Chymotrypsin Inhibitor Research

I am currently trying to test a couple inhibitors of chymotrypsin (Sourced from bovine pancreas).

The experimental protocol for doing this is as follows:

First, a stock solution of 7000nM of the enzyme in phosphate buffer (pH 7.59. Buffer prepared by dissolving 0.44g of dipotassium phosphate in distilled water followed by adding HCl until the pH is reached.) is prepared.
A stock solution of 1.4mM of substrate in pure DMSO is prepared.
A stock solution of 40000nM inhibitor in pure DMSO is prepared.

In a cuvette, varying amounts of the substrate stock solution is added along with some buffer without any dissolved enzyme. 0.5mL of the enzyme solution is added to initiate the reaction. The rate of absorbance change at 410nM is measured over the course of 2 minutes in a spectrophotometer. This is repeated 2 times. The idea is to measure parameters such as maximum velocity and the Km value and see how the presence of the inhibitor affects these parameters.

Substrate



Experimental inhibitor



Inhibitor 2



DMSO is used for the stock solutions of these compounds because they are believed to be almost insoluble in water.

Observation number 1:
It appears that the presence of DMSO causes the enzyme to misfold. I had the thought to run a series of control tests where some amount of substrate solution is added and then some DMSO to top it off. At 40% DMSO by volume (Not accounting for any volume contraction that might be taking place), enzyme activity at 0.28mM substrate concentration plummets by around 60% compared to the same substrate concentration but at 20% DMSO by volume.

Observation number 2:
Mixing 0.2mL inhibitor solution and 0.2mL substrate solution followed by 0.1mL of enzyme-less phosphate buffer causes the entire mixture to turn yellow, which happens to be the color of the product of the degradation of the substrate by the enzyme. However, adding the 0.5mL enzyme solution caused the mixture to once again turn clear before slowly turning yellow due to enzyme activity.

Observation number 3:
Relationship between enzyme concentration and rate of absorbance increase is linear for the range of enzyme concentrations tested (350-3500nM)

In light of these things, how can I accurately determine the inhibitory activity of the experimental inhibitors? Would making a set of plots for rate of absorbance change vs substrate concentration at fixed amount of DMSO without inhibitor and comparing these to what is observed in the presence of inhibitor for the same amount of DMSO work?
What is the explanation for observation #2?
Based on the inhibitor structures, can they expected to act as suicide inhibitors of chymotrypsin or not?
Does anyone know if the denaturation of enzymes in the presence of DMSO is time-dependent?

[Edited on 25-11-2022 by Falvin]

Quote: Originally posted by UC235

N-acetyl-L-tyrosine Ethyl ester is water soluble so you can omit the DMSO. A more conventional chymotrypsin assay uses N-benzoyl-L-tyrosine ethyl ester in 50% methanol which is then added 50:50 to buffer for a final concentration of 25% methanol. Esterase activity (which parallels amidase activity) is monitored in the UV.