MEASURE OF DEHALOGENASE ACTIVITY 1 Estimation of free chloride ions

An estimation of free chloride ions was used to indicate dehalogenase activity in liquid cultures (Section 2.1.1) and enzyme assay studies (Section 2.3.2).

Free chloride ions were assayed using a Marius Chlor-O-Counter (F.T. Scientific, U.K.). A base counting solution (25 ml), containing in ml double-glass-distilled water: glacial acetic acid, 100: Cone.Nitric Acid, 8.0; NaCl (0.5M), 1.0 and gelatin-thymol blue indicator solution, 1.0 was placed into a small beaker. The indicator solution consisted of, in mg 100 ml ^ double-glass-distilled water: white powder gelatin, 600; thymol, 10.0 and thymol blue pH indicator, 10.0, made up as a 10-fold concentration and stored at 4°C until required then diluted using double-glass-distilled water.

The background chloride, due to the NaCl solution, was titrated and samples (1.0 ml) were added and free chloride was titrated against silver ions, generated coulometrically, resulting in the precipitation of insoluble silver chloride. The titration end-point was detected ampherometrically by silver electrodes measuring the appearance of free silver ions. The time of the titration was directly proportional to the chloride ion concentration and was represented by a time based numerical read-out.

For enzyme studies the base counting solution was changed after each set of 4 readings in order to account for changes in counts due to the state of the counting solution. However, for free chloride ion counts in batch cultures, 12 to 15 samples could be counted before the solution required changing.

2.3.2 Assay procedure for dehalogenase activity

Samples (400 or 800 ml) of late exponential cultures were harvested by centrifugation at 5000g for 15 min at 4°C, and the pellet washed and resuspended in refrigerated (4°C) Tris-sulphate buffer (0.02M), pH 7.9. Cell-free extracts were prepared by disrupting the whole organisms by two passages through a French pressure cell (American Inst. Co. Ltd., Maryland) at 83 MPa (ca 11,500 lbs sq.in ^), and the remaining whole organisms and cell debris removed by centrifugation at 30,000g for 45 min at 4°C. The supernatant was decanted and dithiothreitol added to give a final concentration of l.OmM. Dithiothreitol cleaves

disulphide bonds and protects sulphydryl groups, so stabilising enzymes especially if sulphydryl groups are found in the enzymes' active site.

If cell-free extracts were not to be assayed immediately for dehalogenase activity they were stored at -20°C, at which temperature they remained active for several months. Immediately before assaying the extracts were thawed and stored on ice (4°C).

The assay mixture of 5.5 ml contained, in double-glass-distilled water: Tris-sulphate buffer (100 mM), pH 7.9; NaCl (0.005 mM); chlorinated alkanoic acid (0.1 to 0,2«w.l) and 0.1 to 1.0 ml cell free extract. The chlorinated substrates were added as one of 10% wv ^ stock solutions, pH 7.0: MCA (156 y*); DCA (215 yz); 2MCPA (179 yz) and 22DCPA (236 yz). The buffer was added to overcome the acidic products of the enzyme reaction which in the buffers absence would inhibit further enzymic activity.

The assay mixture, including the cell-free extract, but minus the chlorinated substrate, was equilibrated at 30°C in a water bath for 10 min. The reaction was initiated by adding the substrate. Chloride release

was measured in samples (1 ml; Section 2.3.1) taken at intervals during the first 20 min of reaction.

Once extracts were known to possess dehalogenase activity the NaCl was not included in the assay mixture, the volume being made up with double-glass-distilled water, as the additional counts resulting from the presence of the NaCl only added to the time taken to count the free chloride ions resulting from the dehalogenase activity.

The results were expressed as relative dehalogenase activities which were the ratios of rates of chloride release from DCA, 2MCPA and 22DCPA standardised to the rate of chloride release from MCA, the rates being determined from plots of Chlor-O-Counter counts (1 ml assay mixture)’1 against assay time. A second expression of dehalogenase activity was as enzyme specific activity. This was determined for each substrate by converting the chloride ion counts toywolCl’ released, in accordance with the calibration of the counter - 100 counts on range 10 represents l^mwlCl". The final expression of specific activity also involved determining the protein concentration of the cell free extract and had units of umol substrate converted (mg protein)’1 min’1.

2.3.3 Modifications to the enzyme assay procedure for further characterisation of the enzyme systems

To investigate the effect of the pH of the assay mixture on dehalogenase activity the assay mixture buffer and strength were changed. The buffer was TTA buffer which was chosen because it was effective over the complete range required, pH 6.0 to 11.0. TTA buffer (0.2M) contained in gz’1 double-glass-distilled water: Trizma base, 24.24; N tris (Hydroxymethyl)methyl-2-amincflthane sulphonic acid (Tes), 45.84 and glacial

acetic acid, 19.8. This produced a pH of approximately 45, which was adjusted to the required pH by the addition of concentrated NaOH.

The assay procedure was as described in Section 2.3.2. However no water was added to the assay mixture, the additional volume being made up with additional buffer. The buffer was adjusted to values of pH 6.0 to 11.0 in whole pH units and cell-free extracts were assayed at each pH value against MCA, DCA, 2MCPA and 22DCPA. The stability of each substrate was investigated at the highest and lowest pH values over a time period equivalent to the assay period.

The substrate specificity of the dehalogenation reactions were also studied using the assay procedure previously described (Section 2.3.2). For this the following substrates were prepared as 10% (wv stock solutions and used as described: trichloroacetic acid (TCA); 3hionochloropropionic acid (3MCPA); 2monochlorobutyric acid (2MCBA); 3monochlorobutyric acid (3MCBA); 4monochlorobutyric acid (4MCBA); 2monobromopropionic acid (2MBPA); monobromoacetic acid (MBA) and 2rnonobromobutyric acid (2MBBA).

2.3.4 Determination of protein concentration

The protein concentration of the cell-free extracts was

determined using the Biruet Method of Gornall et a l . (1948), with bovine serum albumin (BSA) as the reference protein. The Biuret assay solution contained, in 500 ml double-glass-distilled water: CuSO^.SHgO, 1.5g and COOK. (CH0H)2C00Na.4H20, 6.0g, to which 300 ml of autoclaved NaOH

(10% wv’1) was added. The NaOH was prepared as a 60% (wv’1) stock _2

solution and autoclaved at 10 lb sq.in for 10 min to remove the carbonates. The removal of the carbonates was necessary because they

interfere with the colorimetric response. The 800 ml solution was made up to 1.0* with double-glass-distilled water and stored in a screw- cap plastic bottle. A plastic bottle was used because the solution is unstable when stored in glass.

A standard curve was prepared using 0.5 to 8.0 mg BSA by adding the necessary volume of a 10 mg ml 1 stock solution to 1 ml of double­ glass-distilled water.

The Biuret solution (4 ml) was added and mixed, then left to stand at room temperature for exactly 30 min, after which the absorbance was measured at 550 nm using a spectrophotometer (SP1700 - Pye-Unicam), in 3.0 ml glass cuvettes with a 1.0 cm light-path. A reagent blank was used as a zero BSA concentration.

The protein concentration of the cell-free extracts was determined after dilution to 0.5 or 0.25 full strength in 1.0 ml double-glass- distilled water and the Biruet solution added and absorbance measured as before. The protein concentrations were then determined by reference to the standard curve.

2.4 ELECTROPHORETIC STUDIES OF THE DEHALOGENASE SYSTEMS