Evidence for and partial characterization of three major and three minor chromatographic forms of human neutrophil myeloperoxidase.
Myeloperoxidase (MPO) is an enzyme found in the azurophil granules of neutrophils. Cation-exchange chromatography on carboxymethyl-cellulose previously has been used to demonstrate the heterogeneity of the peroxidase enzymes isolated from human neutrophils. In this study, fast protein liquid chromatography (FPLC) was used to separate and purify three major (I, II, and III) and three minor (IIa, IIIa, IIIb) forms of MPO from isolated neutrophil granules. Purity was confirmed by polyacrylamide gel electrophoresis in the presence of cetyltrimethylammonium bromide (CETAB-PAGE), by crossed immunoelectrophoresis, and by spectral characteristics. All three major forms were indistinguishable by immunodiffusion against rabbit antiserum, scanning spectrophotometry, and amino acid composition. They differed in their elution from a cation-exchange resin, inhibition by 3-amino-1,2,4-triazole, migration rate in CETAB-PAGE, and subunit molecular weight. Subunit molecular weight was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All three major forms appeared to consist of heavy (H), intermediate (M), and light (L) peptides. The M peptide appeared to be derived from the H subunit. All L subunits exhibited a molecular weight of 14,500. The molecular weights for the H subunits varied, and were 60,000, 59,000, and 57,000 for MPO I, II, and III, respectively. The molecular weights for the M peptides were 44,100, 43,000, and 42,000 for MPO I, II, and III, respectively. The treatment of neutrophils, granules, and extracts with protease inhibitors and sodium azide did not block the appearance of three major forms of MPO. Thus, neither protease activity nor MPO autooxidation during extraction and purification procedures is responsible for the appearance of multiple chromatographic forms of MPO derived from human neutrophils.
D000596:Amino Acids / Q000097:blood; D000640:Amitrole / Q000494:pharmacology; D000818:Animals; D001386:Azides; D002417:Cattle; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D002845:Chromatography / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006595:Hexosamines / Q000032:analysis; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D007784:Lactoperoxidase; D008892:Milk / Q000201:enzymology; D009504:Neutrophils / Q000201:enzymology*; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000097:blood*; D019810:Sodium Azide; D013053:Spectrophotometry
D000596:Amino Acids; D001386:Azides; D002593:Cetrimonium Compounds; D006595:Hexosamines; D019810:Sodium Azide; D007784:Lactoperoxidase; D009195:Peroxidase; D000077286:Cetrimonium; D000640:Amitrole