Enzymatic Assay of Catalase (EC 1.11.1.6)
1. OBJECTIVE
To standardize a procedure for the enzymatic assay of catalase.
2. SCOPE
The scope of this procedure is for all products that have a specification for catalase or catalase impurity.
3.1. Purified Water - water from a deionizing system, resistivity ~18MΩ•cm @ 25ºC
3.2. Unit definition - One unit will decompose 1.0 μmole of H2O2 per minute at pH 7.0 at 25C, while the H2O2 concentration falls from 10.3 mM to 9.2 mM. The rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240 nm.
4. DISCUSSION
2 H2O2 Catalase >2 H2O + O2
5. RESPONSIBILITIES
It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.
6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS:
T = 25°C, pH = 7.0, A240nm, Light path = 1 cm
7.2 METHOD:
Continuous Spectrophotometric Rate Reduction
7.3 REAGENTS:
7.3.1 50 mM Potassium Phosphate Buffer pH 7.0 at 25°(Buffer)
Prepare a 11.4 mg/ml solution in purified water using Potassium Phosphate, Monobasic, such as Sigma-Aldrich Product Number P5504. Adjust to pH 7.0 at 25°C using 1 M KOH.
7.3.2 0.036% (w/w) Hydrogen Peroxide Solution (H2O2)
Prepare in Reagent 7.3.1 (Buffer) using Hydrogen Peroxide, 30% (w/w) such as Sigma Product Number H1009. Determine the A240nm of this solution using Reagent 7.3.1 (Buffer) as a blank. The A240nm must be between 0.550 and 0.520 absorbance units. If necessary, add hydrogen peroxide to increase the absorbance and Reagent 7.3.1 (Buffer) to decrease the absorbance.
7.3.3 Catalase Solution (Enzyme)
7.3.3.1 For catalase products supplied as a lyophilized powder:
7.3.3.1.1 Prepare an initial solution of 10 mg/ml in cold Reagent 7.3.1 (Buffer) (this solution will be slightly hazy).
7.3.3.1.2 Immediately before use dilute to approximately 100 units/ml in cold Reagent 7.3.1 (Buffer).
7.3.3.2 For catalase products supplied as a crystalline suspension:
7.3.3.2.1 Incubate a retainer sample of bulk at 37°C for one hour to obtain complete dissolution.
7.3.3.2.2 Then prepare, using either a cut pipette tip or a wide mouth pipette tip, an initial dilution of approximately 1000 units/ml in 37°C Reagent 7.3.1 (Buffer).
7.3.3.2.3 Ensure “swirling” of the initial dilution is not present. If, after mixing, the initial dilution does not appear homogeneous, incubate initial dilution for one hour at 37°C to obtain complete dissolution.
7.3.3.2.4 Immediately before use, perform a secondary dilution to approximately 100 units/ml in 37°C Reagent 7.3.1 (Buffer).
7.3.3.3 For catalase impurities: Immediately before use, prepare a 30 mg/ml solution in cold Reagent 7.3.1 (Buffer). If the catalase activity is greater than 100 Units/ml, then dilute to 100 Units/ml using cold Reagent 7.3.1.
7.4 TEST METHOD
7.4.1 7.4.1 Using a suitable thermostatted spectrophotometer, blank against a cuvette containing Reagent 7.3.1(Buffer).
7.4.2 Pipette (in milliliters) the following reagent into a suitable cuvette:
| Test | |
| Reagent 7.3.2(H2O2) | 2.90 |
7.4.3 Please note that each test cuvette will need to be run one at a time, so do not prepare the next test cuvette until after the preceding cuvette’s run is complete.
7.4.4 For each cuvette monitor the A240nm until constant then add:
| Reagent 7.3.3(Enzyme) | 0.10 |
7.4.5 Immediately mix well by inversion. The Initial A240nm will exceed 0.45 Absorbance units. Start timing the reaction when A240nm reaches 0.45 absorbance units and record the time required for the A240nm to decrease from 0.45 to 0.40 absorbance units. Take one reading per second for about 180 seconds.
7.4.6 For samples that have very small amounts of catalase impurity, start timer upon addition of the enzyme, if the decrease in A240nm to 0.45 takes longer than 25 minutes, then use 25 minutes for the time in the calculation. If the decrease to A240nm takes less than 25 minutes, then allow the reaction to proceed with the recording of A240nm at 1-minute intervals until 0.40 is obtained. The minimal time required to decrease from 0.45 to 0.40 is 25 minutes (< or = to 0.002 /min, limit of detection).
7.5 CALCULATIONS
| 7.5.1 | Units/ml enzyme = | (3.45)(df) |
| (min)(0.1) |
3.45 = Corresponds to the decomposition of 3.45 micromoles of hydrogen peroxide in a 3.0 ml reaction mixture producing a decrease in the A240nm from 0.45 to 0.40 absorbance units
df = Dilution factor
min = Time in minutes required for the A240nm to decrease from 0.45 to 0.40 absorbance units
0.1 = Volume (in milliliter) of enzyme used
| 7.5.2 | Units/mg solid = | Units/mL enzyme |
| mg solid/mL enzyme |
| 7.5.3 | Units/mg protein = | Units/mL enzyme |
| mg protein/mL enzyme |
7.6 FINAL ASSAY CONTENTRATION:
In a 3.00 mL reaction mix, the final concentrations are 50 mM potassium phosphate 0.036% (w/w) hydrogen peroxide, and approximately 10 units of catalase.
8.1 Beers, R.F. Jr. and Sizer, I.W. (1952) Journal of Biological Chemistry 195, 133-140
8.2 Stern, K.G. (1937) Journal of Biological Chemistry 121, 561 572.
8.3 Replaces SPHYDR02 and SPHYDR11.
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.
