Enzymatic Assay of Peroxidase
1. OBJECTIVE
To standardize a procedure for the enzymatic determination of Peroxidase by Pyrogallol units.
2.1 This procedure applies to all products that have a specification for Peroxidase Activity by Pyrogallol units
2.2 Products using this method include, but are not limited to, P0889, P1014, P1709, P1834, P2088, P2649, P4794, P6140, P6782, P8125, P8170, P8250, P8375, P8415, P8651 and P8992
2.3 This assay procedure is not to be used to assay Peroxidase, Insoluble Enzyme attached to Beaded Agarose, Sigma-Aldrich Product Number P3912 or Peroxidase, Sigma-Aldrich Product Number P1432
3.1 Unit Definition: One unit will form 1.0 milligram of purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20ºC. This purpurogallin (20 second) unit is equivalent to approximately 18 µM units per minute at 25ºC.
3.2 Purified Water: Water from a deionizing system, resistivity > or = 18MΩּcm @25°C
3.3 RZ: Reinheitszahl, A403nm/A275nm value. This value is an expression of the ratio of hemin to protein content
4. DISCUSSION
H202 + Pyrogallol Peroxidase> 2H20 + Purpurogallin (donor) (oxidized donor)
5.1 It is the responsibility of all trained Analytical Services laboratory personnel to follow this procedure as written
6.1 Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions
6.2 Hydrogen Peroxide, Sigma-Aldrich Product number H1009, is a H=3* chemical and an oxidizer as well. Please use proper caution.
6.3 Pyrogallol, Aldrich Product Number 254002, is a H=3* chemical. Please use proper precautions when weighing/handling this material.
7.1 CONDITIONS: T = 20ºC, pH = 6.0, A420nm, Light path = 1 cm
7.2 METHOD: Continuous Spectrophotometric Rate Determination
7.3 REAGENTS
7.3.1 100 mM Potassium Phosphate Buffer, pH 6.0 at 20ºC (Buffer)
7.3.1.1 Prepare a 13.6 mg/ml solution in purified water using Potassium Phosphate, Monobasic, Anhydrous, Sigma Aldrich Product Number P5379.
7.3.1.2 Adjust to pH 6.0 at 20ºC with 1.0 M KOH.
7.3.1.3 After pHing, put on ice.
7.3.2 0.50% (w/w) Hydrogen Peroxide Solution (H2O2)
7.3.2.1 Prepare 5 ml of a 1:60 solution in purified water using Hydrogen Peroxide, 30% (w/w) Solution, Sigma-Aldrich Product Number H1009
.7.3.2.2 PREPARE FRESH and store the solution in a capped 4 dram vial on ice to reduce exposure to air
7.3.2.3 Several dilutions of the Hydrogen Peroxide will need to be made throughout the assay to ensure the diluted reagent is at the proper concentration.
7.3.3 5% (w/v) Pyrogallol Solution (Pyr)
7.3.3.1 Prepare a 50 mg/ml solution in purified water using Pyrogallol, Aldrich Product Number 254002.
7.3.3.2 PREPARE FRESH AND KEEP FROM LIGHT. Keep this solution on ice.
7.3.4 Peroxidase Enzyme Solution (Enzy)
7.3.4.1 Prepare a 10 mg/ml solution of peroxidase in COLD Reagent 7.3.1 (Buffer). This solution, at this concentration, is stable for 15 minutes.
7.3.4.1.1 Be sure to keep this 10 mg/ml solution for the 7.5 RZ FACTOR DETERMINATION step to follow the activity assay.
7.3.4.2 Immediately before use, prepare a solution containing 0.4 0.7 unit/ml of Peroxidase in COLD Reagent 7.3.1.
7.3.4.3 Please note that the peroxidase sample should be a homogeneous, fluffy powder. Manufacturing will be running this material through a screen prior to release to ensure a homogeneous sample. If the material is clumpy, sticky, in chunks, or is otherwise not homogeneous, please contact your supervisor.
7.4 ASSAY
7.4.1 Pipette (in milliliters) the following reagents into suitable cuvettes:
| Test | Blank | |
| Purified Water | 2.10 | 2.10 |
| Reagent 7.3.1 (Buffer) | 0.32 | 0.32 |
| Reagent 7.3.2 (H2O2) | 0.16 | 0.16 |
| Reagent 7.3.3 (Pyr) | 0.32 | 0.32 |
7.4.2 Mix by inversion and equilibrate for 10 minutes in the spectrophotometer to 20°C. Monitor the A420nm until constant, using a suitably thermostatted spectrophotometer. Then add:
| Test | Blank | |
| Reagent 7.3.1 (Buffer) | ---- | 0.10 |
| Reagent 7.3.4 (Enz) | 0.10 | ---- |
7.4.3 Immediately mix by inversion and record the increase in A420nm at a rate of 1 reading/second for approximately 3 minutes. Obtain the ΔA420nm/20 seconds using the maximum linear rate for both the Test and Blank.
7.4.4 The enzyme concentration may have to be modified in order for the rate, ΔA420nm/20 seconds, to be within the specified absorbance change rate of 0.16-0.28.
7.5 RZ FACTOR DETERMINATION
7.5.1 Pipette (in milliliters) the following reagents into a quartz cuvette:
| Test | |
| Reagent 7.3.1 (Buffer) | 2.90 |
7.5.2 Record the absorbance at 403nm and 275nm using a suitable spectrophotometer. Then add:
| Reagent 7.3.4 (Enz) | 0.10 |
| 7.6.1 | Units/ml enzyme= | (ΔA420nm/20 sec Test-ΔA420nm/20 sec Blank)(3)(df) |
| (12) (0.1)) |
sec = seconds
3 = Volume (in milliliters) of assay
df = Dilution factor
12 = Extinction coefficient of 1 mg/ml of Purpurogallin at 420 nm (determined by Sigma-Aldrich)
0.1 = Volume (in milliliters) of enzyme used
| 7.6.2 | Units/mg solid = | units/ml enzyme |
| mg solid/ml enzyme |
| 7.6.3 | Units/mg protein= | units/ml enzyme |
| mg protein/ml enzyme |
| 7.6.4 | RZ Factor = | (A403nm Test - A403nm Blank) |
| (A275nm Test – A275nm Blank) |
7.7 FINAL ASSAY CONCENTRATIONS
In a 3.00 ml reaction mix, the final concentrations are 14 mM potassium phosphate, 0.027% (w/w) hydrogen peroxide, 0.5% (w/v) pyrogallol and 0.04 - 0.07 unit peroxidase.
8.1 Chance, B. and Maehly, A.C. (1955) Methods in Enzymology, II, 773-775
8.2 Shannon, L. M., Kay, E. and Lew, J.Y. (1966) Journal of Biological Chemistry, 241, 2166-2172
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