Single-step immunoaffinity purification and characterization of dodecylmaltoside-solubilized human neutrophil flavocytochrome b.
Flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that serves as the central component of an electron transferase system employed by phagocytes for elimination of bacterial and fungal pathogens. This report describes a rapid and efficient single-step purification of Cyt b from human neutrophil plasma membranes by solubilization in the nonionic detergent dodecylmaltoside (DDM) and immunoaffinity chromatography. A similar procedure for isolation of Cyt b directly from intact neutrophils by a combination of heparin and immunoaffinity chromatography is also presented. The stability of Cyt b was enhanced in DDM relative to previously employed solubilizing agents as determined by both monitoring the heme spectrum in crude membrane extracts and assaying resistance to proteolytic degradation following purification. Gel filtration chromatography and dynamic light scattering indicated that DDM maintains a predominantly monodisperse population of Cyt b following immunoaffinity purification. The high degree of purity obtained with this isolation procedure allowed for direct determination of a 2:1 heme to protein stoichiometry, confirming previous structural models. Analysis of the isolated heterodimer by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allowed for accurate mass determination of p22(phox) as indicated by the gene sequence. Affinity-purified Cyt b was functionally reconstituted into artificial bilayers and demonstrated that catalytic activity of the protein was efficiently retained throughout the purification procedure.
Department of Microbiology, Montana State University, 109 Lewis Hall, Bozeman, MT 59717-3520, USA.
Ross M
RM
Taylor
James B
JB
Burritt
Thomas R
TR
Foubert
Meagan A
MA
Snodgrass
Kim C
KC
Stone
Danas
D
Baniulis
Jeannie M
JM
Gripentrog
Connie
C
Lord
Algirdas J
AJ
Jesaitis
D001798:Blood Proteins / Q000302:isolation & purification*; D003573:Cytochrome b Group; D004795:Enzyme Stability; D005960:Glucosides / Q000494:pharmacology*; D006418:Heme / Q000032:analysis; D006801:Humans; D026901:Membrane Transport Proteins; D009252:NADPH Dehydrogenase / Q000737:chemistry; D019255:NADPH Oxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D009504:Neutrophils / Q000201:enzymology*; D010750:Phosphoproteins / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001798:Blood Proteins; D003573:Cytochrome b Group; D005960:Glucosides; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D021122:Protein Subunits; D006418:Heme; C040358:dodecyl maltoside; C036888:cytochrome b558; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase