Very dilute enzyme solutions can easily lose activity due to surface adsorption. This could result in poor reproducibility. The material of the container can also be an important factor.

My suggestion is to perform the incubations at different pH and temperature with reasonably concentrated enzyme solutions (e.g., 1 mg/mL) in polypropylene tubes (such as eppendorf tubes). Be careful to handle all the solutions in the same way, particularly when mixing. Avoid vigorous vortex mixing. Mix gently by inversion of the tubes several times.

Pay attention to optimization of reaction variables and ensure linearity of the reaction curve moving forward using the right assay methods. You should also consider factors such as temperature, enzyme and substrate concentrations.

Adam B Shapiro Lawrence Ekebafe Thank you for your answers! Before the stability assay, I already performed assays to determine the optimum pH and temperature, so I use those settings for the enzyme reaction.

Please let me ask some more things about the reaction setup.

a. For the reaction assay, I use the master mix approach. I first mix the buffer (adjusted to the optimum pH) and substrate (both at a higher concentration), then add water to achieve the desired final concentration. When testing other parameters (such as the effect of glucose or NaCl), I can simply adjust the water volume without altering the concentration of the buffer or substrate.

b. I place the microplate on ice while pipetting the substrate and enzyme, and after finish pipetting I put the plate in the incubator. I want to ensure that the reaction starts at approximately the same time in all wells.

Is my approach correct, or is there anything I need to improve in my enzyme reaction setup? Thank you very much.

You are on the right track, however, there are few necessary precautions to be noted in the course of the study which may include: consequential adjustment of enzyme concentration to ensure the reaction continues at a quantifiable rate within the linear range of the assay. The concentration of the substrate also matters; it should be such that it is okay to saturate the enzyme to prevent inhibition of the product. Time and accuracy for determining formation of products for linearity is crucial, based on the initial speed of the enzyme reaction. The use of controls will do a lot of easiness in the study such as no enzyme and substrate reactions.

You mentioned placing the assay plate on ice, then moving it to an incubator. This may be a source of variability because all the wells of the plate may not warm up at the same rate, with the outermost wells warming the fastest, probably. I suggest preparing the various solutions and performing the incubations and enzyme reactions all at ambient temperature to avoid such problems.

Of course, with storage temperature a variable, you will have to return the enzyme solutions to ambient temperature before starting the assay. To do this, you could incubate the enzymes in tubes, then transfer the tubes to a room temperature water bath to equilibrate the temperature before starting the reactions.

You also mentioned that you dilute the master mix with water to adjust the concentration. I recommend diluting with the assay buffer because diluting the buffer can cause the pH to change. All reagents should be prepared in the assay buffer if possible.

If your assay buffer does not contain any detergent, I strongly recommend adding some to keep the protein from sticking to the plate. I always include 0.01% high-purity Triton X-100 (Thermo-Fisher Scientific) in my enzyme assay buffers.

Adam B Shapiro Thank you very much for your suggestions.

If I dilute the master mix with assay buffer, wouldn't that change the concentration of the buffer? I use 50 mM acetate buffer with 0.3 M NaCl at pH 5. For example, when I don’t add any test substances, I add 180 µL of assay buffer. But when I need to add certain test substances to the reaction mix to observe their effect on enzyme activity (such as glucose or EDTA), the volume of assay buffer is reduced to make space for the test substance, which means the concentration of acetate in the reaction is also reduced. Is that correct?

And for the detergent, should I test the effect of detergent to my enzyme activity first?

I’m sorry for asking such basic questions — I’m just not very confident about how to handle this properly. Thank you!

Lawrence Ekebafe Thank you for your answers.

Based on the suggestions you mentioned regarding important factors to be aware of, is this a correct workflow to follow after purifying the enzyme?

Does the sequence of steps make sense, or is there anything that should be rearranged?

I am sorry for asking such basic questions — I’ve tried to plan and carry out the enzyme assays out several times, but I keep getting confused.

Thank you very much.

Are you expecting linearity after incubation of enzyme assay at different time extending from 1 to 10 hrs.

This is rare as over extended period of enzyme assays the substrate may become limiting

Product inhibition may occur

Your have to choose the time period of incubation in between linearity at fixed substrate concentration , enzyme concentration, pH , temperature etc.

Similarly keep all parameters fixed vary one and optimise the reaction rate linearity

Amarjit K Nath Thank you for your response.

I was studying the effect of pH and temperature on enzyme stability, so the enzyme was incubated alone for 10 hours. After mixing the enzyme with substrate, I incubated the reaction mixture for only 10 minutes.

Imagine you want to test 2 concentrations of glucose: 0 and 100 mM. There are a various ways you could set things up so that the assay buffer concentration is the same for both samples. Here are 2 ways:

1. You dissolve glucose in the assay buffer to some multiple of 100 mM, let's say 200 mM, or twice the final concentration.

You then add one volume of assay buffer or 1 volume of 200 mM glucose in assay buffer to one volume of 2X master mix, resulting in 1X master mix containing 0 or 100 mM glucose.

2. You make a concentrated glucose solution in water at, let's say 1 M, or 10 times the final concentration. You prepare a master mix which is 1.11 times the final concentration of enzyme and 1.11 times the final concentration of assay buffer. You then add 1 volume of water or 1M glucose to 9 volumes of 1.11X enzyme master mix containing so that the final buffer and enzyme concentrations are 1X.

By the way, I think this master mix idea comes from PCR. I think of things differently. I prepare highly concentrated stock solutions of each ingredient, if possible, dilute them all in the assay buffer, and then combine them to start the reaction, adding the enzyme last. If there is an enzyme, a substrate mixture and a test substance, I prepare 3X solutions of each component by diluting the stock solutions in the assay buffer and mix them together in equal volumes. The assay buffer is slightly diluted, because the stock solutions are usually in water, except for the enzyme, which is in storage buffer, but the dilution factor is negligible. When I can't make sufficiently concentrated stock solutions, so that the dilution of the assay buffer is not negligible, I start with a more concentrated assay buffer to account for the dilution.

I would suggest you test the effect of detergent on your enzyme activity. In many cases, the enzyme activity appears to be enhanced by detergent because it prevents the enzyme from being adsorbed to the plate surface, where it loses activity. The detergent works by sticking to the hydrophobic plastic surface by its hydrophobic part, leaving its hydrophilic part facing into the solution, thereby converting a hydrophobic surface into a hydrophilic one. As I mentioned before, 0.01% high-purity Triton X-100 works very well for this. This concentration is below the critical micellar concentration (CMC) of Triton X-100, so the detergent does not form micelles.

People sometimes use bovine serum albumin (BSA) as a "blocker" to coat the plate surface instead of detergent. I prefer detergent because I mostly want to test the effect of small drug-like molecules on the enzyme activity. Such molecules can be bound by BSA.

Wynne if you want to study the effect of different temperature and pH on enzyme stability

you need to incubate enzyme at different temperatures for a particular period say 10 min 20 min 30 min and so on bring it to cold temperature

Then perform assay at optimum conditions and see activity In that case you need to fix same time for assay

Similarly incubate enzyme at different pH for optimum time period till enzyme is stable and shows linearity

perform assay at optimum conditions