vis to actually measure protein unfolding curves given the right system. (5) Scattering of light due to aggregation will also alter absorption spectra. (6) Finally, if the amide backbone is your only chromophore or if precise concentrations need to be measured at low concentration, 205 nm can always be used instead. (7) It's no wonder that UV-Vis is the first technique analytical technique most biochemistry students apply to their research.

2 - Disulfide Bonding

Oxidation will take us all one day, so remember to drink up the next time you buy a fancy smoothie. But it doesn't have to ruin your assays and UV-vis is a great way to make sure that it doesn't. Using the Ellman's Assay (8) one can react free cysteine in equimolar amounts with a chromogenic dye of your choosing to see if -given a known concentration of protein- what percentage of the free cysteines have reacted to form disulfides. This may sound niche, but for particularly reactive cysteines in enzyme active sites or in close proximity at metal binding sites it is one of the few ways to give a quantitative assessment of the concentration of active site or of properly folded material in a protein sample. For all of you crazy organic chemists out there, this works across thiols as well. For example, I have used it to check that our reduced glutathione in the freezer hadn't gone bad. Recent work was conducted measuring the molar absorptivity values in for Ellman's reagent in various organic solvents. (9) I'm sure someone will put that to good use as well.

3 - Metal Binding

I could talk about this one for hours, but I'll try to keep it brief. Metal binding proteins are ubiquitous in biology with their metal binding sites often impart superb structural stability and functionality. (10, 11) A fun quirk of coordination chemistry is that interactions between metals and their ligands induces a charge transfer with two major absorbance bands for metals bound to proteins: the LMCT (Ligand-metal-charge-transfer) around 300 nm and the d-d transition in the 200-500 range. (12) The later is highly metal and ligand dependent; good luck trying to measure the molar absorptivity of ~50 M-1 cm-1 d-d transition of a His3 cobalt substituted carbonic anhydrase. The former typically has an absorbance in the 1000's M-1 cm-1 although these values are hard to find citations for as the noise from the adjacent Tyr/Trp peak at 280 nm can drown it out. The molar absorptivity of these transitions are highly dependent on the coordination residues, but this makes every metal a very quiet reporter of the holo state of a metalloprotein. UV-Vis can immediately identify tertiary contacts at a metal binding site and be used to obtain the protein's binding affinity for that metal. (13, 14) UV-Vis is also an excellent way to analytically determine the concentration of metal in a protein solution from expressed proteins using the so-called zincon assay. (15) Many metal salts often come in the form of mixed hydrates making an analytical determination of cobalt or zinc concentration pretty tricky without a UV-Vis.

4 - Free Amines

This is a little obscure, but I think it's worth adding. Amines are some of the most nucleophilic functionalities on a protein, this makes them a prime site for post translational modification. Just like how the Ellman's assay can turn a free cysteine into a chromogenic reporter with a known absorptivity, a day long incubation can turn any lysine or terminal residue into a chromogenic reporter via reaction with 2,4-pentanedione. (16) While this compound will also react slowly with Arg residues, careful timing and pH control can ensure selective labeling of lysine. This is an excellent test for identifying particularly nucleophilic amines on proteins in the field of protein design. (17, 18) Another similar reaction used is the Ninhydrin test, however this is extremely sensitive to the exact identity of the free amine. The exact context of the dye formed from the reaction must be considered as well when performing analytical titrations as it is pH sensitive. For this reason, it is better for a qualitative assessment of the presence of an amine. (19) This is also just one proteinaceous example of the kind of functionalization used in analytical chemistry to identify a diverse array of chemical functionalities, which is maybe a good topic for another article.

5 - Enzyme Activity

This is an extremely broad topic with new fluorogenic and chromogenic probes being developed all of the time. (20) With the advent of designer enzymes tailored to specific reactions their diversity will only increase further in the future. Here are three classical chromogenic reporters for three common industrially relevant biocatalytic reactions:

P-nitrophenol Acetate - Esterase Activity

Release of a p-nitro phenol anion is a common chromogenic probe generally for reaction kinetics, so keep an eye out for it in future papers. However, keep an eye out because its molar absorptivity is very pH sensitive and this needs to be accounted for in enzyme assays. (21, 22)

P-nitrophenyl-aldehyde - Aldol/Retro-Aldol Reaction

This works on the same principle shown above, but in this case the p-nitro phenyl aldehyde decrease or production can be used to monitor aldol or retro-aldol reactivity (respectively). The de novo aldolase literature is expansive (17, 18) and worth a read. It is one of the most versatile carbon-carbon bond forming reactions know.

NAD+ In Enzyme Coupled Assays

No chromophore in your substrates or products? No problem. The depletion of NADH/NADPH (23) cofactors can be monitored at 340 nm if your enzyme uses it. It is also a common reporter in so-called "enzyme-coupled-assays." Given that the enzyme of interest uses use ATP as as an energy source instead of NADH/NADPH and is much slower than Lactate Dehydrogenase and Pyruvate Kinase, then its activity can be measured via the depletion of NADH/NADPH as it uses up ATP.

?? Intermission ??

How many times has this happened to you? You're trying a new assay for the first time and, after trying 100 different procedural tweaks, the data looks terrible. Your go-to protocols are insufficient to reproduce a beautiful figure from an old paper. It's going to be tough to figure out which assumption that you (or the authors) made was incorrect. Below are the most common reasons why a biochemical assay can be challenging to reproduce and how the UV-Vis spectrometer is my go-to tool to directly investigate these potential problems.

Part II - Assay Troubleshooting

6 - Chemical Purity

This is another classical use of UV-Vis in nearly every biochemistry lab. Most commonly coupled with HPLC or FPLC, a UV-Vis spectrometer alone can be used to determine chemical purity. This is most common for DNA mini-preps where the A280/A260 can be used to determine the purity of a sample of DNA or RNA, but this can be equally helpful for confirming the purity of a protein sample that strongly absorbs at two distinct wavelengths. Here's an example: the hypothetical peptide YFFFFFF. This would be a challenging peptide synthesis and the purity of the product is dependent on the ratio of A280 (where Tyr absorbs strongly) and A260 (where Phe absorbs maximally). Any impurities with a disproportionately high or low number of Tyr per Phe residues would have a drastically different A280/A260 ratio. These same calculations can be performed with any strongly absorbing compound -at any wavelength- and the available spectral databases allow for homologous chromophores to assist with predictions for these constants. (2, 3)

7 - Assay pH

Look, I won't disrespect a really nice pH meter. It's the best tool to measure the pH of any assay buffer. But what do you do when your precious protein isn't behaving properly after mixing together 5 buffer components? Even worse, sometimes you need to check the pH of 10,000 assay wells to make sure that each of your 10,000 inhibitors aren't just inhibiting your protein of interest by curdling it like paneer. A pH probe just won't cut it for that kind of high throughput optimization. The solution (as-stated before) is the UV-Vis spectrometer. The standard universal pH Indicator is made up of a number of chromophores with various isoelectric points, known molar absorptivities, and absorbance maxima. Any single component can be used to measure the pH of an assay within its transition range to be absolutely confident that this important chemical feature is maintained or to prevent any off-target effects.

For each species, there is a known molar absorptivity which can translate directly to the [Conjugate Base] or [Conjugate Acid] and allow the precise analytical determination of the pH of a solution via UV-Vis and this standard can even be added directly to an existing well as an internal standard, if the assay conditions are amenable.

8 - Ionic Strength

See above for a long-winded justification for why a single pH/Ionic Strength probe isn't always the ideal method for confirming ionic strength. As they say, if I had more time then I would have written a shorter letter. Similarly, if you find that you need an internal standard for ionic strength, congo red is very sensitive to the concentration of salt in solution. If I can be totally honest, it wasn't clear from the paper as to what causes this effect. Whether it is cis-trans isomerization of the azo compound, or some kind of aggregation effect. Regardless, it works! (24) If you find you need an internal standard for ionic strength, the absorbance maximum of congo red is very sensitive to the concentration of salt in solution.

Conclusion

While this list certainly isn't comprehensive, it hopefully gave you a better appreciation for the broad uses of your UV-Vis spectrometer for protein bioanalytical chemistry. If it can save you some time optimizing your next bioassay optimization, then that would be great too. For all of the experts out there, please get in touch with any obvious UV-Vis use cases that I haven't included and I can add them. Hopefully this article can be a living document to help us all get out of lab a little earlier and get every assay to work on the first try :)