No abstract

Myeloperoxidase and eosinophil peroxidase catalyzed the oxidation of bromide ion by hydrogen peroxide (H2O2) and produced a brominating agent that reacted with amine compounds to form bromamines, which are long-lived oxidants containing covalent nitrogen-bromine bonds. Results were consistent with oxidation of bromide to an equilibrium mixture of hypobromous acid (HOBr) and hypobromite ion (OBr-). Up to 1 mol of bromamine was produced per mole of H2O2, indicating that bromamine formation prevented the reduction of HOBr/OBr- by H2O2 and the loss of oxidizing and brominating activity. Bromamines differed from HOBr/OBr- in that bromamines reacted slowly with H2O2, were not reduced by dimethyl sulfoxide, and had absorption spectra similar to those of chloramines, but shifted 36 nm toward higher wavelengths. Mono- and di-bromo derivatives (RNHBr and RNHBr2) of the beta-amino acid taurine were relatively stable with half-lives of 70 and 16 h at pH 7, 37 degrees C. The mono-bromamine was obtained with a 200-fold excess of amine over the amount of HOBr/OBr- and the di-bromamine at a 2:1 ratio of HOBr/OBr- to the amine. In the presence of physiologic levels of both bromide (0.1 mM) and chloride (0.1 M), myeloperoxidase and eosinophil peroxidase produced mixtures of bromamines and chloramines containing 6 +/- 4% and 88 +/- 4% bromamine. In contrast, only the mono-chloramine derivative (RNHCl) was formed when a mixture of hypochlorous acid (HOCl) and hypochlorite ion (OCl-) was added to solutions containing bromide and excess amine. The rapid formation of the chloramine prevented the oxidation of bromide by HOCl/OCl-, and the chloramine did not react with bromide within 1 h at 37 degrees C. The results indicate that when enzyme-catalyzed bromide or chloride oxidation took place in the presence of an amine compound at 10 mM or higher, bromamines were not produced in secondary reactions such as the oxidation of bromide by HOCl/OCl- and the exchange of bromide with chlorine atoms of chloramines. Therefore, the amount of bromamine produced by myeloperoxidase or eosinophil peroxidase was equal to the amount of bromide oxidized by the enzyme. Bromide was preferred over chloride as the substrate for both enzymes.

No abstract

A B S T R A C T Isolated neutrophilic leukocytes were incubated with primary amines and related nitrogenous compounds. Stimulation of neutrophil oxygen (02) metabolism with phorbol myristate acetate or opsonized zymosan resulted in production of hydrogen peroxide (H202), myeloperoxidase-catalyzed oxidation of chloride (Cl-) to hypochlorous acid (HOCI), and the reaction of HOC1 with the added compounds to yield nitrogen-chlorine (N-Cl) derivatives. Formation of NCl derivatives of low lipid solubility resulted in accumulation of the derivatives in the extracellular medium. These oxidizing agents were identified and measured on the basis of their absorption spectra and their ability to oxidize 5-thio-2-nitrobenzoic acid to the disulfide form. The yield of N-Cl derivatives was in the order: taurine > Tris > spermidine > spermine > glucosamine > putrescine > guanidinoacetate. Accumulation of N-Cl derivatives was also observed in the absence of added amines, owing to the reaction of HOC1 with endogenous taurine and other amines that were released from the cells into the medium.In the presence of compounds that yield lipophilic N-Cl derivatives, little or no accumulation of oxidizing agents was observed. Instead, these compounds inhibited the accumulation of N-Cl derivatives that was obtained with taurine, and their effect was competitive with taurine. Inhibition was in the order: methylamine > ethanolamine > phenylethylamine > p-toluenesulfonamide > ammonia > guanidine. Formation of lipophilic N-Cl derivatives also resulted in inhibition of 02 uptake and glucose metabolism. Inhibition was prevented by adding catalase to eliminate H202, dapsone to inhibit myeloperoxidase, taurine to compete for reaction with HOC1, or compounds that are rapidly oxidized by HOC1 or N-Cl derivatives, to reduce these Received for publication 19 January 1983 and in revised form I April 1983. oxidizing agents. The results indicate that: (a) formation of N-Cl derivatives that do not penetrate biological membranes can protect leukocytes against the cytotoxicity of HOC1 and lipophilic N-Cl derivatives, and (b) formation of membrane-permeable N-Cl derivatives in the absence of target cells or readily oxidized substances results in oxidative attack by the NCl derivatives on leukocyte components and inhibition of leukocyte functions.

Myeloperoxidase-catalyzed oxidation of chloride (Cl-) to hypochlorous acid (HOCl) resulted in formation of mono- and dichloramine derivatives (RNHCl and RNCl2) of primary amines. The RNCl2 derivatives could undergo a reaction that resulted in incorporation of the R moiety into proteins. The probable mechanism was attack of RNCl2 or an intermediate formed in the decomposition of RNCl2 on histidine, tyrosine, and cystine residues and on lysine residues at high pH. Incorporation of radioactivity from labeled amines into stable, high molecular weight derivatives of proteins was measured by acid or acetone precipitation and by gel chromatography and electrophoresis. Whereas formation of RNCl2 was favored at low pH, the subsequent incorporation reaction was favored at high pH. Up to several hours were required for the maximum amount of incorporation, which was less than 10% of the label in RNCl2. For the amines tested, incorporation was in the order histamine greater than 1,2-diaminoethane greater than putrescine greater than taurine greater than lysine greater than glucosamine greater than leucine greater than methylamine. Initiation of the reaction required HOCl, and oxidized forms of bromide, iodide, or thiocyanate did not substitute. Inhibitors of incorporation fell into three classes. First, ammonia or amines competed with the labeled amine for reaction with HOCl, so that larger amounts of HOCl were required. Second, readily oxidized substances such as sulfhydryl or diketo compounds or thioethers (methionine) reduced RNCl2. Third, certain compounds competed with protein as the acceptor for the incorporation reaction. The amount required to block incorporation into protein depended on protein concentration. Among these inhibitors were imidazole compounds (histidine), phenols (tyrosine), and disulfides (glutathione disulfide, GSSG). Low yields of derivatives of histidine, tyrosine, and GSSG were detected by thin-layer chromatography. Acid-precipitable derivatives were obtained by reacting RNCl2 with polyhistidine or polytyrosine, and to a lesser extent with polylysine at high pH, but not with other poly(amino acids). Precipitable derivatives were also obtained by incubating MPO-containing extracts from leukocyte granules with hydrogen peroxide, Cl-, and labeled amines. The extracts were found to have a high content of substances with primary amino groups, which competed for incorporation. The results account for oxidative incorporation of amines into proteins in leukocytes and provide evidence that HOCl and nitrogen-chlorine (N-Cl) derivatives are formed in these cells. The characteristics of the incorporation reaction suggest that it would not contribute significantly to the antimicrobial activity of myeloperoxidase (MPO). Nevertheless, the reaction may provide a sensitive method for studying MPO action in vivo.

Human salivary lactoperoxidase (HS-LP) is synthesized and secreted by the salivary glands, whereas myeloperoxidase (MPO) is found in PMN leukocytes, which migrate into the oral cavity at gingival crevices. HS-LP levels vary with changes in salivary gland function, but increased numbers of MPO-containing leukocytes indicate infection or inflammation of oral tissues. To determine the contribution of each enzyme to the peroxidase activity of mixed-saliva samples, activity was assayed at pH 5.4 with tetramethylbenzidine as the substrate, with and without the inhibitor dapsone (4,4'-diaminodiphenylsulfone). Dapsone blocked the activity of HS-LP but not MPO. The enzymes were also separated and partially purified from the soluble portion of saliva samples and from detergent extracts of the saliva sediment. Chromatographic properties of the proteins were similar to those of LP from bovine milk (BM-LP) and MPO from human leukocytes. The identity and amounts of the enzymes were confirmed by the absorption spectra and by immunoblotting with antibodies to BM-LP and human MPO. Eosinophil peroxidase (EPO), a distinct enzyme found in eosinophilic leukocytes, was not detected by chromatography or with antibodies to human EPO. On average, 75% of the activity in samples from normal donors was due to MPO and 25% to HS-LP. When corrected for the lower specific activity of HS-LP in this assay, the average amount of MPO (3.6 micrograms/mL) was twice the amount of HS-LP (1.9 micrograms/mL). The amount of MPO corresponded to 1 x 10(6) PMN leukocytes/mL of saliva. The enzymes were distributed differently: Eighty-nine percent of the HS-LP was in the soluble portion of saliva, and 78% of the MPO was in the sediment, which contained 51% of the total activity. In contrast to results obtained with PMN leukocytes from blood, detergent was not required for MPO activity to be measured in saliva, indicating that the enzyme was accessible to peroxidase substrates. The results indicate that MPO is responsible for a large portion of peroxidase-catalyzed reactions in mixed saliva. The unique function of HS-LP may be carried out within the salivary glands, prior to secretion into the oral cavity.

No abstract

In secreted fluids, the enzyme lactoperoxidase (LP) catalyzes the oxidation of thiocyanate ion (SCN-) by hydrogen peroxide (H202), producing the weak oxidizing agent hypothiocyanite (OSCN-), which has bacteriostatic activity. However, H202 has antibacterial activity in the absence of LP and thiocyanate (SCN-). Therefore, LP may increase antibacterial activity by using H202 to produce a more effective inhibitor of bacterial metabolism and growth, or LP may protect bacteria against the toxicity of H202 by converting H202 to a less-potent oxidizing agent. To clarify the role of LP, the antibacterial activities of H202 and the LP-H202-SCNsystem were compared by measuring loss of viability and inhibition of bacterial metabolism and growth. The relative toxicity of H202 and the LP system to oral streptococci was found to depend on the length of time that the bacteria were exposed to the agents. During incubations of up to 4 h, the LP system was from 10 to 500 times more effective than H202 as an inhibitor of glucose metabolism, lactic acid production, and growth. However, if no more H202 was added, the concentration of the inhibitor OSCNfell because of slow decomposition of OSCN-, and when OSCNfell below 0.01 mM, the bacteria resumed metabolism and growth. In contrast, the activity of H202 increased with time. H202 persisted in the medium for long periods of time because H202 reacted slowly with the bacteria and streptococci lack the enzyme catalase, which converts H202 to oxygen and water. After 24 h of exposure, H202 was as effective as the LP system as an inhibitor of metabolism. H202 also caused a time-dependent loss of viability, whereas the LP system had little bactericidal activity. The concentration of H202 required to kill half the bacteria within 15 s was 1.8 M (6%) but fell to 0.3 M (1%) at 2 min, to 10 mM (0.03%) at 1 h, and to 0.2 mM (0.0007%) with a 24-h exposure. The results indicate that if high levels of H202 can be sustained for long periods of time, H202 is an effective bactericidal agent, and the presence of LP and SCNprotects streptococci against killing by H202. Nevertheless, the combination of LP, H202, and SCNis much more effective than H202 alone as an inhibitor of bacterial metabolism and growth.