Enzymatic Assay of Chloramphenicol Acetyltransferase

1. Objective

To standardize a procedure for the determination of the enzymatic assay of Chloramphenicol Acetyltransferase.

2. Scope

This procedure applies to products that have a specification for the enzymatic activity of chloramphenicol acetyltransferase.

3. Definitions

3.1 Acetyl CoA - Acetyl Coenzyme A

3.2 CAT - Chloramphenicol Acetyltransferase

3.3 CoA - Coenzyme A

3.4 DTNB - 5,5’-Dithio-bis (2-Nitrobenzoic Acid)

3.5 TNB - 5-Thio-2-Nitrobenzoic Acid

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

4. Discussion

Acetyl CoA + Chloramphenicol    CAT   > CoA + Chloramphenicol 3-Acetate
CoA + DTNB       >TNB + CoA derivative
The reaction of DTNB with the –SH group on CoA results in an increase in A412nm due to the TNB anion.

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. Safety

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

7. Procedure

7.1 CONDITIONS:
T = 25°C, pH = 7.8, A412nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1    100 mM Tris Buffer, pH 7.8 at 25°C (Buffer)
            Prepare a 12.1 mg /ml solution of Trizma Base, Sigma-Aldrich Product Number such as T1503, in purified water. Adjust pH to 7.8 at 25°C with 1N HCl.

7.3.2    2.5 mM 5,5’-Dithio-bis(2-Nitrobenzoic Acid) Solution, pH 7.8 at 25°C(DTNB)
            Prepare a 1.0 mg/ml solution of DTNB, Sigma-Aldrich Product Number such as D8130, in Reagent 7.3.1 (Buffer).

7.3.3    5.0 mM Acetyl Coenzyme A Solution (Acetyl CoA)
            Prepare a 4.68 mg/ml solution of Acetyl CoA, Sodium Salt, Sigma-Aldrich Product Number such as A2056, in purified water.

7.3.4    0.3% (w/v) Chloramphenicol Solution (Substrate)
            Prepare a 3 mg/ml solution of Chloramphenicol, Sigma-Aldrich Product Number such as C0378, in purified water. Facilitate dissolution by first dissolving in 0.1 ml of methanol, Sigma-Aldrich Product Number such as M1775.

7.3.5    10 mM Tris Buffer, pH 7.8 at 25°C(Enzyme Diluent)
            Prepare a 1.21 mg /ml solution of Trizma Base, Sigma-Aldrich Product Number such as T1503, in purified water. Adjust pH to 7.8 at 25°C with 1N HCl.

7.3.6    Chloramphenicol Acetyltransferase Enzyme Solution (Enzyme)
            Immediately before use, prepare a solution of 500 unit/ml of Chloramphenicol Acetyltransferase in cold Reagent 7.3.5 (Enzyme Diluent).

7.4 TEST METHOD

7.4.1    Pipette ( in milliliters) the following reagents into suitable cuvettes:

  Test Blank
Reagent 7.3.1 (Buffer) 2.70 2.70
Reagent 7.3.2 (DTNB) 0.10 0.10
Reagent 7.3.3 (Acetyl CoA) 0.10 0.10
Reagent 7.3.4 (Substrate) 0.05 0.05

 

7.4.2    Mix by inversion and equilibrate to 25°C using a suitable thermostatted spectrophotometer. Monitor the A412nm until constant. Then add:

Reagent 7.3.5 (Enzyme Diluent) ----- 0.05
Reagent 7.3.6 (Enzyme) 0.05 -----

 

     Immediately mix by inversion and record the increase in A412nm for approximately 5 minutes. Obtain the ΔA412nm / minute using the maximum linear rate for both the Test and the Blank.

7.5 CALCULATIONS

7.5.1 Units/ml enzyme = (ΔA412nm/min Test - ΔA412nm/min Blank)(3)(df)
(0.0136)(0.05)

 

where:
    3 = Total volume (in milliliters) of assay
    df = Dilution Factor
    0.0136 = Micromolar extinction coefficient for TNB at 412nm
    0.05 = Volume (in milliliters) of enzyme used

7.5.2 Units/mg solid = Units/ml enzyme
mg solid/ml enzyme

 

  Units/mg protein = Units/mg solid
mg protein/mg s enzyme

 

7.6 UNIT DEFINITION
In One unit will convert 1.0 nanomole of chloramphenicol and acetyl coenzyme A to chloramphenicol 3-acetate and coenzyme A per minute at pH 7.8 at 25°C.

7.7 FINAL ASSAY CONCENTRATION :
In a 3.00 ml reaction mix, the final concentrations are 94 mM Tris, 0.083 mM DTNB, 0.19 mM acetyl CoA, 0.005% (w/v) chloramphenicol and 25 units chloramphenicol acetyltransferase.

8. References & Attachments

8.1 Replaces SPCHLO01.

8.2 Silverstein, R.M. Anal. Biochem. 63, 281 (1975).

8.3 Hash, J.H. Methods in Enzymology, XLIII, 737.

9. Approval

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

Materials

     
Related Links