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Myeloperoxidase was isolated from leucocytes obtained from the blood of patients suffering from chronic granulocytic leukaemia. The enzyme was purified 850 fold and was homogeneous in ultracentrifuge and free boundary electrophoresis. The molecular weight of the enzyme was found to be about 160,000. The enzyme forms a spectrally characteristic complex with hydrogen peroxide with absorption maximum at 458 mμ. The spectrum of the native enzyme has absorption maxima at 430 and 570 mμ. The reduced enzyme is characterized by a spectrum with absorption maxima at 472 and 637 mμ. The investigated myeloperoxidase catalysed oxidation of amino acids by hydrogen peroxide. Products of the oxidation of amino acids were ammonia, carbon dioxide, and an aldehyde corresponding to the oxidized amino acid. The observed reaction of deamination and decarboxylation is activated by chloride ions. In the presence of the chloride ions the optimum of the reaction is shifted toward the higher pH values. The velocity of the reaction was found to be dependent on the concentration of the amino acid studied. Km values for various amino acids increased in the range 3.4 × 10−4 to 10−3 M in proportion to rising hydrophobic properties of the substrates. Taurine was found to be a competitive inhibitor in the examined reaction, and Ki values were in the range of 2 to 3 × 10−4 M, for different amino acids.

The chlorination of dipeptides by the myeloperoxidase/H20~/C1-system takes place at the N-terminal amino group, whereas no chlorination of the amide nitrogen of the peptide bond can be observed. The N-terminal amino group is chlorinated to N-monochloroamine or/and N-dichloroamine. N-Monochloropeptides were the main products at higher pH values, at lower pH a mixture of N-monochloropeptides and N-dichloropeptides was formed owing to the dismutation of N-monochloroamine to N-dichloroamine.N-Monochloropeptides decompose, yielding NH3 and the corresponding N-(2-oxoacyl)amino acids. N-Dichlorodipeptides decompose faster but to nitriles and the free C-terminal amino acids. N-Dichloroglycyl-amino acid decomposes through a relatively stable intermediate (cyano-formylamino acid) to hydrogen cyanide, cyanogen chloride and the free C-terminal amino acid. lnsulin chlorination also yields N-terminal glycyl and phenylalanyl N-monochloro derivatives, which deaminate to glyoxylyl and phenylpyruvyl residues.Myeloperoxidase of polymorphonuclear neutrophilic granulocytes catalyses the oxidation of CIions to HOCl by hydrogen peroxide [1-31. The chlorinating ability of myeloperoxidase seems to be important for the bactericidal mechanisms of neutrophils [4-61. The physiological role of the chlorination process was emphasized by its discovery in granulocytes undergoing phagocytosis [7]. Peptides and proteins may be target substrates for chlorination during phagocytosis. The incorporation of 36Cl into bovine albumin by purified myeloperoxidase was reported [7].From the chemical point of view, many different functional groups in protein are susceptible to HOCl attack : e.g. amino, imidazolium, guanidinium, indole residues, phenolic rings and the amide nitrogen of peptide bonds. In the enzymatic reaction, however, HOCl production by myeloperoxidase is impaired when the chlorination or oxidation of the HOClreactive substrate is slower than that of the enzyme itself. In such cases autodestruction of niyeloperoxidase occurs.Abbreviations. AcAla, N-acetyl-alanine; CIGly-Leu, N-monochloroglycyl-leucine; CIAla-Ala, N-monochloroalanyl-aianine; ClLeu-Gly, N-monochloroleucyl-glycine; ClzCly-Leu, N-dichloroglycyl-leucine.Enzyme. Myeloperoxidase (EC 1.11.1.7).It was established that amino compounds are effective acceptors of C1' ions formed in myeloperoxidase-mediated reactions [2,8] and protect myeloperoxidase against HOCl attack [9].Preliminary examination of bovine albumin chlorination by the myeloperoxidase system showed that 36Cl incorporated into protein was to a great extent K1-reactive, i.e. N-CI bonds were formed. These results focussed attention on the chlorination of the protein amino groups (N-terminal and 8-lysine amino groups) and the amide nitrogen of peptide bonds.It seemed reasonable to start the systematic elucidation of protein chlorination by examining the reactions of simple dipeptides without side-chain functional residues. This made it possible to check the reactions only of N-terminal amino groups and of the peptidebond ami...

Two pH-dependent spectral forms of myeloperoxidase have been described. Their absorption maxima in the Soret region were at 432 nm and 426 nm for acid and neutral pH, respectively. Chloride exerts an influence on the spectrum of peroxidase, mainly in acidic medium. Dissociation constants for the myeloperoxidase * chloride complex and K , values for chloride in the chlorination reaction catalyzed by myeloperoxidase were determined. It was found that the affinity of chloride ions to the enzyme decreases with an increase of pH.Catalytic activity of myeloperoxidase in the reaction of incorporation of 36Cl into aliphatic amino compounds such as taurine, 8-alanine, ethanolamine and diethanolamine was investigated. Primary amino groups were chlorinated to N-mono-or dichloramines, depending on the pH of the medium. The method of determination of 36C1-labelled chlorocompounds in the presence of excess Na3%I was described.The role of myeloperoxidase as chlorinating enzyme in the mechanism of the bactericidal effect on bacteria phagocytized by neutropllilic granulocytes is discussed.

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