Purification and identification of placental histaminase
Smith, James Kemp
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Saline extracts were made from desanguinated human placentae, and histaminase was purified from these extracts by salt fractionation, ion-exchange chromatography on cellulosic adsorbents, and by gel filtration on Sephadex G-200. The Specific Activity of the preparation increased approximately 800- fold from Stage 1 to Stage 6, part of this increase being due to the removal of enzyme inhibitors. Further attempts to resolve Stage 6 enzyme by ion-exchange chromatography and re-cycling gel filtration removed some of the remaining contaminants, but did not result in further increases in Specific Activity. Starch gel electrophoresis of the purest preparation revealed that the enzyme had not been separated from high-molecular weight haptoglobin-methaemoglobin species. A new spectrophotometric test for histaminase was developed from Kapeller-Adler 's (1951) indigo test, which proved unsuitable for the present work. The new test was about 50 times more sensitive than any previous method measuring a common product of oxidative deamination of all substrates. Extinction changes after 24. hr. incubation were linearly related to enzyme concentration. Zeller's criticisms of indigo tests (1956, 1965) could not be substantiated. The new indigo test gave very similar results to the method of Holmstedt and Tham (1959) in almost all applications. Although the oxidation of substrate and the production of hydrogen peroxide proceeded immediately, indigometric assays were subject to an initial delay in indigo oxidation, lasting several hours. The oxidation of indigo by synthetic hydrogen peroxide was not subject to any delayj the mechanisms of indigo oxidation by synthet c hydrogen peroxide and by hydrogen peroxide produced in the enzyme-substrate reaction were not the same. More than the stoichiometric amount of indigo was oxidised in tests using synthetic or enzymic hydrogen peroxide, due to oxidation by atmospheric oxygen, catalysed by substrates and products of the indigo test. No way was found to eliminate, or correct results for, hyper-stoichiometric indigo oxidation, and therefore rates of oxidation of indigo could not be translated into conventional expressions of enzymic activity. The specificity of the purest preparations of placental histaminase was found to resemble that of hog kidney and pea seedling DAO, With supporting evidence from mixed-substrate experiments, it was concluded that histaminase oxidised not only the aliphatic diamines, but also agmatine, benzylamine and histamine. Activity towards histamine could not have been due to any known contaminant. The Michaelis constants for the oxidation of several substrates by placental histaminase were determined. Km for putrescine, and the appearance of an optimal substrate concentration, varied with the assay method employed. The histamine concentration giving optimal activity was lower than that for hog kidney DAO. The pH optima for the activity of placental histaminase towards several substrates were determined. The inhibitor specificity of placental histaminasa resembled that of hog kidney and pea seedling DAO, but was distinct from that of benzylamine oxidase or monoamine oxidase (MAO). Activity towards the aliphatic diamines, but not towards histamine, tended to diminish as the enzyme was purified, unless EDTA wa3 added to the working buffers. The possible significance of metal ions in the activation and inhibition of histaminase was discussed, and it was suggested that discrepancies in earlier reports on the specificity of hog kidney DAO might be due to contamination of enzyme solutions by metal ions. Contamination of the purest preparation by other proteins precluded detailed study of the cofactors of placental histaminase, but the possible involvement of copper and pyridoxal phosphate was discussed. The molecular weight of histaminase x*as probably at least 200,000. The enzyme was much more thermolabile than hog kidney MO, but it was extremely stable at low temperatures in dilute borate buffer, pH 8.6. Priorities for future work were considered to be: (a) Development of a satisfactory assay method for oxidative deamination, (b) Separation of histaminase from haptoglobin-methaemoglobin. (c) Study of the metabolism of aliphatic diamines in normal and pregnant human subjects.