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.
MW = ca. 59000 in reduced SDS-PAGE indicating two protein subunits.
MW = ca. 120000 on S200 chromatography.
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
- 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.
- 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.
- 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.
- 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.
- Adjust the spectrophotometer to 510 nm and the cell temperature to 25°C.
- 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.
- 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
- 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
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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