Enzyme Analyses

All assays were performed on either Hitachi U3210 or Kontron 940 dual-beam spectrophotometers at 30°C in a final volume of 1ml. Each enzyme was assayed in triplicate and values accepted if they were within 15% of each other. All chemicals were from Sigma Chemical Company and Boehringer Mannheim.

2.5.1 Citrate Synthase (CS)

e s is a mitochondrial matrix enzyme which was used as an indicator of mitochondrial mass in preparations as it has not been found to be altered in disease states (Cooper JM Personal Communication). Respiratory chain activities were expressed as CS ratios to correct for variation in purity of mitochondrial preparations or to correct for the mitochondrial mass in tissue homogenates. The assay is based on the method of Coore et al (Coore 1971). The enzyme catalyses the condensation of acetyl-CoA and oxaloacetate to form citrate, producing CoA whose free thiol group combines with 5-5’-dithiobis- nitrobenzoic acid (DTNB), resulting in an increase in absorbance at 412nm. Two cuvettes were set up containing: lOOmM Tris-HCI buffer pH8.0, 200pM Acetyl-CoA, 200pM DTNB, 0.1% (v/v) Triton-X-100 and sample in a final volume of 1ml. The reaction was initiated by the addition of lOOpM oxaloacetate and the increase in absorbance at 412nm measured. Citrate synthase activity was calculated using the molar extinction coefficient of 13.6 x 10^ for the DTNB-CoA- SH complex and activity expressed as nmol/min/mg protein.

2.5.2 NADH-CoQiOxidoreductase (complex I activity)

The assay is based on the method of Ragan (1987) and measures the rotenone sensitive CoQ, dependant oxidation of NADH at 340nm. The CoQI was a gift from the Eisai Chemical Co, Japan. A dilution of the stock CoQ^ was made in ethanol and the absorbance of the CoQi at 275nm noted. An excess of sodium borohydride was added to the reference cuvette to completely reduce the quinone to quinol and the absorbance change used to calculate the CoQ^ concentration using a molar extinction coefficient of 2.25x10^ (Redfearn 1967). Two identical cuvettes were set up containing 20mM potassium phosphate buffer pH 7.2 with 8mM IVIgClg(see Appendix 1), 150pm NADH, ImM KCN , 2.5mg/ml BSA and sample. The reaction was initiated in the test cuvette by the addition of 50pM CoQi. The rate of NADH oxidation was monitored by the change in absorbance at 340nm. After 10 minutes lOpM rotenone was added to the test cuvette and the rotenone insensitive rate measured. Complex I activity was defined as the rate sensitive to rotenone. Calculation of activity used a molar extinction coefficient of 6.81x10^ for NADH to allow for the contribution of reduced CoQi to the absorbance at 340nm. Enzyme activity was expressed as nmol/min/mg and also as a ratio with citrate synthase (CS).

2.5.3 Succinate cytochrome c oxidoreductase (complex 11/111)

This assay determines the activity of both complex II and III and is based on the method of King (King 1967). It detects the antimycin A sensitive, succinate dependant reduction of cytochrome c at 550nm.

Two identical cuvettes were set up containing: 0.1M potassium phosphate buffer pH 7.4, O.SmM potassium EDTA (Kg-EDTA), O.ImM cytochrome c. Two separate eppendorfs were set up containing ImM KCN, 20mM succinate and sample, these were incubated at 30°C for 5 mins to fully activate the enzyme. This was then added to the cuvettes to initiate the reaction. The change in absorbance was monitored at 550nm. After 10 minutes 20}iM antimycin A was added. The complex I I/I 11 activity was calculated as the rate which was sensitive to antimycin A using the molar extinction coefficient of cytochrome c (e=19.2x10^). Activity was expressed as nmol/min/mg and as a CS ratio.

2.5.4 Succinate-ubiquinol oxidoreductase (complex II)

This assay is based on the method of Hatefi et al (1976).The activity measures the reduction of the dye 6,6-dichlorophenolindophenol (DCPIP) at SOOnm in the presence of succinate and ubiquinone-2 (C0Q2). Enzyme activity was taken as the rate inhibited by 2-thenoyltrifluoroacetone (TTFA).

Two identical cuvettes were set up containing: 50mM potassium phosphate buffer pH7.4, O.ImM Kg-EDTA, 20mM sodium succinate, 74pM DCPIP, ImM KCN, lOpM rotenone and sample. 50pM of ubiquinol-2 was added to initiate the reaction, after 10 minutes ImM TTFA was added to inhibit the reaction. Activity was calculated using the molar extinction coefficient of DCPIP (s=2.1x10^) and expressed as nmol/min/mg and as a CS ratio.

2.5.5 Complex III (Ubiquinol-cytochrome c reductase)

This assay is based on the method of Birch-Machin et al (Birch-Machin 1989). This enzyme catalyses the oxidation of ubiquinol and reduction of cytochrome c which is measured at 550nm. The reaction rate is dependant upon the concentrations of both ubiquinone-2 and cytochrome c, and the concentrations of both are therefore determined pre-assay.

Cytochrome c concentration was determined by addinglSpM of cytochrome c to ddHgO (final volume 1ml) in two identical cuvettes, a few granules of ascorbate was added to the reference cuvette to reduce the cytochrome c. The absorbance change was noted and the concentration of cytochrome c calculated (e =19.2).

Ubiquinol-2 was prepared by taking of ubiquinone-2 (10mM) in ethanol acidified to pH 2 with MCI. The volume was made up to 1ml with ddHgO and a few granules of sodium borohydride added to reduced the quinone to quinol. The quinol was extracted into 3ml of diethylether:cyclohexane (2:1 v/v) and the upper phase collected. 1ml of 2M NaCI was added, and the upper diethylether phase collected which was then evaporated to dryness under a stream of nitrogen gas. The final residue was dissolved in 1ml of ethanol, acidified to pH2 with HOI and aliquoted and stored at -20°C under nitrogen gas to prevent oxidation.

To determine the concentration of ubiquinol-2, lOpI of 5M KOH was added to 10 pi of ubiquinol-2 and 980pl of ethanol. The increase in absorbance, due to oxidation of ubiquinol-2 to ubiquionone-2, was used to calculate the concentration of the ubiquinol-2 using 8=12.25.

In this assay there is a non-enzymatic reduction of cytochrome c which is dependant upon the concentrations of ubiquinone-2 and cytochrome c. This rate was determined prior to assaying the samples and was subtracted from the observed sample rate. Identical cuvettes were set up containing all assay solutions as below except ubiquinol and sample. The non-enzymatic rate was determined by the addition of ubiquinol-2 into the test cuvette and the rate of absorbance change at 550nm noted. The non-enzymatic activity was calculated as below.

To analyse complex III activity the reaction contained 35mM potassium phosphate buffer pH7.2, ImM Kg-EDTA, 5mM MgClg, 2mM KCN, 5pM rotenone and 15pM cytochrome c. Ubiquinol-2 (15pM) was added to the test cuvette to initiate the reaction which was recorded at 550nm for 5 minutes.

Calculation of the pseudo first-order rate constant (k)

This was performed by extrapolation of the absorbance back to time=0 and determination of the change in absorbance at various time points up to 2mins. The non-enzymatic rate of absorbance was also calculated thus and subtracted from these sample absorbance values. K/min/ml was calculated by:

{In0.288-ln(0.288-change in absorbance at time t) x 1000/sample vol.(pi) x dilution factor}, where 0.288 represents the absorbance of cytochrome c fully reduced.

The k/ml values for 5 time points (0, 0.5, 1.0, 1.5 and 2 mins) were plotted against time and the gradient of the line calculated using linear regression analysis (k/min/ml). The final results were expressed as k/min/mg protein and as a CS ratio.

2.5.6 Cytochrome c oxidase (complex IV)

This assay is based on the method of Wharton and colleagues (Wharton et al 1967) which monitors the oxidation of reduced cytochrome c at 550nm.

Preparation of reduced cytochrome c

100ml of 1% solution of cytochrome c (horse heart) was made up in lOmM potassium phosphate buffer. This was reduced by adding an excess of ascorbate (13mg), mixed and checked that it was fully reduced at 550nm. This was done by adding 50pl of the cytochrome c solution to each of two cuvettes containing 950pl lOmM potassium phosphate buffer. To the sample cuvette was added lOpI of freshly made saturated ascorbate solution and the change in absorbance noted. If the change in absorbance was positive (ie. the cytochrome c could be further reduced by the addition of ascorbate) then the cytochrome c solution needed reducing further. After this check the reduced cytochrome c solution was dialysed to remove the ascorbate. This was achieved using size 1 dialysis tubing (Medicell International Ltd..London) and dialysing against 5L of lOmM potassium phosphate buffer pH7.0 at 4°C overnight. To check that no excess ascorbate remained , oxidised cytochrome c was added to the dialysed reduced sample. There should be no change in absorbance showing the ascorbate had been removed.

Calculation of concentration of reduced cytochrome c:

Two identical cuvettes were made up with lOOpI of lOOmM potassium phosphate buffer pH7, 850pl of ddHgO and 50pl of reduced cytochrome c. To the reference cuvette was added lOpI of 0.1M Ferricyanide to oxidise the reduced cytochrome c. The change in absorbance was noted and the concentration calculated by the following (where 50pM cyt c produces an absorbance of 0.96): 0.96/Abs X 50 = volume of stock cyt c required for 50pM solution.

Assay:

Two identical cuvettes were set up containing lOmM potassium phosphate buffer pH7 and 50pM reduced cytochrome c. lOpI of lOOmM Ferricyanide was added to the reference cuvette to oxidise the cytochrome c. The initial absorbance was noted (0.96 = 50pM cytochrome c) and the reaction initiated by the addition of sample to the test cuvette and monitored at 550nm. The pseudo first order rate constant k was calculated as described above for complex III . Complex IV activity (k/min/ml) was expressed as k/min/mg protein and as a CS ratio.

2.5.7 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

This was assayed according to the method of Heinz and Freimuller (1982). The assay measures the rate of oxidation of NADH as 3-phosphoglyceric acid (3- PGA) forms glyceraldehyde-3-phosphate, and is measured at 340nm. The reaction contained (final concentration in 1ml) 83mM triethanolamine, 3mM L- Cysteine, 2mM magnesium sulphate, O.ImM NADH, I.Im M ATP, 0.9mM Kg- EDTA, 5 units of 3-phosphoglyceric phosphokinase, and sample (from 10-50pl). The reaction was initiated by the addition of 6.7mM 3-phosphoglyceric acid (3- PGA) to the sample cuvette and the change in absorbance measured at 340nm. GAPDH activity was calculated using the molar extinction coefficient for NADH (8=6.22 X 10^) and expressed as nmol/min/mg protein.

2.5.8 Aconitase

This assay measures the activity of aconitase which catalysed the isomérisation of citrate to isocitrate which forms a-ketoglutarate with the reduction of NADP which the reaction measures at 340nm.

Two identical cuvettes were set up containing: 50mM Tris-HCI pH7.4, 0.4mM NADP, 5mM sodium citrate, O.SmM MgClg, 1% (v/v) Triton X-100 and 2 units of isocitrate dehydrogenase. Sample was added to the test cuvette only. Both cuvettes were incubated at 30°C for 30 minutes pre-incubation, the absorbance change at 340nm measured for 15 minutes was monitored and aconitase activity calculated using the molar extinction coefficient for NADP (8=6.22 x 10^).