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Lactoperoxidase - Assay

Source:
Bovine Milk
CAS:
9003-99-0
EC:
1.11.1.7
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Lactoperoxidase Assay

Method

The assay procedure is a modification of that described by Putter and Becker [Meth. Enz. Anal., III, 286 (1983)]. The increase in the absorbance at 405 nm resulting from the oxidation of 2,2-azino-di(3-ethyl-bnzothiazoline-6-sulphonic acid), diammonium salt, ABTS. Two moles of ABTS are oxidized for every mole of peroxide that is reduced.

Reagents

0.08 M Sodium-Potassium Phosphate Buffer pH 6.0: Prepare by adjusting the pH of 0.08 M sodium phosphate, dibasic, to 6.0 using 0.08 M potassium phosphate, monobasic.

Sodium Potassium Phosphate Buffer, containing 1 mg/ml Bovine Serum Albumin: Dissolve BSA at a concentration of mg/ml in an aliquot of the above buffer. This will be used as enzyme diluent.

0.0017 M Hydrogen peroxide. Prepare by diluting 1 ml of 30% hydrogen peroxide to 100 ml with reagent grade water. Further dilute 1 ml of this solution to 50 ml with reagent grade water. Prepare fresh daily.

0.051 M ABTS: Dissolve ABTS in 0.08 M sodium phosphate buffer, pH 6.0

Enzyme

Dissolve at one mg/ml in sodium-phosphate buffer. Immediately prior to use, dilute further in phosphate buffer with BSA to obtain a rate of 0.008–0.025 DA/minute.
Keep all dilutions on ice.

Procedure

Set spectrophotometer at 25°C and 405 nm. Pipette into each cuvette 2.65 ml sodium- phosphate buffer, 0.15 ml hydrogen peroxide, and 0.10 ml ABTS. Incubate in spectrophotometer at 25°C for 3-5 minutes to achieve temperature equilibration and establish a blank rate, if any. Add 0.10 ml diluted enzyme and record the increase in A405 for 4-6 minutes. Calculate the rate from the maximum initial linear portion of the curve. The reaction is linear for approximately two minutes.

Calculation

18.6= extinction coefficient of ABTS at 405nm
2 ABTS:1 H2O2

Lactoperoxidase Products

Description
Activity
Code
Cat. #
Size
Price
Description
Lactoperoxidase
Source:
Bovine Milk
Chromatographically purified. A lyophilized powder.
Store at -20°C.
…
Activity
≥35 units per mg dry weight ABTS
Code
LPO
LS000150
10 mg
$89.00
LS000151
50 mg
$360.00
LS000152
Bulk
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The minimum amount for bulk packaging/pricing for this product is 1000 x 1 mg. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.