TITLE: Mitochondrial Enzyme Activities Protocol
AUTHORS: Jack Devine

[DESCRIPTION]
Mitochondrial enzymatic activity assays developed in the Picard lab at Departments of Psychiatry and Neurology, Robert N Butler Columbia Aging Center, Columbia University Irving Medical Center, New York, NY, USA

[STEPS]
SECTION: Homogenization buffer
1.  
 
 
SECTION: Homogenization procedure for Mouse Tissue:
2. ·      Weigh tissue samples by transferring into pre-weighed 1.5ml Eppendorf tube 
·      Subtract empty tube weight from weight of tube containing sample to get sample weight
·      Add 180µl of homogenization buffer per mg of tissue (1mg:180µl, weight:volume)
·      Add 2 Tungsten beads (Qiagen cat# 69997) / tube
SECTION: Homogenization procedure for Mouse Tissue:
3. ·       Move tubes to pre-chilled Tissue Lyser (Qiagen cat # 85300) rack and run at 30 cycles/sec for 1 min
·       Put rack back on ice and set a timer for 5 min
·       Repeat homogenization in Tissue Lyser for 1min
·       Quick spin in pre-cooled microcentrifuge to remove liquid from cap of tubes
·       Place samples in -80 oC freezer until frozen
·       Once frozen, thaw samples in room temperature water bath. Move racks around slowly in water bath to ensure circulation of water 
·       Repeat freeze-thaw cycle 2X. Final thaw of samples is performed on the day assays are run
·       Vortex samples for 2 seconds to ensure homogeneity, transfer samples to 96-well block
SECTION: CITRATE SYNTHASE (CS) ENZYMATIC ACTIVITY ASSAY
4. Assay Principle:
Citrate synthase (CS) enzymatic activity is determined by the change in absorbance of DTNB (Ellman’s reagent) measured at 412 nm. Thiol groups (R-SH) reach with DTNB, cleaving the disulfide bond to generate 2 NTB- molecules, which ionizes to an NTB2- dianion in water at neutral and alkaline pH. This NTB2- ion has a yellow color and absorbs light at 412 nm. The reaction requires oxaloacetate to
proceed and generate thiol groups. The enzymatic activity of CS is determined by quantifying the rate of conversion of DTNB into the yellow NTB2-. The molar extinction coefficient of DTNB is 13.6 L x mol-1 x cm-1.

 



 
 
 
SECTION: CITRATE SYNTHASE (CS) ENZYMATIC ACTIVITY ASSAY
5. Buffers and Reagents
Buffer Tris 200 mM
 
Adjust pH at 7.4 @ room temperature with 10M HCl (~ 5ml), store at 4oC.
Aliquot into 40 ml. Store at -30
 
Acetyl CoA 10 mM
 
Make aliquots and store at -80oC indefinitely. (6.2ml per
50mg bottle). Aliquot into 1.4 ml.
 
DTNB (Ellman’s reagent) 10
mM
 
Make aliquots and store at -80oC indefinitely. Aliquot into 1.4ml.
 
Oxaloacetic acid 2 mM
 
Adjust pH to ~7.3 to 7.4 with KOH – this is difficult, be gentle. Make
aliquots and store at -80oC. Aliquot into 1.6 ml.
 
Triton X-100 10% 
 
 
 
 
Preparation of assay buffer:
 
 
 
SECTION: CITRATE SYNTHASE (CS) ENZYMATIC ACTIVITY ASSAY
6. Spectrophotometer settings:
  Assays are recorded on a Spectramax M2 (Spectramax Pro 6, Molecular Devices)
  The assay is run at 30 oC
  Read absorbance at 412 nm
  Set spectrophotometer to read only half of the plate at a time (to maximize reading time)
SECTION: CITRATE SYNTHASE (CS) ENZYMATIC ACTIVITY ASSAY
7. Running the assay:
·       In a 96-well plate, pipet 10 ml of Oxaloacetate ([final] = 100mM).
·       Pipette 10ml of sample homogenate.
·       Add 200ml of CS assay mix to each well.
·       Quickly put plate in spectrophotometer and read.
·       Record slopes for each sample
·       The negative control is the assay mix + samples without oxaloacetate; the slope should be ~0.
·       Obtain a measure of each sample in duplicates
The enzymatic activity is determined with the molar extinction coefficient of DTNB = 13.6 mM-1
SECTION: COMPLEX II (SUCCINATE-UBIQUINONE OXIDOREDUCTASE) ENZYMATIC ACTIVITY ASSAY
8. Principle of assay:
Succinate dehydrogenase (SDH, also succinate-ubiquinone reductase or Complex II of the electron transport system – EC 1.3.99.1) activity is determined by the change in absorbance of DCIP measured at 600nm. Oxidation of succinate and decylubiquinone by complex II reduces DCIP, which decreases its absorbance at 600nm. Rotenone and KCN are used to inhibit electron flow through complexes I and IV, respectively.
Addition of malonate (an inhibitor of SDH) at the end of the assay allows to measure DCIP reduction independently from SDH. SDH enzymatic activity is then computed from the difference between the two slopes. The molar extinction coefficient of DCIP is 16.3 L x mol-1 x cm-1.

Buffers and Reagents
Buffer KPi-EDTA (KPi same as for COX assay + EDTA)
 
 
 
 
 Take 2.5 L of K2HPO4 and acidify by adding KH2PO4 (approx 50-100ml) until pH 7.8 to give ➊
 Take 2.5 L of ➊ and add 1.461 g EDTA (FW: 292.24) for a [final] of 2 mM, pH will go to 7.4
 Heat for 10 min on hotplate set to 200°C to dissolve EDTA, buffer temperature was at 40°C
 Aliquot into 125 ml.  Store at -30

2,6-dichloroindophenol –
DCIP (20mM)
 
Aliquot into 420ul, Store at -30
Sodium-malonate (500mM)
 
Aliquot 7ml into 350 ul; Store at -30
Succinate-Tris (500mM)
 
Tris Base 2M is made by adding 9.688g of Tris Base (Fisher #152-1, FW:121.1) in 40ml ddH2O. Then take 25ml of this, and add succinate. Then QSP to 28ml in graduated cylinder. Aliquot into 1.4 ml, Store at -30
 
Decylubiquinone (20mM)
 
For a 25mg bottle, add 3.877ml of DMSO for a [final] 20 mM.  Aliquot into 700 ul; Store at -30
 
Potassium cyanide – KCN (50mM)
 
Aliquot 14ml into 700ul; Store at -30
Rotenone (500 µM)
 
Note: Dilute 9.8mg in 1ml of DMSO for 25 mM stock. Then 1:50 dilution (0.4ml 25mM in 19.6ml) in
DMSO to get 500uM. 
Aliquot into 850 ul; Store at -30
 
5-Adenosine Triphosphate ATP
(50 mM)
 
Aliquot 6ml into 300 ul; Store at -30 
 
Antimycin A (400 µM)
 
Aliquot 3ml into 150 ul. Freeze 3 x 3ml aliquots.
 
Bovine Serum Albumin – BSA
(50mg/ml)
 
Aliquot into 10 ml.
 
Preparation of buffer and
samples: 
 
 
Take 33ml of the total 66ml, and add 330ul of Malonate for Malonate+, and 330ul of KPi-EDTA for Malonate-.

 
SECTION: COMPLEX II (SUCCINATE-UBIQUINONE OXIDOREDUCTASE) ENZYMATIC ACTIVITY ASSAY
9. Spectrophotometer settings:
  Assays are recorded on a Spectramax M2 (Spectramax Pro 6, Molecular Devices)
  The assay is done at 30 oC
  Read absorbance at 600 nm
  Set spectrophotometer to read only half of the plate at a time to maximize reading time (read every 17 sec)
 
Running the assay:
·        Set the spectrophotometer to read absorbance at 600nm, in Kinetics mode
·        Pipette 10ml of homogenate in each well.
·        Add 200ml of SDH assay mix to each well.
·       Read plate for 30 min.

·        The negative control is the assay mix with
Malonate, an inhibitor of Complex II
·       Obtain a measure of each sample in duplicates
·       The enzymatic activity is determined with the molar extinction coefficient of DCIP = 16.3 L x mol-1 x cm-1.
 
SECTION: COMPLEX I (NADH-UBIQUINONE OXIDOREDUCTASE, EC 1.6.99.3) ENZYMATIC ACTIVITY ASSAY
10. Principle of assay :
The activity of Complex I of the electron transport system (also NADH-Ubiquinone Oxidoreductase EC 1.3.99.3) is determined by the change in absorbance of DCIP, which accept electrons from CI.
Oxidation of NADH and decylubiquinone by the enzyme leads to a decrease in the
absorbance of DCIP as it is reduced. Antimycin A and KCN are used to inhibit electron
flow through complexes III and IV, respectively. Addition of Rotenone and
Piericidin A (inhibitors of Complex I) allows measurement of non-specific NADH
oxidation. Specific Complex I enzymatic activity is then computed from the
difference between the two slopes. The molar extinction coefficient of DCIP is
16.3 L x mol-1 x cm-1.
The molar extinction coefficient of NADH is
6.22 L x mol-1 x cm-1.
This
assay was modified based on the method of Janssen et al. Clin Chemistr 2007;
53(4):729-734. [BSA] was increased from 1 to 3.5mg/ml, and DCIP 1000uM was
added.
 
 
Buffers and Reagents
NADH
(80mM)
 
Aliquot 3.5ml into 175 ul; Freeze 3.5ml.
Piericidin
A (200mM)
 
Piericidin A comes as 100ug in 100ul solution. Take three vial (3), and add 1.103ml of DMSO to each for a final of 200uM. Combine the three for a final of 3.609ml. Piericidin A is 4X more potent than Rotenone, [final] will be 1/4. 
Aliquot into 175ul
Products already prepared under the heading of other assays, and aliquoted in double amounts.
  250 ml of Buffer KPi (50mM)
  BSA (50mg/ml) 
  Decylubiquinone (20mM)
  KCN (20 mM)
  Antimycin A (400 µM)
  Rotenone (200 µM)

 
Preparation of buffer and
samples:
 
 
Take 33ml of the total 66ml, and add 264ul of Rot (500uM) and 165ul of Pier A (200uM) for Rot/PierA+, and 429ul of DMSO for No-inhibitor. 
Be careful not to introduce inhibitors in the non-inhibited mix.
 
Spectrophotometer settings:
  Assays are recorded on a Spectramax M2 (Spectramax Pro 6, Molecular Devices)
  The assay is run at 37oC
  Read absorbance at 600 nm

Running the assay:
·       Set the spectrophotometer to 37oC to read absorbance at 600nm,
in Kinetics mode.
·       Pipette 10ml of homogenate in each well
·       Add 200ml of assay buffer to each well.
·       Quickly put plate in spectramax and read. 
·       The adequate negative control is the assay mix with Rotenone and Piericidin A (Complex I inhibitors)
·       Obtain a measure of each sample in duplicates
·       The enzymatic activity is determined with the molar extinction coefficient of DCIP = 16.3 L x mol-1 x cm-1.

 
 
 
 
 
 
 
 
 
 
 
 
 
SECTION: COMPLEX IV (COX – CYTOCHROME C OXIDASE) ENZYMATIC ACTIVITY ASSAY
11. Assay Principle:
Cytochrome c oxidase (COX, Complex IV of the electron transport system) enzymatic activity is determined by the decrease in absorbance of cytochrome c (cyt c) induced through its oxidation by COX. For the assay to work, it is essential to provide purified reduced cyt c. This is accomplished by preparing cyt c with dithionite, and then by purifying it from dithionite with filter tubes. Enzymatic activity is obtained by quantifying the rate of conversion from reduced to oxidized cyt c. The molar extinction coefficient of reduced cyt c is 29.5 L x mol-1 x cm-1. 

 

 
SECTION: COMPLEX IV (COX – CYTOCHROME C OXIDASE) ENZYMATIC ACTIVITY ASSAY
12. Buffers and Reagents
Buffer KPi 50mM
 
 
 
  Take 700
ml of K2HPO4 and acidify by adding KH2PO4
(about 100-200 ml) until pH 7.5. 
  KEEP KH2PO4 to acidify SDH
buffer

Aliquot into 35 ml for both COX and Complex I assays. Store at -30
 
n-Dodecylmaltoside 10% w/v
 
Add 10ml to 1g bottle and rock slowly because this product emulsifies
easily. Aliquot into 700ul. Store at
-30
 
 
SECTION: COMPLEX IV (COX – CYTOCHROME C OXIDASE) ENZYMATIC ACTIVITY ASSAY
13. Buffers and Reagents
Buffer KPi 50mM
 
 
 
Take 700 ml of K2HPO4 and acidify by adding KH2PO4 (about 100-200 ml) until pH 7.5. 
KEEP KH2PO4 to acidify SDH buffer
Aliquot into 35 ml for both COX and Complex I assays. Store at -30
 
n-Dodecylmaltoside 10% w/v
 
Add 10ml to 1g bottle and rock slowly because this product emulsifies easily. Aliquot into 700ul. Store at -30
SECTION: COMPLEX IV (COX – CYTOCHROME C OXIDASE) ENZYMATIC ACTIVITY ASSAY
14. Preparation
of buffer and samples: 
 
 
 
Spectrophotometer settings:
  Assays are recorded on a Spectramax M2 (Spectramax Pro 6, Molecular Devices)
  The assay is run at 30oC
  Read absorbance at 550 nm
 
Running the assay:
·       Set the spectrophotometer to read absorbance at 550nm, in Kinetics mode
·       Pipette 10ml of homogenate in each well.
·       Add 200ml of COX assay mix to each well.
·       Quickly put plate in spectrophotometer and read for 10min. 
·       The negative control is the assay mix without tissue homogenate; the slope should be 0.
·        Obtain a measure of each sample in duplicates.
·        The enzymatic activity is determined with the molar extinction coefficient of Cyt c = 29.5 L x mol-1 x cm-1.
SECTION: PREPARING REDUCED CYTOCHROME C FOR COX ASSAY
15. PREPARING REDUCED CYTOCHROME C FOR COX ASSAY
Buffers and Reagents (Potassium Phosphate – KPi 50mM)
 
 
 
Take 250 ml of K2HPO4 and acidify by adding KH2PO4 (about 75ml) until pH 7.5
 
For the final COX assay mix, 1 ml of reaction buffer requires 2-6ml of the final concentrated cyt c. When measuring the reaction in a 96-well plate, each well will contain 200ml; so you
need 0.5-1.2ml of concentrated cyt c per well. For example, if you are running 50 different samples measured in triplicates, you will need about 360ml of cyt c: [1.2ml x (50 samples x 6 measures) = 360ml]. Make sure there is enough in the freezer before you start.
 
NOTE: the final usable volume for the assay will be about 20-25% of
that prepared from the beginning. For example, preparing 20 ml will yield about
9-10 ml of concentrated reduced cyt c.
SECTION: PREPARING REDUCED CYTOCHROME C FOR COX ASSAY
16. Dilution and reduction
 
To prepare 20ml (initial) of cyt c, take 100ml of KPi buffer (50mM) and adjust pH at 7.0

 
The extra 80 ml are for the washing steps later in the protocol


Add 20ml of KPi buffer 50mM to a 5 g bottle of cytochrome c (Sigma C7752-5g) (250mg/ml)

 
Transfer to a 50ml Falcon tube and put on ice in the dark
·       Add 150 (300mg was added) mg of sodium bisulfite (dithionite) (7.5 mg/ml) (Sigma 157953, MW: 174.11)
·       Incubate for 5 minutes on ice and protect from light
·       Keep at 4oC from that point on
 
Purification 
·       Put 500 ml of the mix (KPi buffer + cyt c + dithionite) in 1.5 ml size ultracentrifugation filter tubes 10K MWCO (Amicon Ultra-0.5 mL 10kD Centrifugal Filters – Millipore UFV2BGC10[[1]]) [Note: 3kD will also work]
o   Vivaspin 2, # VS0291 (3000MWKO)
·       Centrifuge for 10 minutes at 10 000g for 1.5mltubes. The goal is to drain about 75% of the added volume. The sodium dithionite leaves through the filter with the buffer but cyt c is too big to go
through and remains in the tube. [Note: 3kD columns will need to be spun down faster and for much longer]
·       Re-fill the filter tube with ice cold buffer (KPi 50mM) to re-suspend and rinse the cyt c.
·       Repeat centrifugation and rinse steps for a total of 4 washes and about 40 minutes of centrifugation.
·       After the last centrifugation, invert filters in new tube bottoms provided in the kit. Spin these tubes at 1000g for one minute to retrieve the concentrated, purified cyt c. For a starting volume in each tube of 600ml, you should extract 100-200ml at the end.
·       Pool all collected product in one tube so that you have an homogenous mix. Quickly vortex. 
·       Estimate the volume and leave on ice and protected from light.
 
Measuring the concentration
and purity of reduced cytochrome c
·       Prepare two tubes for quantification
o   1ml of purified cyt c to 0.5 ml of KPi buffer (1:500)
o    2ml of purified cyt c to 0.5 ml of KPi buffer (1:250)
o   4ml of purified cyt c to 0.5 ml of KPi buffer (1:125)
·      Use the spectrophotometer in Absorbance/Spectrum mode to quantify optical density

 ·       
Set “Blank” wells in plate layout (to correct
for background) with only KPi buffer
·       
Load 200ul of each sample in duplicates
·       

 
E.g.:
 ·       Run a Spectrum between 500 and 600nm (5nm intervals) to obtain the absorbance of cytochrome c at different wavelength
·       The purity of reduced cytochrome c is calculated by the ratio of OD550(reduced cyt c) et OD565
(oxidized cyt c). Oxidized cytochrome c will not contribute to the reaction rate. The ratio OD550/OD565 should be > 6-10.
·       The concentration of reduced cytochrome c is determined by OD550 x 500[[1]] / (29.5[3] L x mol-1 x cm-1[4]). Use the concentrated cyt c at a [final] of 100mM for the COX assay. Determine the amount to add per ml of buffer to arrive at 120mM of reduced cyt c. This should be between 2-10ml of cyt c per ml of buffer.