Catalase

Application and Principle

This procedure is used to determine the catalase activity, expressed as Baker Units, of preparations derived from Aspergillus niger var, bovine liver, or Micrococcus lysodeikticus. The assay is an exhaustion method based on the breakdown of hydrogen peroxide by catalase and the simultaneous breakdown of the catalase by the peroxide under controlled conditions.

 

Reagents and Solutions
Ammonium Molybdate Solution (1%) Dissolve 1.0 g of ammonium molybdate [(NH4)6MoO24·4H2O] (Merck, Catalog No. 1182) in water, and dilute to 100 mL.
0.250 N Sodium Thiosulfate Dissolve 62.5 g of sodium thiosulfate (Na2S2O3·5H2O) in 750 mL of recently boiled and cooled water, add 3.0 mL of 0.2 N sodium hydroxide as a stabilizer, dilute to 1000 mL with water, and mix. Standardize as directed for 0.1 N Sodium Thiosulfate (see Solutions and Indicators), and, if necessary, adjust to exactly 0.250 N.

Peroxide Substrate Solution Dissolve 25.0 g of anhydrous dibasic sodium phosphate (Na2HPO4), or 70.8 g of Na2HPO4·12H2O, in about 1500 mL of water, and adjust to pH 7.0 ± 0.1 with 85% phosphoric acid. Cautiously add 100 mL of 30% hydrogen peroxide, dilute to 2000 mL in a graduate, and mix. Store in a clean amber bottle, loosely stoppered. The solution is stable for more than 1 week if kept at 5°in a full container. (With freshly prepared substrate, the blank will require about 16 mL of 0.250 N Sodium Thiosulfate. If the blank requires less than 14 mL, the substrate solution is unsuitable and should be prepared fresh again. The sample titration must be between 50% and 80% of that required for the blank.)

 

Procedure
Pipet an aliquot of not more than 1.0 mL of the sample, previously diluted to contain approximately 3.5 Baker Units of catalase, into a 200-mL beaker. Rapidly add 100 mL of Peroxide Substrate Solution, previously adjusted to 25, and stir immediately for 5 to 10 s. Cover the beaker, and incubate at 25 ± 1 until the reaction is completed. Stir vigorously for 5 s, and then pipet 4.0 mL from the beaker into a 50-mL Erlenmeyer flask. Add 5 mL of 2 N sulfuric acid to the flask, mix, then add 5.0 mL of 40% potassium iodide solution, freshly prepared, and 1 drop of Ammonium Molybdate Solution (1%), and mix. While continuing to mix, titrate rapidly to a colorless endpoint with 0.250 N Sodium Thiosulfate, recording the volume, in milliliters, required as S. Perform a blank determination with 4.0 mL of Peroxide Substrate Solution, and record the volume required, in milliliters, as B.

[Note: When preparations derived from beef liver are tested, the reaction is complete within 30 min. Preparations derived from Aspergillus and other sources may require up to 1 h. In assaying an enzyme of unknown origin, run a titration after 30 min and then at 10-min intervals thereafter. The reaction is complete when two consecutive titrations are the same.]

 

Calculation
One Baker Unit is defined as the amount of catalase that will decompose 264 mg of hydrogen peroxide under the conditions of the assay.
Calculate the activity of the sample by the equation
Baker Units/g or mL = 0.4(B S) × (1/C)

in which C is the milliliters of aliquot of original enzyme preparation added to each 100 mL of Peroxide Substrate Solution, or when 1 mL of diluted enzyme is used, C is the dilution factor; B is the volume, in milliliters, as defined above; and S is the milliliters of 0.250 N Sodium Thiosulfate, as defined above.

 

CERTIFICATE OF ANALYSIS

 

Packaging and storage

Solid food packaging bag, 25 kg / barrel.

 

Catalase should be stored in dry, cool and well ventilated places away from the sun, heat.

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