Spectral and enzymatic properties of human recombinant myeloperoxidase: comparison with the mature enzyme.
Human recombinant myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary (CHO) cell line, has been characterized and compared to the mature enzyme isolated from polymorphonuclear leukocytes. Both molecules appear essentially similar in physicochemical enzymatic terms according to the following observations. 1. The unprocessed recombinant protein displays the characteristic light absorption spectra of ferric mature MPO and exhibits its typical spectral changes in the presence of dithionite or hydrogen peroxide. 2. The addition of 14C-labeled 5-aminolevulinic acid, a heme precursor, to the culture medium of recombinant CHO cells yields labeled recMPO, indicating the presence of a heme-like structure in the molecule. 3. Like mature MPO, recMPO has a peroxidatic activity and catalyzes the oxidation of chloride ions in the presence of hydrogen peroxide, producing hypochlorous acid as measured by the monochlorodimedon assay. For both enzymes, the chlorinating activity optimally occurs around pH 5.0 at about 100 microM of hydrogen peroxide and is strongly inhibited by methimazole. 4. Diethylpyrocarbonate significantly reduces the enzymatic activity of both molecules, suggesting that histidine residues may be of prime importance in the active site of the enzymes. 5. According to infrared spectroscopy data, both enzymes present a very similar secondary structure organization. In conclusion, the data suggest that the processing of the precursor enzyme (recMPO) into the mature form occurs without major structural and functional consequences.
Jacquet
A
A
Applied Genetics, Université Libre de Bruxelles, Nivelles, Belgium.
Deby
C
C
Mathy
M
M
Moguilevsky
N
N
Deby-Dupont
G
G
Thirion
A
A
Goormaghtigh
E
E
Garcia-Quintana
L
L
Bollen
A
A
Pincemail
J
J
D000818:Animals; D016466:CHO Cells; D002713:Chlorine / Q000378:metabolism; D006224:Cricetinae; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006418:Heme / Q000378:metabolism; D006801:Humans; D009504:Neutrophils / Q000201:enzymology; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013055:Spectrophotometry, Infrared; D013057:Spectrum Analysis
D011994:Recombinant Proteins; D006418:Heme; D002713:Chlorine; D009195:Peroxidase; D004047:Diethyl Pyrocarbonate