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Human Eosinophil Peroxidase
welcome to
product specification
of
human eosinophil peroxidase
Index:
0 Company Headquater
1 General Product Information
2 Detailed Unit Definition
3 UV / VIS Spectrum
4 MALDI-TOF Spectrum
1
2
3
4
4
0 Company Headquater
Planta Natural Products
Vertriebs GmbH
Erlgasse 48
A-1120 Wien
Vienna / Austria
fax: + 43-1-8105366-13
email: info@planta.at
http://www.peroxidase.at
in cooperation with
Metalloprotein Research Group
Department of Chemistry
BOKU - University of Natural Resources and Applied Life Sciences, Vienna
Muthgasse 18
A-1190 Wien
Vienna / Austria
fax: +43-1-36006-6059
email: Christian.Obinger@boku.ac.at
http://www.chemie.boku.ac.at/3735.html
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Product Information Sheet Page 1 of 4
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Human Eosinophil Peroxidase
1 General Product Information
Product name:
Eosinophil Peroxidase from human eosinophils
Description:
Haem protein that catalyses oxidations by H2O2, including EPO-bromidemediated killing of helmintic parasites, metazoan pathogens and cancer cells,
and the enzyme is associated with the majority of tissue damage in asthma.
Form:
Brown solid, lyophilized from 5 mM phosphate buffer, pH 7.0.
Solubility:
Reconstitution of 100 µg with 800 µl distilled water gives approximately 1.7µM
EPO calculated with a molar extinction coefficient at 413 nm of 110000 M 1cm -1.
Storage:
Stored below 0°C this lyophilized product is stable for years. Storage of the
reconstituted form at 4°C for 2 weeks has no effect on enzyme activity. If
solutions must be stored for extended periods of time, protein concentration
should be kept above 1 mg/ml, and the activity should be determined prior to
use.
Source:
Prepared from human blood that has been shown by certified tests to be
negative for HBsAg and HIV antibodies.
FOR RESEARCH ONLY!
NOT FOR HUMAN OR DRUG USE!
CAS Number:
9003-99-0
EC Number:
1.11.1.7
Molecular weight:
~ 70 500 Da ( see MALDI-TOF Spectrum )
Purity:
> 99 %
Reinheitszahl:
A413 / A280 > 0.9 ( see UV / VIS Spectrum )
Specific activity:
270
1520
– 300
– 1590
units per mg (assay 1, Guaiacol)
units per mg (assay 2, MCD)
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Product Information Sheet Page 2 of 4
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Human Eosinophil Peroxidase
2 Detailed Unit Definition
Assay 1 (Guaiacol):
One unit will produce an increase in absorbance at 470 nm of 1.0 per minute
at pH 7.0 and 25°C, calculated from the initial linear rate of reaction using
guaiacol as substrate for the peroxidatic activity.
Total reaction volume:
1 ml
Final assay concentrations:
100 mM phosphate buffer, pH 7.0
100 µM guaiacol
100 µM hydrogen peroxide
50 nM EPO
References:
Desser, R.K., Himmelhoch, S.R., Evans, W.H., Januska, M.,
Mage, M., and Shelton, E. (1972) Arch. Biochem. Biophys.
148, 452.
Assay 2 (MCD):
One unit will produce a decrease in absorbance at 290 nm of 1.0 per minute
at pH 7.0 and 25°C, calculated from the initial linear reaction rate using
monochlorodimedon (MCD) and bromide as substrate for the brominating
activity.
Total reaction volume:
1 ml
Final assay concentrations:
100 mM phosphate buffer, pH 7.0
100 µM MCD
10 mM NaBr
100 µM hydrogen peroxide
10 nM EPO
References:
Hager, L.P., Morris, D.R., Brown, F.S., and Eberwein, H.
(1966) J. Biol. Chem. 241, 1769.
Kettle, A.J., and Winterbourn, C.C. (1988) Biochim. Biophys.
Acta 957, 185.
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Product Information Sheet Page 3 of 4
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Human Eosinophil Peroxidase
3 UV / VIS Spectrum
1.2
Absorbance
0.9
RZ > 0.9
0.6
0.3
0
250
400
550
700
850
Wavelength (nm)
UV/VIS spectrum of 9 µM EPO in 5mM phosphate buffer pH 7.0 recorded against distilled water on a diodearray spectrophotometer Zeiss Specord S-10
4 MALDI-TOF Spectrum
MALDI-TOF spectrum of 100 µg freshly reconstituted EPO in 800 µl distilled water.
The MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) was carried out
on a DYNAMO MALDI-TOF-MS (Thermo BioAnalysis, Santa Fe, New Mexico) with sinapinic acid matrix. Spectra
were recorded in the dynamic extraction mode (setting 0.1) and calibrated externally using bovine serum albumin.
The protein sample was mixed with a 1% solution of the matrix in 70% acetonitrile. 1 µl of this mixture was
deposited to a probe, air dried and inserted into the mass spectrometer to acquire a spectrum. The inset shows the
SDS polyacrylamide gel electrophoresis of this preparation (5 µg EPO) in comparison with 5 µg MPO.
Electrophoresis was performed on boiled reduced samples. The first lane shows the migration of standard
proteins.
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Product Information Sheet Page 4 of 4