3.2.1 Assays
3.2.1.1 Alcohol Dehydrogenase
Alcohol dehydrogenase (ADH) activity was assayed according to the method of Bergmeyer (1983) by measuring the rate o f change in absorbance at 340 nm due to the following reaction which is catalysed by ADH;
Ethanol + NAD"^ < Acetaldehyde + N A D H +
The reaction mixture consisted o f 600 mM ethanol, 1.8 mM NAD (Sigma Chemical Co. Ltd., Dorset, U.K.), 1.0 mM glutathione (Sigma Chemical Co. Ltd., Dorset, U.K.), 0.62 mM semicarbazide HCl in 50 mM Tris-HCl buffer pH 8 .8 . The semicarbazide was included to prevent the back reaction, while the glutathione acts as a stabilising agent during dilution.
The reaction was started by adding 25 pL o f sample to 1.5 mL o f reaction mixture in a cuvette and mixing by inversion. The rate o f change o f absorbance was monitored for 60 s a t 5 s intervals using a Beckman DU-650 spectrophotometer (Beckman Instruments,
Buckinghamshire, U.K.). A linear fit on the absorbance data was automatically carried out by the instrument to determine the rate o f change, with the result being deemed acceptable if the linear regression coefficient, r^, was above 0.97. The enzyme activity, E, in the sample was calculated as units o f activity, U, per mL using the following equation:
% o At
V,
(equation 3.1) where AA^^/At is the rate of change o f absorbance at 340 nm, Vf is the final volume o f reaction mixture and added sample, is the sample volume, is the dilution factor and S3 4 0 is the absorptivity o f NAD at 340 nm and equals 6.22 cm^ pmol '. lOOmM potassium dihydrogen phosphate buffer pH 6.5 was used to dilute the samples to give changes in absorbance less than 0.5 per minute.
Measurement variability of the assay, expressed as standard deviation from a mean value, had previously been determined by Deghani (1996) as ±5.4% o f measured ADH activity. Repeat measurements carried out over a range o f activities confirmed that the standard deviation was within this value. In practice samples were assayed in triplicate to give the mean ADH activity.
3.2.1.2 Total Protein
The concentration of protein was determined using Coomassie Blue G-250 reagent (Bio- Rad Laboratories Ltd., Hertfordshire, U.K.). The method is based on the work o f Bradford (1976) and involves binding o f protein to the reagent which suppresses protonation and results in a colour change from pale orange-red to blue.
Samples to be assayed were diluted to within the range o f 200-1000 pg.mL ' protein using 100 mM potassium dihydrogen phosphate buffer pH 6.5. Care was taken to ensure ammonium sulphate levels were below 1.0 M in all cases to prevent interference with the assay. 100 pL o f sample and 2.9 mL o f assay reagent were mixed in a cuvette and the change in absorbance at 595 nm was recorded after 300 s using a Beckman DU-650 spectrophotometer (Beckman Instruments, Buckinghamshire, U.K.). A standard curve
produced using bovine serum albumin (Sigma Chemical Co. Ltd., Dorset, U.K.) enabled the protein concentration to be calculated.
Measurement variability o f the assay, expressed as standard deviation from a mean value, had previously been determined by Deghani (1996) as ±3.6% o f measured protein concentration. Repeat measurements over a range o f concentrations confirmed that the standard deviation was within this value. In practice samples were assayed in triplicate to give the mean protein concentration.
3.2.1.3 Glucose
The concentration o f glucose was determined using Sigma Glucose HK reagent (Sigma Chemical Co. Ltd, Dorset, U.K.). The assay gives quantitative, enzymatic determination o f glucose concentration at 340 nm and is based on the following reactions:
Glucose + ATP G - 6 - P + ADP G - e - P + NAD^ >6 - P G + N A D H
Glucose is first phosphorylated by adenosine triphosphate (ATP) in a reaction catalysed by hexokinase (HK). The glucose-6 -phosphate (G-6 -P) formed is then oxidised to 6 - phosphogluconate (6 -PG) in the presence o f NAD. This reaction is catalysed by glucose- 6 -phosphate dehydrogenase (G-6 -PDH).
10 pL o f sample and 1.5 mL o f assay reagent were mixed in a cuvette and the increase in absorbance at 340 nm due to the reduction o f NAD to NADH was measured after 5 minutes using a Beckman DU-650 spectrophotometer (Beckman Instruments, Buckinghamshire, U.K.). The glucose concentration, Cg,u, was then calculated in mg.L'' from the following equation:
^ ^ 1 ^ A 3 4 0 V f MWg|„
~
7^ '
/ V, 1000
(equation 3.2) where AA3 4 0 is the change in absorbance at 340 nm, MWg|„ is the molecular weight o f glucose and equals 180.16 g.mol'% and / is the lightpath and equals 1 cm.
Measurement variability o f the assay, expressed as standard deviation from a mean value, has previously been determined by Deghani (1996) as ±0.3% o f measured glucose concentration up to a maximum o f ±0.053 g/L. Repeat measurements over a range of concentrations confirmed that the standard deviation was within this value. In practice samples were assayed in triplicate to give the mean glucose concentration.
3.2.1.4 Ethanol
The concentration o f ethanol was determined using Boehringer Mannheim UV method kits (Boehringer Mannheim Ltd, East Sussex, U.K.). The method is based on the following reactions:
Ethanol + NAD ^ ■■> Acetaldehyde + N AD H + H* Acetaldehyde + NAD ^ + H^O— > Acetic acid + N AD H + H^
Ethanol is first oxidised to acetaldehyde in the presence o f the enzyme ADH. The equilibrium o f this reaction lies on the side o f ethanol and NAD. However, it can be completely displaced to the right at alkaline conditions and by trapping o f the acetaldehyde formed. To achieve this the acetaldehyde is oxidised to acetic acid in the presence o f aldehyde dehydrogenase (Al-DH). The change in absorbance at 340 nm due to the reduction o f NAD to NADH in this reaction alone. A,, was measured first and then the change in absorbance at 340 nm due to the reduction o f NAD to NADH from both reactions acting at the same time, Aj, was measured. The absorbance difference was calculated as A? minus A,. The absorbance difference for a blank was then subtracted from that o f the sample, giving the final absorbance difference AA.
100 |iL o f sample or deionised water was mixed with 3 mL o f assay reagent containing Al-DH in a cuvette and the change in absorbance at 340 nm after 180 s was measured using a Beckman DU-650 spectrophotometer (Beckman Instruments, Buckinghamshire, U.K.) to give A,. 50 pL o f assay reagent containing ADH was then added, the cuvette contents mixed by inversion and the change in absorbance at 340 nm measured after 300 s using a Beckman DU-650 spectrophotometer (Beckman Instruments, Buckinghamshire,
U.K.) to give Aj. AA was then determined and the concentration o f ethanol, in g.L‘ calculated using the following equation:
1 AA X MWeth