Enzymatic Assay of Carbonic Anhydrase for Wilbur-Anderson Units (EC 4.2.1.1)

Document History

See CR BIOT-MAJ-1013.

1. Objective

To standardize a procedure for the enzymatic assay of Carbonic Anhydrase (EC 4.2.1.1) for Wilbur-Anderson Units.

2. Scope

This procedure applies to all products that have a specification for Wilbur-Anderson Units relative to Carbonic Anhydrase activity.

3. Definitions

3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC

3.2. Unit Definition - One Wilbur-Anderson unit will cause the pH of a 20 mM Trizma buffer to drop from 8.3 to 6.3 per minute at 0°C. (One Wilbur-Anderson unit is essentially equivalent to one Roughton-Booth unit.)

3.3. SUB = Substrate

3.4. CA = Carbonic Anhydrase

3.5 Comparable Equipment:

Equipment that falls within the scope of this operation procedure’s definition of the replaced equipment, or equipment that has been qualified in accordance with the applicable specifications listed within the FPS of the test sample. Equipment is qualified if it meets at least one of the following conditions:

  1. The equipment has been qualified for use with the test sample and its qualification has been documented and stored in the Biotech Analytical Services “Method Qualifications” location.
  2. Product history demonstrates that the equipment has been used consistently to accurately demonstrate that the test sample has met specifications.
  3. The replacement equipment meets the same specification as the previously used equipment set by the manufacturer. This previous equipment has either been qualified for use with the test sample or the product history demonstrates that the equipment has been used consistently to accurately demonstrate that the test sample has met specifications.

4. Discussion

Carbonic Anhydrase catalyzes 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 + HCO3- 
Zn+2 + H2O <          > H+ + Zn+2-OH

5. Responsibilities

It is the responsibility of all Analytical Services personnel to follow this protocol as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS: T = 0 to 3 ºC, pH = 8.3 to 6.3

7.2 METHOD: Potentiometric

7.3. EQUIPMENT

7.3.1    pH Meter, Model M545, Corning Pinnacle or equivalent

7.3.2    Orion 8203BN “PerPHect ”, “ROSS GLASS”, Semi-micro Electrode with BNC connector, Thermo Electron Corporation or comparable electrode

7.3.3    Digital Stir plate or equivalent

7.3.4    Spinbar, Stirring Bar,Teflon, Flea Micro, 10 x 3 mm, Aldrich Z283878-3 or equivalent

7.3.5    Air displacement pipetters and appropriate plastic tips

7.3.6    Digital thermometer, calibrated

7.3.7    Four dram vials with polyethylene snap-caps.

7.3.8    Digital, NIST Traceable timer, increments in hundredth of a second or equivalent

7.4. REAGENTS

7.4.1    Buffer, reference standard, pH 7.00 +/- 0.01 at 25oC, Sigma-Aldrich Product Number B4770, Buffer, reference standard, pH 4.00 +/- 0.01 at 25°C, Sigma-Aldrich Product Number B5020, and Buffer, reference standard, pH 10.00 +/- 0.01 at 25°C, Sigma-Aldrich Product Number B4895. Use “as is” (“Neat”) (pH REF)

7.4.2    20 mM Trizma Base Buffer, pH 8.3 at 25oC (BUFFER )

7.4.2.1    Prepare at 2.43 mg / ml in purified water using Trizma Base, Reagent Grade, Sigma-Aldrich Product Number T1503.

7.4.2.2    Adjust pH to 8.3 at 25°C with 2 N Sulfuric Acid. Store in a 1 L polypropylene bottle and cover with ice up to the bottom of the lid.

7.4.3    CO2 Water Solution (SUB)
Use “Vess Seltzer, No Sodium Water” as is (Neat). Store in ice up to bottom of lid.

7.4.4    Dry ice.

7.4.4.1    This can be obtained from packaging.

7.4.5    Carbonic Anhydrase Enzyme Solution (CA)

7.4.5.1    Immediately before pipetting into reaction mixture, prepare a solution in cold purified water containing 2.0 mg solid / ml. Then immediately dilute to 60 to 90 units / ml.

7.4.5.2    This concentration corresponds to a reaction time between 10 to 20 seconds.

7.4.5.3    Prepare a fresh sample/control weigh-up and dilution for each run.

7.4.5.4    Prepare Test in duplicate. Always run a minimum of one control weigh-up.

7.5 Enzymatic Assay Method

7.5.1    Place a minimum of 40 four dram vials in the bottom of a freezer.

7.5.2    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 Reagent 7.4.1 (pH REF) into a pre-chilled four dram vial from 7.5.1. 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.

7.5.3    Performing Blank Reaction

7.5.3.1    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
Ice cold Reagent 7.4.2 (Buffer) 3.00
Ice cold Purified water 0.05

 

7.5.3.2    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

7.5.3.3    After the pH has reached the maximum pH (> 8.5), add the following with reverse pipetting:

Ice cold Reagent 7.4.3 (SUB) 2.00

 

7.5.3.4    Record the time (T-Blank) required for the pH to change from 8.3 to 6.3. Make sure Reagent 7.4.3 (SUB) is closed after each addition to a reaction.

7.5.3.5    The general blank time after first opening Reagent 7.4.3 (SUB) is approximately 40 seconds. Record this time as “Blank-1”. If this occurs, transfer reagent 7.4.3 (SUB) back fourth between a 1 L beaker and the “Vess Seltzer” bottle a couple of times. Place Reagent 7.4.3 (SUB) back in ice. Repeat step 7.5.3.1 through step 7.5.3.4. Record all blank times in seconds. The blank times tend to increase with each opening of the bottle containing Reagent 7.4.(SUB). Once a blank time of 65 seconds is reached, proceed with 7.5.4. After all test runs, the final blank average must be in the range of 70 to 100 seconds.

7.5.3.6    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 Reagent 7.4.3 (SUB) and prepare a new Reagent 7.4.2 (Buffer).

7.5.4    Performing Test-Control Reaction

7.5.4.1    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
Ice Cold Reagent 7.4.2 (Buffer) 3.00

 

7.5.4.2    Check temperature of reaction mixture with digital thermometer. If less than 3 oC proceed this step 7.5.4.3, 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.

7.5.4.3    After the pH has reached the maximum pH(> 8.5), add the following with reverse pipetting:

Ice Cold Reagent 7.4.3 (SUB) 2.00

 

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

Reagent 7.4.5 (CA) 0.05

 

7.5.4.5    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.

7.5.4.6    Evaluate the Reagent 7.4.3 (SUB)by repeating Step 7.5.3. 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 Reagent 7.4.3 (SUB) and invert. Allow to equilibrate on ice for five minutes. Repeat Step 7.5.3 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.

7.5.5    Performing Test-Sample-1 Reaction

7.5.5.1    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.

  Test-Sample-1
Ice Cold Reagent 7.4.2 (Buffer) 3.00

 

7.5.5.2    Check temperature of reaction mixture with digital thermometer. If less than 3 oC proceed this step 7.5.4.3, 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. 

WARNING:DO NOT PUT THE LID ON TOO TIGHTLY AFTER THE DRY ICE IS ADDED!! IF IT IS CAPPED TOO TIGHTLY, THIS CAN BUILD UP SUBSTANTIAL PRESSURE AND RESULT IN AN EXPLOSION!

7.5.5.3    After the pH has reached it maximum pH(> 8.5), add the following with reverse pipetting:

Ice cold Reagent 7.4.3 (SUB) 2.00

 

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

Reagent 7.4.5 (CA) 0.05

 

7.5.5.5    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.

7.5.5.6    Evaluate the Reagent 7.4.3 (SUB) by repeating Step 7.5.3. If the Blank is in the range of 70 to 100 seconds, proceed with Sample Test Step 7.5.6. If too high, add small chips of dry ice to Reagent 7.4.3 (SUB) and invert. Allow to equilibrate on ice for five minutes. Repeat Step 7.5.3 until Blank is in the range of 65 to 85 seconds. Recall, the blank time goes up with every opening of the reagent substrate bottle.

7.5.6.    Performing Test-Sample-2 Reaction

7.5.6.1    Repeat steps 7.5.5.1 to 7.5.5.6 for Test-Sample-2

7.5.6.2    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.

7.6 CALCULATIONS

7.6.1    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.

7.6.2    Determine the Carbonic Anhydrase Units / ml as follows:

Units = ( TBlank Average - TTest Average ) (df) / (TTest Average )(0.05)
ml of enzyme

 

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

7.6.3    Determine the Carbonic Anhydrase Units / mg solid as follows:

Units = (Units / ml of enzyme) / (mg solid per ml of enzyme)
mg of solid

 

7.6.4    Determine the Carbonic Anhydrase Units / mg Protein as follows:

Units = (Units / mg solid)(100) / (% Protein)
mg of protein

8. References & Attachments

8.1 Wilbur, K.M. and Anderson, N.G. (1948) Journal of Biological Chemistry. 176, 147-154.

8.2 Worthington, C.C. (1988) in Worthington Enzyme Manual (Worthington, C.C. ed.) pp 57-59, Worthington Biochemical Corporation, Freehold, NJ

8.3 Tripp, B.C., Smith, K. and Ferry, J.G. (2001) Journal of Biological Chemistry. 48615 – 48618.

9. Approval

Review, approvals and signatures for this document will be generated electronically using DocCompliance (QUMAS). Print a “For Use” copy if hardcopy with signature verification is required.

Materials

     
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