Enzymatic Assay of Peroxidase (EC 2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) as a Substrate

Document History

Replaces SPABTS02. Procedure updated to conform to current QUMAS formatting guidelines. Refer to CR SOP-DEK-ENZ42.

1. Objective

To standardize a procedure for the assay of Peroxidase using 2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) as a substrate.

2. Scope

This procedure applies to all products that have a specification for activity of Peroxidase using 2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) as a substrate.

3. Definitions

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

3.2.    Unit Definition - One unit will oxidize 1.0 µmole of 2,2' azino bis (3 ethylbenzthiazoline-6-sulfonic acid) per minute at pH 5.0 at 25°C.

3.3.    ABTS - 2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid). ABTS is a registered trademark of Boehringer Mannheim Gmbh.

4. Discussion

H2O2 + ABTS   Peroxidase   > 2H2O+ oxidized ABTS

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

T = 25°C, pH = 5.0, A405nm, Light path = 1 cm

7.2.    METHOD
Continuous Spectrophotometric Rate Determination

7.3.    REAGENTS

7.3.1.    100 mM Potassium Phosphate, pH 5.0 at 25°C (Buffer) Prepare a 13.6 mg/ml solution in purified water using Potassium Phosphate, Monobasic, Sigma-Aldrich Product Number P5379. Adjust the pH of this solution to 5.0 at 25°C using 1N KOH.

7.3.2. 9.1 mM ABTS (Substrate) Prepare a 5.0 mg/ml solution in Reagent 7.3.1 using ABTS, Sigma-Aldrich Product Number A9941 . Check the pH of this solution and adjust to 5.0 at 25°C as necessary. Sigma-Aldrich Product Number A9941 is supplied as 10 mg tablets. Prepare Fresh.

7.3.3. 0.3% (w/w) Hydrogen Peroxide Solution (H2O2) Prepare a 0.3% (w/w) solution in purified water using Hydrogen Peroxide 30% (w/w) Solution, Sigma-Aldrich Product Number H1009. Prepare Fresh.

7.3.4. 40 mM Potassium Phosphate Buffer with 0.25% (w/v) Bovine Serum Albumin and 0.5% (v/v) Triton X-100, pH 6.8 at 25°C (Diluent) Prepare a solution in purified water containing the following:    5.4 mg/ml Potassium Phosphate, Monobasic, Sigma-Aldrich Product Number P5379.    2.5 mg/ml Albumin, Bovine, Fraction V Powder, Sigma-Aldrich Product Number A4503.    5.0 mg/ml Triton X-100, Sigma-Aldrich Product Number T6878. Triton is a registered trademark of Union Carbide Corp.    Adjust the pH of this solution to 6.8 at 25°C using 1N KOH.

7.3.5.    Peroxidase Enzyme Solution (Enzy)    Prepare a 10 mg/ml stock solution of Peroxidase in cold Reagent 7.3.4.    Immediately before use, dilute to 0.20 – 0.80 units/ml in cold Reagent 7.3.4.


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

  Test Blank
Reagent 7.3.2 (Substrate) 2.90 2.90


7.4.2.    Let the cuvettes equilibrate at 25°C in a suitably thermostated spectrophotometer for approximately five minutes, then add:

Reagent 7.3.3 (H2O2) 0.10 0.10
Reagent 7.3.4 (Diluent) 0.05 -----
Reagent (Enzy) ----- 0.05


7.4.3.    Immediately mix by inversion and record the increase in A405 nm for approximately 3 minutes. The fastest rate is usually in the first minute. If multiple levels of enzyme are to be run, test one at a time.

7.4.4.    Obtain the ΔA405 nm/minute for the maximum linear rate for both the Test and Blank.


7.5.1. Units/mg solid = (ΔA405 nm/min(test) - ΔA405 nm/min(blank)) * 3.05 * DF
36.8 * 0.05

    3.05 = Final volume (in milliliters) of reaction
    DF = Dilution factor of enzyme
    36.8 = Millimolar extinction coefficient of oxidized ABTS at A405 nm
    0.05 = Volume (in milliliters) of enzyme used

In a 3.05 ml reaction mix, the final concentrations are 96 mM potassium phosphate, 8.7 mM 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 0.01% (w/w) hydrogen peroxide, 0.004% (w/v) bovine serum albumin, 0.008% (v/v) Triton X-100 and 0.01 - 0.04 unit peroxidase.

8. References & Attachments

8.1.    Keesey, J. (1987) Biochemica Information, First Edition, pp. 58, Boehringer Mannheim Biochemicals, Indianapolis, IN

8.2.    Pütter, J. and Becker, R. (1983) Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.) 3rd ed., Vol III, pp. 286-293, Verlug Chemie, Deerfield Beach, FL

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.


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