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 KH2PO4 into 0.1 M  K2HPO4 until the pH = 7.0 at 25°C.

 

  1. 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.    
  2. 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.

 

  1. 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.

 

  1. 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.

 

  1. 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

 

 

  1. 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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