PubMed · 4424516
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D Brekke. 1974-09-30. [Letter: Benzidine which disappeared].. https://pubmed.ncbi.nlm.nih.gov/4424516/
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Dapsone (4,4'-diaminodiphenylsulfone) is an antimicrobial substance that also has anti-inflammatory activity, which has been attributed to inhibition of the leukocyte enzyme myeloperoxidase (MPO). We observed that dapsone was a much better inhibitor of the eosinophil peroxidase (EPO) in an assay that measured peroxidase-catalyzed oxidation of tetramethylbenzidine at pH 5.4. To clarify the specificity and pH-dependence of dapsone inhibition of the purified enzymes under more physiologic conditions, we studied peroxidase-catalyzed oxidation of chloride to the antimicrobial and cytotoxic agent hypochlorous acid. Taurine was added as a trap for hypochlorous acid, to prevent inactivation of the enzymes or chlorination of dapsone by hypochlorous acid. Dapsone was much more effective as an inhibitor of both MPO and EPO when chloride rather than tetramethylbenzidine was the substrate. Inhibition of both enzymes was greater at neutral pH than at acid pH (pH 7 vs pH 5), but EPO was more sensitive to inhibition than MPO regardless of pH. Inhibition was increased by lowering chloride, raising hydrogen peroxide, or lowering the enzyme concentration. Inhibition was accompanied by irreversible loss of enzyme activity, which was correlated with loss of the heme absorption spectrum, indicating chemical modification of the enzyme active site. EPO, but not MPO, was partially protected against inactivation by adding physiologic levels of bromide along with chloride. The results suggest that dapsone could prevent MPO- and EPO-mediated tissue injury at sites where the peroxidase enzymes are secreted and diluted into the neutral pH environment of the tissue interstitial space. Dapsone might not inhibit peroxidase-mediated antimicrobial activity, which occurs at high enzyme concentrations in the acid environment of phagolysosomes.
Although Neisseria gonorrhoeae can use haem as the sole exogenous iron source for growth in vitro, the mechanism of haem-iron uptake in the gonococcus is unknown. Two haemin-binding proteins (HmBPs) of 97 and 44 Kda were isolated by batch ligand affinity-chromatography from whole cells or total membranes of gonococci grown under iron-limited conditions but not from those grown under iron-sufficient conditions. Competition binding experiments indicated that the haemin-protein interaction was specific; only haemin or haem-containing proteins, such as human haemoglobin or equine cytochrome c111, but not protoporphyrin IX, iron loaded human transferrin or lactoferrin, could abrogate binding. Identical HmBPs were isolated from three other clinical gonococcal strains, suggesting that these may be interstrain structural and functional homogeneity amongst these polypeptides.
A rapid, simple method is described which permits a three-fold enhancement of the working range of ELISA procedures using TMB as a substrate. This consists of measuring absorbance values at a wavelength away from the absorption maximum of TMB and using a predetermined multiplication factor.