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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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Bacteria preincubated with myeloperoxidase (MPO) are more readily killed upon the addition of H2O2 and Cl- than controls not subject to prior incubation. This effect was evidenced by decreased requirements of MPO and H2O2 (to approximately 33%) for equivalent bactericidal activity. MPO adsorbed onto the bacterial surface is not accessible to other competing substrates such as guaiacol and [1(-14)C] alanine. It appears that when MPO is adsorbed to the bacteria, it carries out a coupled reaction in which the activated chlorine directly attacks the bacterial cell surface.

The course of chlorination in neutrophilic granulocytes has been shown. The process of 36Cl incorporation occurs during and after the engulfment of bacteria by granulocytes. Incorporated radioactivity was found in insoluble fractions.The myeloperoxidase obtained from neutrophils catalyzes chlorination of protein (bovine serum albumin) and bacteria (Staphylococcus epidermidis) in the presence of hydrogen peroxide and chloride. The products of chlorination are insoluble.Chlorination in neutrophils is inhibited by the iodide and myeloperoxidase inhibitors azide and cyanide.A quantitative method of determination of biological chlorination in cells has been devised.In a previous study it was found that myeloperoxidase isolated from neutrophilic granulocytes catalyzes the reaction of chlorination [l]. The mechanism of catalysis involves the oxidation of chloride ions by hydrogen peroxide and the formation of Clf ions [2,3]. These ions spontaneously chlorinate the amino compounds [l, 31.The phagocytosing granulocytes possess all factors needed for the chlorination process, i.e. myeloperoxidase coating the bacteria in the phagosome [4], hydrogen peroxide [5-81, chloride ions [9] and pH 4.7 -6.6 within the phagocytic vacuole [lo, 111 optimal for myeloperoxidase-mediated chlorination [3]. Therefore it is highly probable that chlorination occurs if the phagocytes produce all the factors in the same place and at the same time.The present study reports attempts to show the course of chlorination in phagocytosing granulocytes supported only by glucose and Na36C1. MATERIALS AND METHODSAll chemicals used were of reagent grade. Hydrogen peroxide, glucose, potassium iodide, mercuric chloride were obtained from P.O.CH. (Poland), bovine serum albumin from Biomed. (Poland), taurine from Koch-Light (England), 2-alanine from Reanal (HunEnzyme. Myeloperoxidase (EC 1.11.1.7). gary) and ethyl acetate from Chemapol (Czechoslovakia). Na36C1 (340 mCi/mol) was purchased from Radiochemical Centre (Amersham, England).Myeloperoxidase was isolated from human leucocytes by the method previously described [12]. The ratio A,3,/A280 was 0.6. The specific activity of the enzyme assayed by the method of Maehly [13] was 51 guaiacol units/mg. Isolation of Leucocytes and Preparation of BacteriaHuman leucocytes were obtained from healthy 30-40-year-old males or females. Routinely, 20 ml of blood (A, B, or AB group) were collected with 1 kU of heparin as anticoagulant, and mixed with an equal volume of 0 group plasma in a siliconized burette (1.2 x 64 cm). The mixture was allowed to settle for 1 h at 37 "C. The agglutinated erythrocytes were discarded and the supernatant was transferred to siliconized tubes and centrifuged gently. Then the sedimented leucocytes were suspended in 0.2 ml of autologous plasma. Using this method 8 x lo7 to 1.1 x lo8 leucocytes, of which 65-80 % consisted of neutrophils, were obtained.Staphylococcus epidermidis (no. 2340) was grown in trypticase soy broth (Difco) for 18 h. The bacteria were washed twice with saline and water a...