Скачать или смотреть Carbonic Anhydrase Esterase Activity Assay

An esterase assay is a biochemical test that measures the activity of enzymes called esterases. These enzymes hydrolyze esters into alcohol and carboxylic acid reaction products. When esterase enzymes act on the colorless ester substrate 4-nitrophenyl acetate, the yellow colored hydrolysis product 4-nitrophenol is produced (along with acetic acid). The rate at which the yellow color forms can be used to quantify the enzyme's catalytic efficiency. Some enzymes that are not called "esterases", like certain types of carbonic anhydrase (CA), exhibit esterase activity and it is convenient to measure CA enzyme activity by using the esterase assay.

TECAN (Spectrophotometer) Settings Used in this Video were:
1) Standard program for generating the calibration curve
Incubate the plate for 5 minutes at 25 °C and begin absorbance measurement at 405nm for each well once.

2) Enzyme Test program
Incubate the plate for 5 minutes at 25 °C and begin absorbance measurement at 405nm for each well once every 30 seconds for a total of 30 minutes.

Frequently Asked Questions are:
Q) What should I dissolve my enzyme in for my stock solution
You should dissolve your enzyme in your buffer solution and we suggest you start with a concentration around 3.5 mg/mL. This can be adjusted based on the purity and activity of the enzyme you use.

Q) Do you need a TECAN to do this experiment
No, other plate reader/spectrophotometer instruments are also compatible with the assay method.

Q) How long can you wait after adding the enzyme to the test plate before you put it in the the spectrophotometer
After the enzyme has been added to the solution in the well the reaction will begin. Start the program as soon as you’ve dosed your enzyme solutions and collect all measurements within the 30min-60min reaction window .

Q) What kind of enzyme is required?
This assay is usually used with esterase enzymes (enzymes that hydrolyze ester bonds), like lipases. However, some other enzyme types of enzymes can also hydrolyze ester bonds. In the video, we tested bovine carbonic anhydrase (CA) enzymes.

Q) How much enzyme (mg) is required?
There is not a set quantity of enzyme needed for your stock solution. You just have to change your dilutions to bring the tests to a readable range.

Q) Enzyme Storage
Carbonic Anhydrases is relatively stable at room temperature but to extend enzyme shelf life store enzymes in the refrigerator at 4℃ for solutions and in the freezer for lyophilized powder.

Q) Enzyme Disposal
In solution Carbonic Anhydrase is non toxic but the powder form of the enzyme can cause allergy when inhaled. For disposal, carefully pour enzyme solutions down the sink and avoid aerosolizing the solution. Dispose of used assay plates, pipette tips, and disposal containers in a ziplock bag to prevent residual dried enzymes from dusting into the air.

Q) Enzyme Safety
Enzymes are non toxic but can cause allergies when inhaled. This is not a concern when in solution but when handling a powder remember to work in a way that prevents dusting and wipe up any dust immediately with a wet disposable towel. For extra precaution, use a HEPA filtered laminar flow bench when available. Think of it like working with pollen.

Q) What should you do if your absorption over time graph is not linear
A non linear increase in your absorption over time likely means that your enzyme concentration is too high. Enzymes react too quickly with the substrate and deplete it prematurely. Repeat the experiment with higher dilutions until you get linear readings.

Q) Most common mistakes
Preparing dilutions incorrectly
Be sure that your math is correct for your dilutions
Not diluting your standard and substrate
Remember to dilute your stock solution to 2mM and your substrate to 8mM before dosing.

Q) How does the reaction occur?
The chemical reaction is the hydrolysis of the acetate ester group in 4-nitrophenyl acetate (NPA). During ester hydrolysis, a water molecule will break the ester bond producing a carboxylic acid (in this case acetic acid) and an alcohol (4-nitrophenol, NP). When only water is present, the "control" reaction occurs slowly. The reaction speeds up when an esterase enzyme is present. The esterase enzyme binds to the ester “substrate” molecule making it easier for water to perform a nucleophilic attack on the carbonyl carbon of the ester that causes the ester bond to break. The hydroxyl group (-OH) of water forms a new bond with the carbonyl carbon of the acetate group, and the proton (H+) from water forms a new bond with the oxygen of NP. Because the starting material (NPA) is colorless and the product (NP) has a yellow color, this “colorimetric” reaction is a convenient way to measure how efficient an enzyme is (its “activity”) at catalyzing the reaction. Many carbonic anhydrases (CA) are capable of catalyzing NPA ester hydrolysis. Therefore, this assay can be used to quantify (assign a numerical value to) the CA activity.