Ask a Chemist: How Colorimetric Assays Work

One of my colleagues, a very good molecular biologist, told me that the only time she uses chemistry is when she needs to calculate molarities. I, of course, scoffed at this statement, and tried to remind her of all the chemistry she uses daily. True, I may be a bit biased since I am a chemist, but surely, all the chromophores, fluorophores, and imaging probes, etc., involve some knowledge of chemistry and chemical reactions. She shrugged, and replied that they’re all supplied in kits.

So, in defence of all chemistry nerds out there, here is my rebuttal: even the most traditional of molecular biologists perform chemical reactions nearly every day. From purification by ion exchange resins and affinity columns, to calculating protein concentrations and coupling bioconjugates, chemistry is behind it all. For this article I will focus on a couple of colorimetric assays and hopefully convince you that knowledge of these chemical reactions is helpful in performing these assays and understanding the results, as well as in troubleshooting when things go awry.

By taking advantage of the electronic properties and specific wavelengths of chromophores, we can deduce a great deal of information.  Colorimetric assays help us to distinguish a particular enzyme from another, to quantitate catalytic activity, as well as inhibition of this activity, to generate proliferative and toxicity profiles, and even to determine the concentration of proteins in solution. They are a simple and convenient way to visualize biological processes. As a former PI of mine used to say, in his thick German accent, while pointing to his eyes, “Nature has provided us with our own spectrophotometers, no?”

Even more appealing, many colorimetric assays are commercially available as kits, usually with a detailed protocol. In fact, colorimetric assays are used so regularly in chemistry and biology, that to cover them all would take an entire book.  So, for simplicity’s sake, this article will focus on two phosphatase assays.

Kinases and phosphatases mediate a variety of cellular processes like metabolism, gene transcription and translation, protein-protein interactions, and apoptosis, through protein phosphorylation and dephosphorylation.  For this reason, many different technologies, including various colorimetric assays, exist to detect these enzymes and their activity.

para-Nitrophenylphosphate

Assay Mechanism

para-Nitrophenylphosphate (pNPP) is widely used as a synthetic substrate to measure the catalytic activity of various phosphatases.

The phosphate group of pNPP is cleaved by the enzyme to yield p-nitrophenol, which is also colorless since the wavelength maximum for the electronic excitation of p-nitrophenol in water is 318 nm. However, under alkaline conditions, the p-nitrophenol is converted to the p-nitrophenolate anion, resulting in a bathochromic shift (a change in absorbance of a compound to a longer wavelength based on solution conditions) to around 400 nm. This is at the blue edge of the electromagnetic spectrum, but since we see the color of the reflected light (as opposed to the absorbed light), the solution looks yellow.

Things to Keep in Mind…

One of the great selling points of the pNPP assay is its simplicity, so it’s easy to see why one might forget that this bathochromic (a great trivia word!) shift is a crucial element to the assay. What if, for example, the enzymatic activity requires an optimum pH in the acidic range, as so happens to be the case with acid phosphatase.  In that instance, the end point addition of a strong base (such as sodium hydroxide or potassium hydroxide) is necessary in order to obtain the desired yellow color.

Malachite Green

Assay Mechanism

The para-nitrophenylphosphate assay is great, but what if your favourite phosphatase doesn’t efficiently metabolize pNPP? Another commercially available phosphatase assay is the malachite green assay. This simple assay method is based on the complex formed between malachite green, ammonium molybdate, and free orthophosphate (aka inorganic phosphate, Pi) under acidic conditions.

 

Orthophosphate, liberated from a phosphorlyated substrate upon cleavage by the phosphatase, forms a complex with ammonium molybdate in a solution of sulfuric acid.  The formation of the malachite green phosphomolybdate complex, measured at 620-650 nm, is accordingly directly related to the free orthophosphate concentration. Therefore, it is possible to quantify phosphorylation and phosphate release from protein phosphatase substrates.

Things to Keep in Mind…

This assay measures only inorganic free phosphate; organophosphates (lipid-bound or protein-bound phosphates) must first be hydrolyzed and neutralized prior to measurement.  Knowledge of the different classes of organophosphates and their respective different free energies of hydrolysis aids in the optimization of assay conditions.  Higher-energy organophosphates (acetal phosphates, phosphoanhydrides, etc ) possess acid-labile phosphate groups that can be released into solution by incubation at low pH. On the other hand, lower-energy organophosphates (phosphoesters) are stable in acidic solution and require much harsher conditions (such as thermal decomposition) to be detected by this method.

In the presence of a large excess of malachite green, the 3:1 ion associate ((MG⁺)₃(PMo₁₂O₄₀ ^3-)) can easily form and precipitate in the acidic aqueous solution. To stabilize the 1:1 ion associate in the aqueous solution, polyvinylalcohol is added to the solution.

Another thing to take into consideration during the assay is the possibility of any redox reactions that could interfere with the assay. Molybdenum is a transition metal and so exists in many oxidation states. In the molybdate anion, it has an oxidation state of +6.  Reduction of the acidified Mo(VI) solution by organic compounds, like ascorbic acid and reducing sugars (i.e. glucose), or by inorganic compounds, like SnCl₂, creates the Mo(IV) species, which is blue in color.

These two examples barely skim the surface of the colorimetric assays. Just think of all the catalase assays for the detection of hydrogen peroxide (like Sigma’s quioneimine derived assay), and of course, the very popular MTT-based assays for measuring cytotoxicity.  A better understanding of the chemistry involved in these assays helps to more deeply understand the reactions, and any possible troubleshooting procedures, as well, and will be discussed at a later time.

Colorimetric assays are just one of the many ways in which chemistry and biology work hand in hand in scientific research.  I think it’s fair to say that the multi-disciplinary approach is now recognized as a vital way to move forward in the drug discovery process. My chemical biology professor commented that in the ‘olden days’ it was enough to study medicinal chemistry and that the pharmaceutical companies would teach you the rest. Now, medicinal chemists are expected to understand pharmacology, cell biology, and molecular biology techniques.  It takes an entire team of people, involving many specialities, in order to be successful in developing safe and effective drugs.  But it is not enough simply to involve many collaborators; I would argue that researchers on all sides need to learn to occasionally think alike.  So hopefully the next time my aforementioned colleague performs a colorimetric assay she can don her ‘chemistry hat’ in addition to her ‘molecular biology hat’, and remember that drug research is an interdisciplinary art.