Measuring Enzymatic Assay of

CA
IN WILBUR-ANDERSON UNITS

Equipment required
pH meter (semi-micro electrode with BNC connector)
Digital Stir plate or equivalent
Spinbar, Stirring Bar
Air displacement pipetters and appropriate plastic tips
Digital thermometer, calibrated
Four dram vials with polyethylene snap-caps.
Digital, NIST Traceable timer, increments in hundredth of

a second or equivalent

Reagents required
Buffer, reference standard, pH 7.00 +/- 0.01 at 25 oC,

Sigma-Aldrich Product Number B4770, Buffer, reference

standard, pH 4.00 +/- 0.01 at 25C,
Sigma-Aldrich Product Number B5020, and Buffer, reference
standard, pH 10.00 +/- 0.01 at 25C,
Sigma-Aldrich Product Number B4895. Use as is (Neat) (pH
REF)
20 mM Trizma Base Buffer, pH 8.3 at 25oC
CO2 Water Solution
Carbonic Anhydrase Enzyme Solution

Catalysis using Carbonic Anhydrase

Carbonic Anhydrase catalyses the reversible hydration of

CO2. This reaction involves a two-step mechanism. The first

step is the nucleophilic attack of a zinc-bound hydroxide ion
on CO2. The second step is the regeneration of the active site
by ionization of the zinc-bound water molecule and removal
of a proton from the active site.
CO2 + H2O < Carbonic Anhydrase > HCO3- + H+
Zn+2-OH- + CO2 < > Zn+2 + HCO3Zn+2 + H2O < > H+ + Zn+2-OH

Enzymatic Assay Method

Place a minimum of 40 four dram vials in the bottom of a freezer.
Place a large suitable container of ice on top of the digital stirrer along

with a small four dram rack. Place approximately 10 ml of each

reference buffer (pH REF) into a pre-chilled four dram vial. Allow
each pH reference buffer and the pH electrode to equilibrate to less
than 3 oC. Check temperature of each reference buffer with a digital
thermometer. Also, preset the pH meter at a maximum of 3 oC.
Calibrate the pH meter with each pH reference buffer.

Enzymatic Assay Method (continued)

Performing a blank reaction:
Immediately prior to performing the assay on the blank, pipette (in milliliters)

the following reagents into a pre-chilled four dram vial with a micro stir bar.

Blank

Icecoldtrizmabase
buffer.

3.00

IcecoldPurifiedwater

0.05

Check temperature of reaction mixture with digital thermometer. If less than

3 oC proceed with step 7.5.3.3, If not, dry ice can be used to expedite cooling,
however, be careful not to freeze the solution. Then place the pH electrode in
the solution, with stirring, at approximately 300 rpms.
After the pH has reached the maximum pH (> 8.5), add the following with

reverse pipetting:
IcecoldC02-water
solution

2.00

Enzymatic Assay Method (continued)

Record the time in seconds (T-Test) required for the pH to change from 8.3

to 6.3. If the time is not in the 10 to 20 second range, a more concentrated

enzyme must be prepared.
Evaluate the substrate (CO2 water solution) by repeating blank test. If the
Blank is in the range of 70 to 100 seconds, proceed with Sample Test Step
7.5.5. If too high, add small chips of dry ice to substrate and invert. Allow to
equilibrate on ice for five minutes. Repeat blank test until Blank is in the
range of 70 to 100 seconds. Recall, the blank time goes up with every
opening of the reagent substrate bottle.
Even though, the first blank may be in the range of 70 to 100 seconds, the
number of seconds for the pH to decrease from 7.0 to 6.3 must be less than
8 seconds. Since the pKa of Trizma is 8.1, the buffering capacity of the
reaction mixture has decreased. After running three blanks, if the average
time required to decrease from pH 7.0 to 6.3 is greater than 8 seconds, then
replace substrate and prepare a new buffer solution.

Enzymatic Assay Method (continued)

Performing Test-Control Reaction:
Immediately following performing of the assay on the blank, pipette (in milliliters) the

following reagents into a pre-chilled four dram vial with a micro stir bar.
Test-Control
IceColdbuffer

3.00

Check temperature of reaction mixture with digital thermometer. If less than 3 oC, carry

on. If not, dry ice can be used to expedite cooling, being careful not to freeze the solution.
Then place the pH electrode in the solution, with stirring, at approximately 300 rpms.
After the pH has reached the maximum pH(> 8.5), add the following with reverse
pipetting:
Icecoldsubstrate
(C02water)

2.00

When the pH reaches 8.4 to 8.5, add the following:

CA solution

0.05

Enzymatic Assay Method (continued)

Record the time in seconds (T-Test) required for the pH to change from 8.3

to 6.3. If the time is not in the 10 to 20 second range, a more concentrated

enzyme must be prepared.
Evaluate the substrate (CO2 water solution) by repeating blank test. If the
Blank is in the range of 70 to 100 seconds, proceed with Sample Test Step
7.5.5. If too high, add small chips of dry ice to substrate and invert. Allow to
equilibrate on ice for five minutes. Repeat blank test until Blank is in the
range of 70 to 100 seconds. Recall, the blank time goes up with every
opening of the reagent substrate bottle.
Even though, the first blank may be in the range of 70 to 100 seconds, the
number of seconds for the pH to decrease from 7.0 to 6.3 must be less than
8 seconds. Since the pKa of Trizma is 8.1, the buffering capacity of the
reaction mixture has decreased. After running three blanks, if the average
time required to decrease from pH 7.0 to 6.3 is greater than 8 seconds, then
replace substrate and prepare a new buffer solution.

Enzymatic Assay Method (continued)

Perform Test-Sample-1 reaction
Same method as previous. The blank should come within

range of 65-85 seconds.

Perform Test-Sample-2 reaction
At this time in the procedure, there should be a minimum
of five Blank values with a blank average time in the range
of 70 to 100 seconds.. Also, there should be a value of 10 to
20 seconds for one control and one value of 10 to 20
seconds for each sample test.

Calculations
Calculate the average, standard deviation, and % Coefficient of Variation
for all recorded blanks in the range of 70 seconds to 100 seconds.
% CV = Standard Deviation x 100 /Average
The % CV must be 20%; if not, the control must be within 70% of release
value. If neither criteria is met, the assay procedure must be repeated with
all new reagents.If the criteria is met, the assay system is suitable.
Determine the Carbonic Anhydrase Units / ml as follows:
Units=
mlofenzyme

(TBlankAverage-TTestAverage)(df)
/(TTestAverage)(0.05)

where,

T = Time (in seconds) required for pH to change from 8.3 to 6.3 as per
Unit Definition
df = dilution factor of Reagent 7.4.5 (CA) used
0.05 = volume (in milliliters) of Reagent 7.4.5 (CA) used

Calculations
Determine the Carbonic Anhydrase Units / mg solid

as follows:
Units=
mgofsolid

(Units/mlofenzyme)/(mgsolid
permlofenzyme)

Determine the Carbonic Anhydrase Units / mg

Protein as follows:
Units=
mgofprotein

(Units/mgsolid)(100)/(%
Protein)

References
http://

www.sigmaaldrich.com/technical-documents/protoco
ls/biology/enzymatic-assay-of-carbonic-anhydrase
.html