MY862, Myeloperoxidase

MYELOPEROXIDASE

NO. MY862

Lot 535

DESCRIPTION

Prepared from leucocytes of purulent human sputum by salt fractionation, ion exchange and gel diffusion chromatography.  Supplied as a salt-free, lyophilized, greenish, soluble powder.

Stable 1 year at  5°C.

ANALYTICAL DATA

MW = ca. 59000 in reduced SDS-PAGE indicating two protein subunits.

MW = ca. 120000 on S200 chromatography.

Absorbance Ratio

430 nm/280 nm 0.74 in 0.2 M phosphate buffer pH 7.0 at 25°C.

Units per mg of protein 155.0

1 unit will decompose 1 mmole of H₂O₂ per minute at pH 7.0 and 25°C when using 4-aminoantipyrine as the hydrogen donor.

ASSAY OF ENZYMATIC ACTIVITY

Reagents

1. KPO₄ Buffer; 0.1 M pH 7.0 at 25°C.  Prepare by mixing 0.1 M KH₂PO₄into 0.1 M  K₂HPO₄ until the pH = 7.0 at 25°C.

2. Hydrogen Acceptor; 45.0 mM H₂O₂.  Prepare by dissolving 150 mg Urea-H₂O₂ (Sigma Chemical Co., No. U1753) in 100 ml KPO₄ buffer. Keep cold, prepare fresh daily.    
3. Hydrogen Donor; 2 mM 4-aminoantipyrine in 0.14 M phenol.  Dissolve 1.32 gm phenol in 100 ml KPO₄ buffer.  Add 40 mg 4-aminoantipyrine and stir until completely dissolved.  Store at room temperature in an amber glass bottle.  Discard after 7 days.

4. Enzyme Solution.  Dissolve 1.0 mg per ml in KPO₄ buffer.  Further dilute to 0.1 mg  per ml with KPO₄ buffer.  Keep both solutions in an ice bath.

Procedure

1. Adjust the spectrophotometer to 510 nm and the cell temperature to 25°C.

2. Dispense into the cell 3.0 ml of the hydrogen donor solution and 30ml hydrogen acceptor solution.  Mix well and equilibrate to 25°C in the cell.   Avoid foaming the mixture.

3. Add 5ml of enzyme solution (0.1 mg/ml) and record the increase in absorbency A₅₁₀ at 30 second intervals.  Calculate the increase in absorbency per minute from the linear response, which occurs within the first 2 minutes after addition of MPO.

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TECHNICAL DATA SHEET

MYELOPEROXIDASE

NO. MY862

4. Determine the extinction coefficient Î of  1 mM H₂O₂.  EXAMPLE: Using 3.0 ml hydrogen donor plus 30ml H₂O₂ add 0.01 ml of the 1.0mg/ml MPO, read D₅₁₀ at 30 second intervals. The D₅₁₀ maximum should be approximately 1.20-1.30.   By calculation Î for 1 mM H₂O₂ = D₅₁₀ max./ 0.45 mM.

Calculation of Specific Activity

Vol = 3.035 ml               A=510 nm          T=25°C          Light Path = 1 cm

                                                Units   ₌    (DA/min) (3.035 ml)

                                                 mg              0.0005 mg x Î

Please Note:

All the human sputum leucocyte enzymes are prepared from human sputum shown to be non-reactive for HIV antibody, negative for Hepatitis C antibody.

The MY862 is re-assayed every 6 months in our laboratory to verify activity.When the product leaves EPC we feel it will be stable for 1 year at 5°C

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