Selection of the right detergent

For the three enzymes to be measured in this PhD study, all three standard enzyme assays either used no detergent (for COXand BHAD) [11, 13] or Triton X100 detergent for CS assay. As mentioned previously, there was limited SPIRIT study muscle tissue for analysis of enzyme activity so the aim for this study was to have one protein homogenate that could be used to assay all three enzymes of interest. This meant that to achieve maximal activity, one detergent that did not interfere with enzyme activity had to be found. Six detergents were investigated (Figure 5.4). The results for CHAP and SDS are not shown as COX activity was not detectable, and Tween-80 was very low activity so these detergents were excluded from further experiments when these detergents were used. The other four detergents, Triton X100, Tween-20, and Brij-35 were examined. For this experiment, 5 l of 1% detergent (Triton X100 or Tween-20 or Brij-35) was added to the extraction buffer (section 5.3.1.1), protein was extracted from 20 mg of rat muscle tissue using the optimised procedures discussed in sections 5.3.1.1 and 5.3.1.2, and COX enzyme activity was assayed. In this experiment the 1st pellet from the protein homogenate (1st supernatant) was resuspended in extraction buffer with appropriate detergent and centrifuged again (Figure 5.4). The 2nd supernatant and 2nd pellet along with the 1st supernatant were measured for protein concentration and COX activity (Table 5.3). This experiment was repeated three times. Figure 5.5(a) represents the results for one experiment and shows the activity for the 1st supernatant.

As seen in Table 5.3, Brij-35 and Tween-20 were the detergents that provided the best results having maximum COX activity (0.35 units/ml and 0.31 units/ml respectively for the first supernatant). Extraction buffer containing Brij-35 exhibited greatest concentration of protein. Comparing the results in Table 5.2, where no detergent has been used in the extraction buffer, to those in Table 5.3, where detergent has been used in extraction buffer, it can be seen that extraction of protein from rat skeletal muscle is enhanced in the presence of detergent. It was clear that for all detergents used, the first pellet (Figure 5.4) needs to be processed again for extraction of further protein (Table 5.3). It was extracted by adding 100 l of extraction buffer and after homogenisation and centrifugation the second extract was obtained. The activity assay was performed on the second supernatant. Activity of 0.21units/ml was detected in the 2nd Brij-35 appeared to be the best detergent in obtaining maximal COX activity in rat skeletal muscle tissue

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Figure 5.4: Flow diagram showing optimisation step by step process for the selection of

most suitable detergent for all three assays. Thesecond pellet was resuspended in extraction

buffer containing 1% detergent and syringed 20 times through 25 gauge needle and then spun at 600 g for 15 minutes.

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Table 5.3: Effect of different detergents on protein concentration and COX activity Detergent Used Muscle Protein

Homogenate Protein Concentration (mg/ml) COX activity (units/ml) Brij-35 1st supernatant 2.52 ± 0.31 0.35 ± 0.02 2nd supernatant 0.81 ± 0.02 0.21 ± 0.02 2nd pellet 0.32 ± 0.00 0.01± 0.01 Tween-20 1st supernatant 2.04 ± 0.34 0.31 ± 0.29 2nd supernatant 0.88 ± 0.02 0.04 ± 0.02 2nd pellet 0.044± 0.002 0.036 ± 0.002 Triton X100 1st supernatant 1.86 ± 0.29 0.16 ± 0.12 2nd supernatant 0.25 ± 0.02 0.076 ± 0.02 2nd pellet 0.21 ± 0.02 0.036 ± 0.002

Data expressed as mean ± SD (n = 3). Protein concentration was determined using the standard BCA assay. COX activity at 25 0C was determined spectrophotometrically [11], with absorbance measured every 30 seconds at 550 nm for 10 min. COX activity is expressed as units/ml which represents mol/min/ml/mg protein.

Although rat muscle protein homogenate using Brij-35 was showing best results for COX activity, it was important to establish that Brij-35 did not interfere with the other mtiochondrial enzyme assays of CS and BHAD. The rat muscle protein supernatants prepared for the previous experiment (results shown in Figure 5.5(a) and Table 5.3) were used for determining both CS activity (Figure 5.5(b)) and BHAD activity (Figure 5.8(c)). For each enzyme, the experiment was repeated three times and the assay results for one experiment are shown in Figure 5.5. The mean activities for the three enzymes using the different detergents for extraction of the protein from rat muscle are shown in Table 5.4.

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Table 5.4: The effect of different detergents on the activities of three mitochondrial enzymes in rat skeletal muscle

Detergent Total COX activity (units/ml) Total BHAD activity (units/ml) Total CS activity (units/ml) Brij-35 0.52 0.02 0.015 0.010 0.14 0.01 Tween-20 0.35 0.16 0.009 0.020 0.18 0.01 Triton X100 0.24 0.07 no activity 0.17 0.08

Data expressed as mean ± SD (n = 3); COX represents cytochrome c oxidase; CS represents citrate synthase and BHAD represents beta-hydroxyacyl-CoA dehydrogenase. Enzyme activity has been determined from combined supernatant (The combined supernatant was formed by combining the 1st and 2nd supernatant as shown in Figure 5.4 and is expressed as units/ml which represents mole/min/ml/mg of protein).

As can be seen in Figure 5.5 and Table 5.4 both COX activity and CS activity was detected using all three detergents in the extraction buffer. BHAD activity was only detected in the presence of Brij-35 and Tween-20 indicating that Triton X100 interfered with the BHAD assay. Muscle homogenate with Brij-35 detergent showed greatest BHAD activity results (0.015 0.010) as compared to Tween-20.

For CS activity, Triton X100 appeared to show greater variation between the three experiments. Tween-20 seemed to lead to greater CS activity however the activity when using Brij-35 was still in the same magnitude of activity observed for Tween-20. For BHAD activity Tween-20 in the extraction buffer seemed to lead to a 60% drop in activity compared Brij-35 in the extraction buffer. Due to this, Brij-35 was selected as the best detergent to use in the extraction buffer as all three enzyme activities were able to be measured with good enzyme activity being exhibited.

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Figure 5.5: Determination of most suitable detergent for all three enzyme assays. Since

three different detergents were used, three blanks were prepared for each detergent i.e. the blank was extraction buffer containing 1% of detergent. (a) COX activity [11] was measured at 25 oC every 30 seconds for 10 min with absorbance measured at 550 nm (b) CS activity [12] was measured at 25 oC every 30 seconds for 5 min with absorbance measured at 412 nm. (c) BHAD activity [13] was measured at 25 oC every 30 seconds for 5 min with absorbancemeasured at 340 nm.

(c) (a)

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The last experiment in optimisation of the protein extraction method was to examine how much Brij-35 should be used in the extraction buffer. The different concentrations of Brij-35 used ranged from 1% - 2.5%. The experiment was repeated three times and Figure 5.6 represents the results for one experiment. Table 5.5 presents the protein concentration and COX activity results for this experiment.

Figure 5.6: Determination of suitable concentration of Brij-35 in extraction buffer. COX

activity at 25 0C was determined spectrophotometrically [11], with absorbance measured every 30 seconds at 550 nm for 10 min. Blank was extraction buffer containing 1% of Brij-35.

Table 5.5: Protein concentration and COX activity using different concentrations of Brij-35 in the extraction buffer

Percentage Brij-35 Protein Concentration (mg/ml) COX activity (units/ml) 0% 2.17 0.001 0.037 0.001 1% 2.84 0.001 0.31 0.001 1.5% 2.82 0.002 0.19 0.002 2 % 2.80 0.001 0.17 0.001 2.5% 2.81 0.001 0.019 0.001

Data expressed as mean ± SD (n = 3). Protein concentration was determined using the standard BCA assay. COX activity at 25 0C was determined spectrophotometrically [11], with absorbance measured every 30 seconds at 550 nm for 10 min.COX activity is expressed as units/ml which represents mole/min/ml/mg of protein.

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 200 400 600 800 absor b ance -units 550nm time-seconds a=blank a1 = without Brij-35 a2 = 1% Brij-35 a3 =1.5% Brij-35 a4=2 % Brij-35 a5 = 2.5%Brij-36

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The results in Table 5.5 show that 1% Brij-35 is a suitable concentration of the detergent to use for extraction of protein from the muscle and measurement of optimal COX activity.