UV-VIS Anti-cholesterol esterase assay

UV-VIS Anti-cholesterol esterase assay (1 sample)

Measuring the inhibition of cholesterol esterase activity using UV-Vis spectrophotometry is a valuable method for evaluating compounds or substances that may have anti-cholesterol esterase properties. Cholesterol esterase is an enzyme that catalyzes the hydrolysis of cholesterol esters. Here's a general protocol for conducting an anti-cholesterol esterase assay using UV-Vis spectrophotometry:

Materials and Reagents:

Cholesterol esterase enzyme
Substrate solution (e.g., p-nitrophenyl acetate)
Inhibitor substance or sample extract
Appropriate buffer solution (e.g., phosphate buffer, pH 7)
Deionized water
Positive control inhibitor: Orlistat
Pipettes and pipette tips
Test tubes or microcentrifuge tubes
Vortex mixer or shaker
Timer or stopwatch
Lint-free tissue for cuvette cleaning

Procedure:

Prepare a Blank Solution:

Use a UV-grade quartz cuvette and fill it with the buffer solution (without any inhibitor or cholesterol esterase). This will serve as your blank reference for baseline correction.

Calibrate the Spectrophotometer:

Turn on the UV-Vis spectrophotometer and allow it to warm up.
Set the wavelength to the appropriate value for monitoring the enzymatic reaction. The exact wavelength may depend on the specific assay conditions, but it's typically around 405-410 nm for p-nitrophenyl acetate.
Adjust the spectrophotometer's baseline using the blank solution, so that it reads zero absorbance at the chosen wavelength.

Prepare the Reaction Mixture:

In a test tube or microcentrifuge tube, mix the following components in the specified order:
A known volume of cholesterol esterase enzyme solution
A known volume of substrate solution (e.g., p-nitrophenyl acetate)
A known volume of inhibitor substance or sample extract
Buffer solution to reach the desired total volume
Ensure that the enzyme and substrate concentrations are consistent between experiments.

Incubate the Reaction Mixture:

Incubate the reaction mixture at an appropriate temperature (typically around 37°C) for a specific amount of time. The incubation time may vary depending on the enzyme kinetics and the specific assay conditions.

Measure Absorbance:

After the incubation, take a small volume (usually 1 mL) of the reaction mixture and transfer it to a quartz cuvette.
Wipe the cuvette with a lint-free tissue to remove any fingerprints or smudges.
Place the cuvette in the spectrophotometer and record the absorbance at the chosen wavelength.

Calculate Cholesterol Esterase Inhibition Percentage:

Compare the absorbance of the sample with the inhibitor to the control sample without the inhibitor (blank)