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Kinetic data for the hydrolysis of potassium pacetylphenyl sulphate by fresh rat liver suspensions suggested that the arylsulphatase activity so observed could be attributed to a single enzyme (Dodgson, Spencer & Thomas, 1953) which was later found to be localized mainly in the microsomes of the liver cell (Dodgson, Spencer & Thomas, 1954a). Preliminary attempts to purify the microsome enzyme (unpublished data) were handicapped by its insolubility in various buffers and salt solutions of widely differing pH even after treatment of the liver by acetone-drying, alternate freezing and thawing, or use of the tissue disintegrator (Mickle, 1948). Sulphate and phosphate ions had little effect on the enzyme. These results contrast sharply with the arylsulphatase pattern reported for ox liver (Roy, 1953a, b; 1954). Using dipotassium 2-hydroxy-5nitrophenyl sulphate as the assay substrate, Roy was able to separate two arylsulphatases from the soluble material of acetone-dried ox liver and both enzymes were strongly inhibited by sulphate and phosphate ions. With the same substrate the arylsulphatase activity of mouse liver was found to be mainly concentrated in the mitochondria (Roy, 1953a). The present work, a preliminary report of which has already appeared (Dodgson, Spencer & Thomas, 1954 b), shows how the occurrence of three different arylsulphatases in mammalian livers enables these contrasting findings to be reconciled. METHODS Arylsulphates. Potassium p-acetylphenyl sulphate (APS) and potassium p-nitrophenyl sulphate (NPS) were prepared by the method of Burkhardt & Lapworth (1926) and dipotassium 2-hydroxy-5-nitrophenyl sulphate (nitrocatechol sulphate, NCS) according to Roy (1953a). Measurement of arylnulphata8e activity. The method previously used for APS (Dodgson, Lewis & Spencer, 1953) was also adopted for NPS but using the Am.. and e,,. determined for p-nitrophenol (Dodgson & Spencer, 1953). The use of NPS was restricted to liver extracts, as recoveries of p-nitrophenol from fresh liver suspensions are low (Dodgson & Spencer, 1953). The faint turbidity of the final solutions was the same in both test and controls and duplicate determinations were satisfactory. Turbidity only inter

The present study describes the biotransformation of 2,4,6-trinitrotoluene (TNT) (220 μM) by using anaerobic sludge (10%, vol/vol) supplemented with molasses (3.3 g/liter). Despite the disappearance of TNT in less than 15 h, roughly 0.1% of TNT was attributed to mineralization (14CO2). A combination of solid-phase microextraction–gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry identified two distinctive cycles in the degradation of TNT. One cycle was responsible for the stepwise reduction of TNT to eventually produce triaminotoluene (TAT) in relatively high yield (160 μM). The other cycle involved TAT and was responsible for the production of azo derivatives, e.g., 2,2′,4,4′-tetraamino-6,6′-azotoluene (2,2′,4,4′-TA-6,6′-azoT) and 2,2′,6,6′-tetraamino-4,4′-azotoluene (2,2′,6,6′-TA-4,4′-azoT) at pH 7.2. These azo compounds were also detected when TAT was treated with the anaerobic sludge but not with an autoclaved sludge, suggesting the biotic nature of their formation. When the anaerobic conditions in the TAT-containing culture medium were removed by aeration and/or acidification (pH 3), the corresponding phenolic compounds, e.g., hydroxy-diaminotoluenes and dihydroxy-aminotoluenes, were observed at room temperature. Trihydroxytoluene was detected only after heating TAT in water at 100°C. When 13CH3-labeled TNT was used as the N source in the above microcosms, we were unable to detect13C-labeled p-cresol or [13CH3]toluene, indicating the absence of denitration or deamination in the biodegradation process. The formation and disappearance of TAT were not accompanied by mineralization, suggesting that TAT acted as a dead-end metabolite.

I956 the mixing chamber before the reagents are emptied into the reaction vessel. The accuracy of mixing exceeds the limit of resolution of the spectrophotometer. 3. The reaction vessel consists of a cylindrical quartz tube held in position with unstretched nylon bushings in a brass block adjacent to the cuvette housing unit. The flow of material is controlled by a needle valve at the exit of the reaction-vessel housing unit. Approximately 6 ml. of fluid is required to flush out the reaction vessel. 4. Temperature control is maintained with a water jacket round the four syringes and with cooling channels within the reaction-vessel housing unit. Steady-state temperature is reached in the syringes in a maximum of 6 min. 5. The signal from the density-scale slide-wire potentiometer of the spectrophotometer is fed through a potential divider into a Brown stripchart recorder. As the operator tracks the changes in optical density of the reaction mixture the percentage transmission is recorded continuously. By means of a mixing switch the temperature of the reaction vessel measured with a thermopile is recorded at intervals during the reaction time. 6. The percentage error (standard deviation) of the velocity constant of decomposition of hydrogen peroxide by bacterial catalase measured with this apparatus is less than ± 2 %.

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