Myeloperoxidase, Recombinant, Human (MPO, pANCA)

Myeloperoxidase (MPO) is a heme-containing enzyme belonging to the XPO subfamily of peroxidases. It is an abundant neutrophil and monocyte glycoprotein that catalyzes the hydrogen peroxide-dependent conversion of chloride, bromide, and iodide to multiple reactive species (1). Post-translational processing of MPO involves the insertion of a heme moiety and the proteolytic removal of both a propeptide and a 6 aa internal peptide (2). This results in a disulfide-linked dimer composed of a 60 kD heavy and 12kD light chain that associate into a 150 kD enzymatically active tetramer. The tetramer contains two heme groups and one disulfide bond between the heavy chains (2). Alternate splicing generates two additional isoforms of MPO, one with a 32 aa insertion in the light chain, and another with a deletion of the signal sequence and part of the propeptide (3). Human and mouse MPO share 87% aa sequence identity. MPO activity results in protein nitrosylation and the formation of 3-chlorotyrosine and dityrosine crosslinks (4-6). Modification of ApoB100, as well as the lipid and cholesterol components of LDL and HDL, promotes the development of atherosclerosis (5, 7-9). MPO is also associated with a variety of other diseases (1), and inhibits vasodilation in inflammation by depleting the levels of NO (10). Serum albumin functions as a carrier protein during MPO movement to the basolateral side of epithelial cells (11). MPO is stored in neutrophil azurophilic granules. Upon cellular activation, it is deposited into pathogen-containing phagosomes (2). While mice lacking MPO are impaired in clearing select microbial infections, MPO deficiency in humans does not necessarily result in heightened susceptibility to infections (12, 13).\n\nSource: Human CD33 Signal Peptide (Met 1-Ala 16), Human Myeloperoxidase (Ala 49-Ser 745), HHHHHHHHHH; A DNA sequence encoding the signal peptide from human CD33 joined with the human Myeloperoxidase (Ala 49-Ser 745; Accession # P05164) was fused with a 10x histidine tag at the C-terminus. The chimeric protein was expressed in a mouse myeloma cell line, NS0.\n\nMolecular Mass: \nThe recombinant human Myeloperoxidase starts at Ala 49 and has a calculated molecular mass of approximately 80kD. As a result of glycosylation, the protein migrates as an ~80-90 kD protein in SDS-PAGE under reducing conditions. In gel filtration chromatography, the recombinant protein migrates as a tetramer.\nEndotoxin Level: < 1.0 EU per 1ug of the protein as determined by the LAL method.\n\nActivity: Measured by its ability to oxidize guaiacol in the presence of hydrogen peroxide (Desser, R.K., et al. 1972 Arch.Biochem. Biophys. 148: 452-65). See Activity Assay Protocol on next page for details. The specific activity of the recombinant human Myeloperoxidase is > 5000 pmoles/min/ug as defined in the assay.\n\nStorage and Stability: Lyophilized powder may be stored at -20/-70 degrees C. Reconstitute to nominal volume by adding ddH20 and 0.1% BSA or HSA . Aliquot and store at -20/-70 degrees C. Reconstituted product is stable for 3 months at -20/-70 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

M9760-02R

Size

250ug

Form

Supplied as a lyophilized powder from 20mM Tris, 150mM sodium chloride, pH 7.5. Reconstitute with ddH20 and 0.1% BSA or HSA to 0.5-1mg/ml.

Purity

(same/more than) 90%, as determined by SDS-PAGE and visualized by silver stain. Endotoxin:(same/less than) 1EU/1ug (LAL).

References

1. Klebanoff, S.J., 2005, J. Leukoc. Biol. 77:598. 2. Hansson, M. et al., 2006, Arch. Biochem. Biophys. 445:214. 3. Hashinaka, K. et al., 1988, Biochemistry 27:5906. 4. van Dalen, C.J. et al., 2000, J. Biol. Chem. 275:11638. 5. Hazen, S.L. and J.W. Heinecke, 1997, J. Clin. Invest. 99:2075. 6. Heinecke, J.W. et al., 1993, J. Clin. Invest. 91:2866. 7. Podrez, E.A. et al., 1999, J. Clin. Invest. 103:1547. 8. Bergt, C. et al., 2004, Proc. Natl. Acad. Sci. 101:13032. 9. Hazen, S.L. et al., 1996, J. Biol. Chem. 271:23080. 10. Eiserich, J.P. et al., 2002, Science 296:2391. 11. Tiruppathi, C. et al., 2004, Proc. Natl. Acad. Sci. 101:7699. 12. Aratani Y. et al., 2000, J. Infect. Dis. 182:1276. 13. Kutter, D., 1998, J. Mol. Med. 76:669.