Proprietes des diphenol oxydases extraites des sols

Properties of diphenol oxidases extracted from salts. Salmine and SP-Sephadex C-25 were used to separate the enzyme system associated with humic materials in the neutral extracts of fresh soils (NAFS Extract). Electrophoresis on polyacrylamide gel shows that this preparation is heterogeneous. The elementary analysis of the soil enzyme is C 43·13%; N 5·09%; H 7·21%; O 44·58%. Chromatographic analyses indicate that the soil enzyme contains 53 per cent amino acids, 36 per cent sugars and amino sugars and 10 per cent ammonium and inorganic materials. The soil enzyme has a maximum absorption at 270–280 nm. The soil enzyme degrades the following substrates at the relative rate mentioned in parentheses : d-catechin (298);p-cresol (251); catechol (156); dl-DOPA (100);p-phenylene diamine (59);p-quinol (20) in terms of rate of oxygen absorption. This enzymatic preparation has the properties of an o- and p-diphenol oxidase. The rate of decarboxylation was measured using a radiorespirometer. The following relative values are dl-DOPA-l-¹⁴C (100); dl-tyrosine-l-¹⁴C (35) ; dl-tyrptophan-1-¹⁴C (7); dl-phenylalanine-l-¹⁴C (2). The dl-DOPA-2-¹⁴C was partially degraded to ¹⁴CO₂. The O₂ absorbed and CO₂ (carboxyl) evolved in case of dl-DOPA was in the ratio of 1·8 at 37°C. The activation energy on dl-DOPA was 3·1 and 7·9 kcal/mole/°C for oxygen absorption and decarboxylation respectively. The enzymatic activity on dl-DOPA-l-¹⁴C was optimum in air and inhibited in a N₂ atmosphere. Decarboxylation on dl-DOPA-l-¹⁴C followed the Michaelis-Menten law, from which we found that K_m = 8·3 × 10^?4M for decarboxylation. The oxidative decarboxylation was inhibited by H₂O₂ (74%); KCN (75%); ascorbate (92%); BAL (97%);DIECA(90%).Melanogenesis of dl-DOPA followed first order kinetics. The maximum absorption at 305 nm during melanogenesis shows the formation of dopachrome.