Measurement of mitochondrial respiratory chain enzymatic activities in Drosophila melanogaster samples

Summary Mitochondrial respiratory chain (MRC) dysfunction is linked to mitochondrial disease as well as other common conditions such as diabetes, neurodegeneration, cancer, and aging. Thus, the evaluation of MRC enzymatic activities is fundamental for diagnostics and research purposes on experimental models. Here, we provide a verified and reliable protocol for mitochondria isolation from various D. melanogaster samples and subsequent measurement of the activity of MRC complexes I–V plus citrate synthase (CS) through UV-VIS spectrophotometry. For complete details on the use and execution of this protocol, please refer to Brischigliaro et al. (2021).


SUMMARY
Mitochondrial respiratory chain (MRC) dysfunction is linked to mitochondrial disease as well as other common conditions such as diabetes, neurodegeneration, cancer, and aging. Thus, the evaluation of MRC enzymatic activities is fundamental for diagnostics and research purposes on experimental models. Here, we provide a verified and reliable protocol for mitochondria isolation from various D. melanogaster samples and subsequent measurement of the activity of MRC complexes I-V plus citrate synthase (CS) through UV-VIS spectrophotometry. For complete details on the use and execution of this protocol, please refer to Brischigliaro et al. (2021).

BEFORE YOU BEGIN
This protocol describes step-by-step how to measure the enzymatic activity of the MRC complexes in isolated mitochondria obtained from different Drosophila melanogaster tissues. Firstly, mitochondria are separated from homogenized tissues through differential centrifugation using a sucrosebased buffer. Then, mitochondria membranes are disrupted to allow the substrates to access the MRC complexes. Enzymatic activities of individual MRC complexes are measured using specific kinetic assays and UV-VIS spectrophotometry. The MRC activities measured in this way are often normalized to the activity of the citrate synthase, a mitochondrial matrix enzyme of the Krebs cycle that is considered as a good marker of mitochondrial volume. This detailed protocol builds on previously described methods for the isolation of mitochondria from D. melanogaster tissues (Brischigliaro et al., 2019;Brischigliaro et al., 2021) and on the procedures for measuring MRC enzymatic activities in human samples (Bugiani et al., 2004;Kirby et al., 2007;Spinazzi et al., 2012;Trounce et al., 1996). However, extensive optimization was carried out by the authors in order to provide a detailed and step-by-step procedure easy to follow also for newcomers.
Essential material you must have/prepare: 1. Biological samples: adult files or larvae. 2. Refer to key resources table and materials and equipment to prepare reagents and buffers. Note: 50 mM sodium azide (NaN3) can be used as an alternative.

Sample collection and preparation
Coenzyme Q 1 (4 mM): dissolve 2 mg of Coenzyme Q 1 in 2 mL of 100% ethanol. Prepare 100-mL aliquots and store at À20 C for 6 months.
Malonate (1 M): dissolve 208 mg of malonic acid in 2 mL of distilled H 2 O. Store at À20 C for 6 months. Note: 50 mM sodium azide (NaN 3 ) can be used as an alternative.
Antimycin A (1 mg/mL): dissolve 4 mg of antimycin A in 4 mL of 100% ethanol. Store at À20 C for 6 months.
CRITICAL: antimycin is highly toxic, handle it and dissolve under a fume hood using PPE.
Reagents for complex IV (cytochrome c oxidase) assay Note: 50 mM sodium azide (NaN3) can be used as an alternative.
Oxaloacetate (10 mM): dissolve 2.6 mg of oxaloacetic acid in 2 mL of distilled H 2 O. Prepare fresh before the assay.

Reagents for complex V (ATP synthase) assay
Phosphoenolpyruvate (50 mM): dissolve 21 mg of phosphoenolpyruvic acid in 2 mL of distilled H 2 O. Store at À20 C for 6 months. NADH (5 mM): dissolve 3.7 mg of b-Nicotinamide adenine dinucleotide (NADH) in 1 mL of distilled H 2 O. Prepare fresh before the assay. ATP (25 mM pH 7.0): add 7 mL of distilled H 2 O to 138 mg of adenosine triphosphate (ATP), dissolve by adjusting pH to 7.0 with 2 M NaOH. Adjust the volume to 10 mL. Prepare 1 mL-aliquots and store at À80 C for 6 months. Antimycin A (0.2 mg/mL): dissolve 1 mg of antimycin A in 5 mL of 100% ethanol. Store at À20 C for 6 months.
CRITICAL: antimycin is highly toxic, handle it and dissolve under a fume hood using PPE.

Isolation of mitochondria
Timing: 1-2 h Prior to measurement of mitochondrial respiratory chain enzymatic activities, mitochondria are prepared by differential centrifugation as described below.
CRITICAL: samples must always be kept on ice, even during the homogenization step, using ice-cold buffers to minimize protein degradation.
1. Homogenize samples in 2-3 mL of MS buffer with BSA, with 15-20 strokes at 1,000 rpm in a motor driven Teflon-glass Elvehjem potter on ice. Make sure the samples are always cold to avoid protein degradation.

OPEN ACCESS
Note: if a motor driven potter is not available, use a Dounce glass potter with a loose-fitting glass pestle for adults and third instar larvae and a tight-fitting glass pestle for thoraces and small larvae.
CRITICAL: during the homogenization step, apply the same number of strokes to all the samples to obtain consistent results.
2. Transfer the homogenate to a clean 30 mL centrifuge tube, rinse the potter with 7 mL of MS buffer with BSA and transfer to the tube with homogenate. 3. Centrifuge the homogenate at 1,000 3 g for 10 min at 4 C, to separate the liquid phase from debris, i.e., unbroken cells and nuclei.
CRITICAL: centrifuge steps must be performed at 4 C to minimize sample degradation.

Filter supernatant through a 100 mm strainer and transfer it into a new tube.
Note: filtering step is needed to remove legs, wings and cuticle debris that tend to float after homogenization.
5. Centrifuge samples at 6,000 3 g for 10 min at 4 C to pellet the mitochondrial fraction.
CRITICAL: After the centrifugation steps, lipids are present as a white layer adhering to the tube walls. Make sure to remove lipids during the washing steps to avoid interference during measurement of enzymatic activities.
6. Discard supernatant and resuspend the pellet with 10 mL of MS buffer with BSA. Repeat step 5. 7. Remove the supernatant and resuspend the pellet with 10 mL of MS buffer without BSA. 8. Centrifuge samples at 7,000 3 g for 10 min at 4 C. 9. Discard the supernatant and resuspend the pellet in 300-400 mL of MS buffer without BSA. 10. Measure protein concentration with method of choice (e.g., Bradford, Lowry, or BCA).
Note: protein concentration should be measured in triplicate setting up a standard curve, to achieve good consistence and reliability of the results.
Note: this procedure yields crude mitochondrial fractions. Samples can be stored at À80 C for maximum 3 months. Avoid multiple freeze and thaw cycles of pellets to minimize degradation of protein complexes that can result in artifactual reduction of enzyme activities. Isolated mitochondria preparations can be stored in aliquots to avoid multiple freeze and thaw cycles.
Pause point: Proceed with protocol or store samples at À80 C for up to 3 months.

Disruption of mitochondrial membranes
Timing: 0.5 h Prior to the activity assays, mitochondrial membranes need to be disrupted in order to allow access of the substrates to the MRC enzyme complexes.
11. Pellet isolated mitochondria by centrifuging samples at 7,000 3 g for 5 min at 4 C.
Note: depending on the type and the number of assays to be performed, evaluate the total amount of mitochondria needed for the experiment. For further details on the amounts of mitochondria needed for each assay, check the specific protocols below. 12. Carefully discard supernatant and resuspend the pellet with hypotonic buffer (10 mM Tris-HCl pH 7.6) at 1 mg/mL final protein concentration. 13. Snap-freeze in liquid nitrogen. Thaw at 25 C in a benchtop thermo shaker or, alternatively, in a water bath. Perform three freeze-thaw cycles to enhance mitochondrial membrane disruption.
Note: At this point, mitochondria should be immediately used, do not freeze them. Keep samples on ice during the enzymatic activity assays.

Measurement of mitochondrial respiratory chain enzymatic activities
The general concept behind measurement of the mitochondrial respiratory chain activities is to follow each enzymatic reaction through time. The enzyme kinetics is assessed via spectrophotometry by following variations in the UV-VIS absorbance of colorimetric substrates or reaction products.
To date, most of UV-VIS plate readers and standard cuvette spectrophotometer can be set up to register absorbance during a time period.
If a 96-well plate is used, as described in this protocol, we suggest following kinetic activity by registering absorbance values for 2 min, every 20 or 30 s, depending on the number of samples measured and reading speed of the plate reader at the desired wavelength. A plate reader is highly advised to measure multiple samples at the same time and to save reagents, as assays are carried out in 200 mL of reaction volume. If a standard cuvette spectrophotometer is used, adjust the reaction volume as needed.
Note: the measurement of mitochondrial respiratory chain enzymatic activities should be carried out at 30 C.
Note: allow reaction buffers to warm up at 20 C-25 C before the assays.
Note: enzyme activities should be measured at least in triplicate plus at least one replicate reaction in presence of a selective inhibitor to calculate specific activity.

Measurement of complex I (NADH:ubiquinone oxidoreductase) activity
Timing: 0.5-1 h Complex I is a NADH:ubiquinone oxidoreductase. Complex I is a starting point of the mitochondrial respiratory chain, the other being Complex II and other FADH 2 -dependent enzymes. It oxidizes NADH generated by the Krebs cycle in the mitochondrial matrix, passing two electrons through one flavin mononucleotide (FMN) and a series of iron-sulfur (Fe-S) clusters, to the electron carrier CoQ.
The activity of complex I is measured through a direct method, following the decrease in absorbance at l=340 nm due to the oxidation of NADH, carried out by the enzyme (Estornell et al., 1993;Spinazzi et al., 2012).
14. For each replicate plus one (to ensure there is enough for all the samples), prepare the reaction mix as described in the following  resources table), using a multichannel pipette. 16. Add 10 mL of mitochondrial lysate from step 13 (1 mg/mL) and mix well. 17. Record baseline activity at l=340 nm for 2 min at 30 C. 18. Add 4 mL of 4 mM CoQ 1 (final concentration 80 mM) to each well to start the reaction. Make sure the substrate is mixed well in the reaction mix. 19. Promptly record the absorbance at l=340 nm for 2 min at 30 C. 20. *To assess rotenone sensitive NADH-dehydrogenase activity (specific to mitochondrial complex I), perform parallel reactions in presence and absence of rotenone (complex I inhibitor).
Note: expected results are reported in Figure 1A.

Measurement of complex II (succinate:coenzyme Q oxidoreductase) activity
Timing: 0.5-1 h Complex II is a succinate:coenzyme Q oxidoreductase -succinate dehydrogenase -that oxidizes succinate to fumarate during the sixth step of the TCA cycle in the mitochondrial matrix. It couples the oxidation to the reduction of the electron carrier CoQ. The activity of complex II is measured through an indirect method following the decrease in absorbance at l=600 nm due to the reduction of DCPIP. DCPIP is reduced by CoQ 1 from the transfer of electrons from succinate to CoQ through the activity of complex II (Trounce et al., 1996;Spinazzi et al., 2012).
21. For each replicate plus one, prepare the reaction mix as described in the following table: 22. Add 196 mL of the reaction mix into each well of a 96-well plate (see key resources table), using a multichannel pipette. 23. Add 2 mL of mitochondrial lysate from step 13 (1 mg/mL) and mix well. 24. Shake the plate for 5 s. 25. Pre-incubate the plate at 25 C for 10 min. 26. Record baseline activity at l=600 nm for 2 min at 30 C. 27. Add 2 mL of 4 mM CoQ 1 (final concentration 40 mM) to each well to start the reaction. 28. Shake the plate for 5 s, wait for 5 s. 29. Record absorbance at l=600 nm for 2 min at 30 C. 30. *To subtract the contribution of unspecific reactions, perform parallel reactions in presence and absence of malonate (complex II inhibitor).
Note: During complex II activity assay, baseline activity reflects succinate dehydrogenase (SDH) activity, as complex II is able to reduce DCPIP when CoQ 1 is not present, although at slower rate.
Note: Expected results are reported in Figure 1B.

Measurement of complex III (ubiquinol:cytochrome c oxidoreductase) activity
Timing: 0.5-1 h Complex III, also known as cytochrome bc1 complex, is a ubiquinol:cytochrome c oxidoreductase that catalyzes the electron transfer from ubiquinol (CoQ) to the second mobile electron carrier of the MRC, cytochrome c. The activity of complex III is measured through a direct method, following the increase in absorbance at l=550 nm due to the reduction of cytochrome c, carried out by the enzyme (Trounce et al., 1996;Spinazzi et al., 2012).
31. For each replicate plus one, prepare the reaction mix as described in the following  ), using a multichannel pipette. 33. Add 2 mL of mitochondrial lysate from step 13 (1 mg/mL) and mix well. 34. Shake the plate for 5 s, wait for 5 s. 35. Record baseline activity at l=550 nm within 2 min at 30 C. 36. Add 2 mL of 10 mM decylubiquinol (final concentration 100 mM) to each well to start the reaction. 37. Shake the plate for 5 s, wait for 5 s. 38. Record absorbance at l=550 nm for 2 min at 30 C. 39. *To subtract the contribution of possible unspecific reactions, perform parallel reactions in presence and absence of antimycin A (complex III inhibitor).
Note: It is crucial that the cytochrome c used for this assay is the product with Merk catalog number C7752, or similar (prepared with acetic acid without using TCA). Other kinds of cytochrome c isolated using other methods result in very high rates of unspecific cytochrome c reduction, greatly interfering with the assay.
Note: expected results are reported in Figure 1C.

Measurement of complex IV (cytochrome c oxidase) activity
Timing: 0.5-1 h Complex IV, or cytochrome c oxidase, is the terminal enzyme of the ETC. It catalyzes the oxidation of cytochrome c and the reduction of molecular oxygen (O 2 ) to water (H 2 O). The activity of complex IV is measured through a direct method, following the decrease in absorbance at l=550 nm due to the oxidation of reduced cytochrome c, carried out by the enzyme (Wharton and Tzagoloff, 1967;Trounce et al., 1996;Spinazzi et al., 2012).
40. For each replicate plus one, prepare the reaction mix as described in the following table: 41. Add 198 mL of the reaction mix into each well of a 96-well plate (see key resources table), using a multichannel pipette. 42. Shake the plate for 5 s, wait for 5 s. 43. Record baseline activity at l=550 nm within 2 min at 30 C. 44. Add 2 mL of mitochondrial lysate from step 13 (1 mg/mL), which will start the reaction. Mix well. 45. Shake the plate for 5 s, wait for 5 s. 46. Record absorbance at l=550 nm for 2 min at 30 C. 47. *To subtract the possible contribution of unspecific reactions, perform parallel reactions in presence and absence of KCN (complex IV inhibitor).
Note: expected results are reported in Figure 1D.

Measurement of complex V (ATP synthase) activity
Timing: 0.5-1 h Complex V is a F-type ATP synthase. It is a rotary enzyme that couples proton translocation, driven by the proton motive force (PMF) generated by proton translocation at the level of complexes I, III and IV, with ATP synthesis. It is also known as F O F 1 -ATP synthase.
Note: currently, no kinetic spectrophotometry-based methods are available for directly assessing ATP synthesis by ATP synthase. Therefore, the most commonly used methods are based on the evaluation of the reverse activity (ATP hydrolysis) of the enzyme, which have been proven useful to determine Complex V activity in isolated mitochondrial samples. A luminometric assay for measuring mitochondrial ATP synthesis was described before (Vives-Bauza et al., 2007).
This activity is measured through an indirect method by linking its ATPase activity to NADH oxidation. Firstly, in presence of ADP (resulting from hydrolytic activity of ATP synthase), PEP is converted to pyruvate + ATP by pyruvate kinase. Then, pyruvate is converted to lactate by lactate dehydrogenase, through the oxidation of NADH, which is what this assay determines by measuring the decrease in absorbance at l=340 nm.
48. For each replicate plus one, prepare the reaction mix as described in the following table: 49. Add 177 mL of the reaction mix into each well of a 96 well plate (see key resources table), using a multichannel pipette. 50. Add 3 mL of mitochondrial lysate from step 13 (1 mg/mL) and mix well. 51. Pre-incubate at 25 C for 5 min. 52. Record baseline activity at l=340 nm within 2 min at 30 C. 53. Add 20 mL of 25 mM ATP pH 7.0 (final concentration 2.5 mM) to each well to start the reaction. 54. Record absorbance at l=340 nm for 2 min at 30 C. 55. *To subtract the possible contribution of unspecific reactions, perform parallel reactions in presence and absence of oligomycin (complex V inhibitor).
Note: expected results are reported in Figure 1E. Citrate synthase catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate during the first step of the Krebs cycle in the mitochondrial matrix. The activity of citrate synthase is often used as a reliable method of normalization of MRC activity assays. The activity of citrate synthase is measured through an indirect method, following the formation of a yellow-colored thionitrobenzoate (TNB) derivative of DTNB by measuring the changes in absorbance at l=412 nm. DTNB spontaneously forms TNB by reacting with sulfhydryl groups of free coenzyme A resulting from citrate synthase activity (Srere, 1969;Trounce et al., 1996;Spinazzi et al., 2012).

Measurement of citrate synthase activity
56. For each replicate plus one, prepare the reaction mix as described in the following table: 57. Add 188 mL of the reaction mix into each well of a 96 well plate (see key resources table), using a multichannel pipette. 58. Add 2 mL of mitochondrial lysate from step 13 (1 mg/mL) and mix well. 59. Record baseline activity at l=412 nm within 2 min at 30 C. 60. Add 10 mL of 10 mM oxaloacetate (final concentration 500 mM) to each well to start the reaction. 61. Record absorbance at l=412 nm for 2 min at 30 C.
Note: expected results are reported in Figure 1F.

EXPECTED OUTCOMES
This protocol provides detailed methodology for measuring the activity of each individual MRC enzymatic complex and the F O F 1 -ATPase in isolated mitochondria from Drosophila tissues. The data collected with this procedure describe the kinetic enzyme activity of the MRC, which in turn provides quantitative data about the amount and/or functionality of the individual complexes in biological samples.
This protocol records absorbance variations through time (DAbsorbance/minute -DA/min), which are used to calculate the enzymatic activity (Figure 1). The DA/min is calculated by subtracting the absorbance value at the end of the reaction to the absorbance value at the beginning of the reaction. Data collected can be plotted in Microsoft Excel and linear regression should be performed to evaluate R 2 values of the reactions. Generally, R 2 >0.95 is considered a good result (Figure 1). For R 2 <0.95 we suggest repeating the measurement (see troubleshooting section), as this suggests nonlinear reaction and unreliable assessment of enzymatic activity. Low R 2 values for reactions in presence of inhibitors should not be considered problematic, as background variations in absorbance could contribute when low activities are recorded (Figure 1). Specific activity is assessed by subtracting DA/min values recorded in presence of specific inhibitors of MRC complexes to DA/min values recorded in absence of the inhibitor. Generally, good mitochondrial preparations have very low enzymatic activities in presence of the inhibitor (>85%-90% of activity inhibited), (Figure 1).

Enzyme activity calculations
To obtain quantitative enzymatic specific activity (SA) values, expressed as amount of substrate (nmol) converted per time unit (min) per mass unit (mg) of mitochondrial protein (nmol min -1 mg -1 ), use the following formula: À DA min 3 mL of reaction Á À e 3 mL of sample 3 mg mL of sample protein Á The molar extinction coefficients (e) at the specific wavelengths (l) of the different molecules for which the absorbance is measured in each assay can be found in the following table: For example, the calculations for complex I activities reported in Figure 1A are:

LIMITATIONS
This method relies on the availability of enough amounts of tissue. For instance, if small larvae (e.g., 1 st or 2 nd instar larvae) and thoraces are being used, many individuals will be needed. Furthermore, dissection could be time consuming. In case of a complex experimental setup (e.g., many experimental conditions or genotypes) we suggest to prepare and store mitochondrial preparations before proceeding with the enzyme activity assays.

TROUBLESHOOTING
Problem 1 Low or no enzymatic activity observed for different MRC complexes.

Potential solution
Samples may be degraded, make sure you perform all the procedures on ice using ice-cold buffers, make sure samples are store at -80 for long periods of time and avoid multiple freeze and thaw cycles of the samples. Sample amount may be too low, try to increase sample amount until you observe linear reactions significantly higher than the background.
Problem 2 Non-linear reactions or plateau phase observed.

Potential solution
Excessive amount of sample used for the activity assay. Reduce the amount of sample.

Problem 3
Activity observed during baseline recording.

Potential solution
Contamination of buffers or reagents. Discard old reagents and prepare fresh solutions. Problem 4 Low complex I activity.

Potential solution
Oxidation of NADH. Prepare fresh NADH solution. NADH solution should be colorless or light yellow. Yellow color indicates oxidation of NADH.

Potential solution
Contamination with reticular or cytosolic NADH dehydrogenases. Thoroughly wash the mitochondrial pellets twice by resuspending in the appropriate amount of buffer. Make sure that centrifuge speed is correctly set.

Problem 6
Low complex II activity.

Potential solution
Pre-incubation step was skipped. Pre-incubate mitochondria with assay solution at least 10 min before measuring the activity.

Problem 7
Low or no complex III activity.

Potential solution
Carry over of potassium borohydride used for reduction of decylubiquinone. Avoid excess potassium borohydride. Make sure the decylubiquinol solution stops bubbling after addition of HCl. If bubbling does not stop after addition of 2-3 HCl aliquots, repeat centrifugation step and carefully avoid touching the bottom of the tube.

Problem 8
Low or no complex IV activity.

Potential solution
Carry over of sodium dithionite used for reduction of cytochrome c. Avoid excess dithionite that can counteract complex IV activity.

Problem 9
Low or no complex V activity.

Potential solution
Complex V activity assay relies on pyruvate kinase and lactate dehydrogenase activities. Store enzymes at 2 C-8 C.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Carlo Viscomi (carlo.viscomi@unipd.it).

Materials availability
This study did not generate new unique reagents. Data and code availability This study did not generate/analyze datasets/code.