PRODUCTINFORMATON

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product specification of	human myeloperoxidase

Detailed Unit Definition

Assay 1
(Guaiacol - Sigma)

Assay 2
(Guaiacol)

Assay 3
(TMB)

Assay 4
(MCD)

Assay 5
(TNB)

Assay 2 (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 per haem
References:	Desser, R.K., Himmelhoch, S.R., Evans, W.H., Januska, M., Mage, M., and Shelton, E. (1972) Arch. Biochem. Biophys. 148, 452.

Assay 1
(Guaiacol - Sigma)

Assay 2
(Guaiacol)

Assay 3
(TMB)

Assay 4
(MCD)

Assay 5
(TNB)

One unit will produce an increase in absorbance at 655 nm of 1.0 per minute at pH 5.4 and 25°C, calculated from the initial linear reaction rate using 3,5,3‘,5‘-tetramethylbenzidine (TMB) as substrate for the peroxidatic activity.
Total reaction volume:		1 ml
Final assay concentrations:		100 mM phosphate buffer, pH 5.4 containing 8% (v/v) dimethylformamide 1.4 mM TMB 300 µM hydrogen peroxide 1 nM per haem
References:	Suzuki, K., Ota, H., Sasagawa, S., Sakatani, T., and Fujikura, T. (1983) Anal. Biochem. 132, 345. Marquez, L.A., and Dunford, H.B. (1997) Biochemistry 36, 9349.

Assay 1
(Guaiacol - Sigma)

Assay 2
(Guaiacol)

Assay 3
(TMB)

Assay 4
(MCD)

Assay 5
(TNB)

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 chloride as substrate for the chlorinating activity.
Total reaction volume:		1 ml
Final assay concentrations:		100 mM phosphate buffer, pH 7.0 100 µM MCD 100 mM NaCl 100 µM hydrogen peroxide 200 nM per haem
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.

Assay 1
(Guaiacol - Sigma)

Assay 2
(Guaiacol)

Assay 3
(TMB)

Assay 4
(MCD)

Assay 5
(TNB)

One unit will produce a decrease in absorbance at 412 nm of 1.0 at pH 7.0 and 25°C after a time of 5 minutes in the dark due to the reaction of taurine chloramine (which is produced within 20 minutes reaction time by using taurine and chloride as substrate for the chlorinating activity) with 5-thio-2-nitrobenzoic acid (TNB).
Total reaction volume:		1 ml
Final assay concentrations:
	for the chlorination of taurine:	100 mM phosphate buffer, pH 7.0 10 mM taurine 100 mM NaCl 100 µM hydrogen peroxide 20 nM per haem
	for the oxidation of TNB:	assay for the chlorination of taurine diluted 1:2 (1+1) after a reaction time of 20 minutes in 100 mM phosphate buffer, pH 7.0 200 µM TNB

References:

Weiss, S.J., Klein, R., Slivka, A., and Wei, M. (1982) J. Clin. Invest. 70, 598.
Kettle, A.J., and Winterbourn, C.C. (1994) Methods Enzymol. 233, 502.

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Product name:	Myeloperoxidase from human polymorphonuclear leukocytes
Description:	Haem protein that catalyses oxidations by H₂O₂, including MPO-chloride-mediated killing of microbes and tumor cells, inactivation of chemotactic factors, cross-linking of proteins and iodination of proteins.
Form:	Green solid, lyophilized from 5 mM phosphate buffer, pH 7.4.
Solubility:	Reconstitution of 100 µg with 800 µl distilled water gives approximately 2.0 µM per haem calculated with a molar extinction coefficient at 430 nm of 91000 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. Freezing solutions of myeloperoxidase may cause substantial loss of activity.
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:	~ 145 000 Da ( see MALDI-TOF Spectrum )
Purity:	> 99.5 %
Reinheitszahl:	A₄₃₀ / A₂₈₀ ł 0.84 ( see UV / VIS Spectrum )
Specific activity:	1350 22 1300 4.4 75	– – – – –	1750 27 1700 5.6 95	units per mg (assay 1, Guaiacol according to Sigma) units per mg (assay 2, Guaiacol) units per mg (assay 3, TMB) units per mg (assay 4, MCD) units per mg (assay 5, TNB)

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 rate of reaction using guaiacol as substrate.
Total reaction volume:		3.035 ml
Final assay concentrations:		49 mM phosphate buffer, pH 7.0 99 mM guaiacol 0.0017% (w/w) hydrogen peroxide 0.35 unit MPO
References:	Sigma Quality Control Test Procedure, Enzymatic Assay of Myeloperoxidase, http://www.sigmaaldrich.com