An Assay to Determine NAD(P)H: Quinone Oxidoreductase Activity in Cell Extracts from Candida glabrata

Materials and Reagents

1. 40 ml glass test tube

2. Cotton wads

3. 1.5 ml centrifuge tubes (Tarsons, catalog number: 500010)

4. 15 ml Falcon tubes (Tarsons, catalog number: 546021)

5. 0.5 mm diameter glass beads (BioSpec Products, catalog number: 11079105)

6. 96-well plates (Biofil, catalog number: 011096)

7. Menadione (Sigma-Aldrich, catalog number: M5625)

8. Ethanol (PHARMCO-AAPER, catalog number: 111WORLDUV200)

9. 200 mM phenylmethylsulphonyl fluoride (PMSF, serine proteases inhibitor) (Amresco, catalog number: 0754-5G)

10. 100 mM sodium orthovanadate (NaOVa, Tyr and alkaline phosphatase inhibitor) (Sigma-Aldrich, catalog number: S6508)

11. 200 mM sodium fluoride (NaF, Ser/Thr and acid phosphatase inhibitor) (Sigma-Aldrich, catalog number: 450022)

12. 100× protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8215)

13. Pierce^TM BCA protein assay kit (Thermo Scientific, catalog number: 23227)

14. Nicotinamide adenine dinucleotide (NADH; reduced form of NAD), disodium salt, grade 2 approx. 98% (500 µM) (Roche, catalog number: 10128023001)

15. NAD(P)H: FMN oxidoreductase from Photobacterium fischeri (Roche, catalog number: 10476480001)

16. Culture media: Yeast extract (1%)-Peptone (2%)-Dextrose (2%) YPD (Becton, Dickinson and Company (BD) Diagnostic Systems, catalog number: 242810) (see Recipes)

17. Phosphate Buffer Saline (PBS) (see Recipes)

    1. NaCl (Affymetrix, catalog number: 21618)

    2. KCl (G-Biosciences, catalog number: RC1167)

    3. Na₂HPO₄ (Fisher Scientific, catalog number: 27785)

    4. KH₂PO₄ (Fisher Scientific, catalog number: 13405)

18. Cell lysis buffer (see Recipes)

    1. Tris base (MP Biosciences, catalog number: 808229)

    2. EDTA (Sigma-Aldrich, catalog number: E6635)

    3. Dextrose (Becton, Dickinson and Company (BD) Biosciences, catalog number: 215530)

19. Quinone reductase activity assay buffer (see Recipes)

    1. Tris base (MP Biosciences, catalog number: 808229)

    2. NaCl (Affymetrix, catalog number: 21618)

    3. HPLC grade water (Merck Life Science Pvt. Limited, catalog number: 61765010001730)

Equipment

1. 30°C shaker incubator (New Brunswick Innova, model: 43R)

2. Spectrophotometer/Cell density meter (Biowave-WFA, model: C08000)

3. Fine weighing balance (Mettler Toledo, model: MS205DU)

4. FastPrep 24 homogenizer or Bead beater (MP Biomedicals, model: 19083976)

5. Cooling table top centrifuge (Eppendorf, model: 5424R)

6. Quartz cuvette, 10 mm light path (Hellma Analytics, catalog number: 104-10-40)

7. SpectraMax multimode microplate reader (Molecular Devices, model: M5)

8. Needle and Spatula (Local stores)

Software

1. Excel 2018 (Microsoft)

2. SoftMax Pro Software 7.0

3. GraphPad Prism 7

Procedure

1. C. glabrata culturing

   1. Inoculate one single colony from the C. glabrata-streaked YPD agar plate in a 40 ml glass test tube with 5 ml of YPD medium.

   2. Place the tubes in a plastic beaker, and put cotton wads around the tubes so that these are tightly arranged in the beaker.

   3. Place the beaker in an incubator shaker, and grow the cultures with agitation at 200 rpm (rotation per minute) at 30°C overnight (14 to 16 h).

   4. The next morning, measure the optical density of the cultures at 600 nm (OD₆₀₀) using the spectrophotometer, by diluting 20 times in sterile PBS.

   5. To obtain logarithmic (log)-phase cultures, inoculate the fresh YPD medium (10 ml, in 100 ml flask) with overnight-grown cultures at an OD₆₀₀ of 0.1 and incubate with 200 rpm agitation at 30°C for 4-5 h, until the OD₆₀₀ reaches 0.4-0.6. The OD₆₀₀ may be measured every half an hour after 4 h of incubation.




2. Cell lysis

   1. Transfer 10 ml of log-phase culture (0.4-0.6 OD₆₀₀) to a 15 ml conical centrifuge/Falcon tube.

   2. Centrifuge tubes at 4,700 × g for 5 min at 4°C.

   3. Discard the supernatants and add 10 ml of PBS to the cell pellets.

   4. Centrifuge tubes at 4,700 × g for 5 min at 4°C and discard the supernatants. Add 1 ml of PBS and suspend cell pellets in PBS by gentle pipetting.

   5. Transfer cell suspensions to 1.5 ml microcentrifuge tubes and centrifuge tubes at 2,400 × g for 5 min at 4°C in a refrigerated microcentrifuge.

   6. Remove the supernatant completely using a 1 ml micropipette tip, without disturbing the pellet, add 200 μl cell lysis buffer to each tube, and suspend the cell pellet by gentle pipetting.

   7. Add 0.5 mm glass beads to each tube up to a volume equal to that of the cell suspension, and lyse cells using the FastPrep 24 homogenizer at the maximum speed setting of 6.5 M/s for five cycles of 60 s each, with 3-5 min ice incubations in between cycles. Of note, based on the fungal species, this cell lysis step may require standardization for the optimal number of cycles and bead-beating speed settings.

   8. Take the tubes out from the FastPrep homogenizer and puncture them at the bottom using a needle. Place the cell homogenate-containing punctured tube inside a new 1.5 ml microcentrifuge tube whose lid stays open. Arrange the tubes in a centrifuge with the open lids facing inwards and towards the center of the rotor, and centrifuge at 2,400 × g for 5 min at 4°C. The supernatant will be transferred to the lower tube, while the glass beads will stay in the upper tube. Alternatively, centrifuge the cell homogenate-containing tubes at 21,000 × g for 10 min at 4°C to remove the beads, and transfer the supernatant to a new tube.

   9. Centrifuge tubes again at 21,000 × g for 10-15 min at 4°C to remove cell debris and non-lysed cells, and collect the clear supernatants containing cellular proteins (approximately 150 μl) in new microcentrifuge tubes.

   10. Measure protein concentration in cell lysates using the BCA protein assay kit, following the manufacturer’s instructions. For this, add 11.5 μl sterile water and 1 μl cell lysate per assay. The reaction mixture volume in each well of a 96-well plate will be 12.5 μl. For standard plot generation, add 1 μl to 5 μl of BSA (Bovine serum albumin; 2 mg/ml concentration provided with the kit) in five separate wells of the 96-well plate. Make up the volume of each well to 12.5 μl with sterile water. Keep one well containing 12.5 μl of sterile water as blank, to which no protein sample is added. Add 100 μl of BCA solution (Solution A and Solution B in 50:1 ratio) to each well. Cover the plate with aluminum foil and incubate it at 37°C for 30 min. Measure sample absorbance at 562 nm in a multimode microplate reader. Calculate the protein concentration in cell lysate samples, which usually ranges from 5 to 10 μg/μl, using the BSA standard curve.




3. Setting up of reaction mixture

   1. Start preparing 1 ml total volume of the reaction mixture by placing a 1.5 ml centrifuge tube on ice. It will contain 500 µg protein, 500 µM menadione, and 500 µM NADH.

   2. Add the cell lysate corresponding to 500 µg of protein and 10 μl of 50 mM menadione stock solution (freshly prepared by dissolving 8.609 mg powder in 1 ml absolute ethanol in a 1.5 ml microcentrifuge tube) to the reaction mixture. Of note, menadione is required for this assay, as no enzymatic activity was observed in the absence of exogenous menadione. However, menadione may be replaced with other quinones to check the specificity of different quinone reductase enzymes present in cell lysates. Similarly, since the levels and activities of Fld-LPs may change under various stress conditions, it is important to standardize the assay with 2-3 different concentrations of cell lysates and measure the absorbance values in the linear range.

   3. Make up the reaction volume to 950 µl with the assay buffer.

   4. For each cell lysate, prepare one blank control that contains all reagents.

   5. Set up a positive control by adding 20 μl of NAD(P)H: FMN oxidoreductase (1 Unit; Roche) instead of the cell lysate.

   6. Prepare 10 mM NADH stock solution by dissolving 6.6343 mg of powder in 1 ml HPLC grade water, before proceeding to the absorbance measurement step.




4. Absorbance measurement

   1. Set up the Spectramax M5 plate reader to measure absorbance at 340 nm over a period of 60 s at 10 s intervals.

   2. Perform all the following steps in the dark.

   3. Add 50 µl of 10 mM NADH solution to the reaction mixture, mix well, transfer to a quartz cuvette (1-cm-path-length), and read the absorbance at 340 nm over a period of 60 s at 10 s intervals. Since NADH addition will start the quinone detoxification reaction, this step needs to be performed as quickly as possible. Therefore, it is important to start reactions individually just before the absorbance measurement.

   4. As a negative control, measure the absorbance of the blank sample (without adding NADH) at 340 nm over a period of 60 s at 10 s intervals.

   5. Repeat the above-mentioned steps for all cell lysate and positive control samples.

Data analysis

1. For each sample, a graph displaying absorbance values as a function of time will appear on the SpectraMax M5 multimode microplate system, which is generated using the SoftMax Pro Software 7.0. Save the raw file as pdf and export the raw values to an excel file. This can be done by a right click on the graph and choosing the copy cuvette data option, followed by pasting data in the excel sheet.

2. Arrange the data in a new excel sheet by placing sample absorbance values in an ascending order by time of measurement.

3. Considering the absorbance of the substrate NADH as 100 at 0 s time point, calculate NADH oxidation in each sample by dividing the absorbance at each time point by 0 s absorbance and multiplying the number by 100. The number obtained reflects the NADH (substrate) amount remaining in the reaction mixture at that particular time point.

4. Plot the amount of substrate remaining versus the time point (in seconds) at which the absorbance was measured, using GraphPad Prism 7 or any other software. A representative graph is shown in Figure 3. Perform the experiment in biological triplicates.

   
   

   
   Figure 3. NADH:quinone oxidoreductase activity measurement in C. glabrata cell lysates, as measured using menadione and NADH. The C. glabrata wild-type strain BG2 was grown in the YPD medium to log-phase, followed by cell collection and lysis using glass beads. Cell lysate (500 μg) was incubated with menadione (500 µM) and NADH (500 µM), and absorbance was recorded at 340 nm over a period of 60 s. Absorbance of the substrate NADH was taken as 100 at 0 h time point, and NADH oxidation was calculated using the formula: [(absorbance at each time point/0 h absorbance) × 100]. Data represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences in activity between the wild-type cell lysate and the blank sample, which contained all reagents except NADH. *, P < 0.0332; **, P < 0.0021; Grouped multiple t-test.

   
   

Notes

1. NADH and menadione solutions are to be freshly prepared just before use.

2. The HPLC-grade water was used to prepare reagents and buffer to avoid variations associated with water quality.

3. pH of the cell lysis buffer should be strictly maintained, as enzymatic reactions are standardized for a buffer of pH 7.4-7.5 only.

4. The NADH solution should be wrapped with aluminum foil to minimize exposure to light and stored at 4°C during the enzyme assay.

5. In case the assay is being performed with flavodoxin-like proteins purified from E. coli, add FMN (100 μM) or FAD (100 μM) as cofactors to the purified enzyme and incubate for 1 h at 4°C, followed by the addition of menadione and other reagents to the reaction mixture.

6. The NAD(P)H:quinone oxidoreductase activity can also be measured in cells treated with different quinones. Using this protocol, we have measured NAD(P)H:quinone oxidoreductase activity in log-phase menadione (90 µM)-treated C. glabrata cultures (Battu et al., 2021). Menadione treatment was given by adding 20 μl of menadione stock solution (50 mM) to 10 ml log-phase culture and incubating cultures with 200 rpm agitation for 90 min at 30°C. One culture aliquot was kept as untreated control, to which menadione was not added. Cultures were processed using the steps described above in the protocol [Menadione treatment can be replaced by exposure to any other stressor or growth of a mutant strain].

Recipes

1. Yeast extract-Peptone-Dextrose (YPD) medium (1 L)

   YPD medium can either be prepared using BD Difco^TM YPD Broth (# 242810) or adding the following ingredients to sterile water, followed by autoclaving.

   10 g Yeast extract

   20 g Peptone

   20 g Dextrose

2. Phosphate Buffer Saline (PBS) (1 L)

   137 mM NaCl

   2.7 mM KCl

   10 mM Na₂HPO₄

   1.8 mM KH₂PO₄

   Dissolve 80 g of NaCl, 2 g of KCl, 14.4 g of Na₂HPO₄, and 2.4 g of KH₂PO₄ in water to prepare a 10× solution.

   Adjust the final pH to 7.4 with HCl. Dilute 10× solution to 1× solution with distilled water.

3. Cell lysis buffer (100 ml)

   50 mM Tris-HCl (pH 7.5) (50 ml of 1 M stock solution)

   2 mM EDTA (133.3 µl of 1.5 M stock solution)

   2% dextrose (4 ml of 50% stock solution)

   10 mM Sodium fluoride (NaF)*

   1 mM Phenylmethylsulphonyl fluoride (PMSF)*

   1 mM Sodium orthovanadate (NaOV)*

   1× Protease inhibitor cocktail*

   *Inhibitor stock solutions can be prepared in advance and stored in small aliquots at -20°C. Thaw an aliquot and add to the lysis buffer just before use.

4. Quinone reductase activity assay buffer (10 ml)

   10 mM Tris-HCl (pH 7.4) (100 µl of 1 M stock solution)

   150 mM NaCl (300 µl of 5 M stock solution)

   Make up the volume to 10 ml with HPLC grade water.

Acknowledgments

This work was fully supported by the DBT/Wellcome Trust India Alliance Senior Fellowship to RK (IA/S/15/1/501831; www.indiaalliance.org/). Research in the Kaur laboratory is also supported by grants from the Department of Biotechnology (BT/HRD/NBA/37/01/2014; http://www.dbtindia.gov.in/) and Science and Engineering Research Board, Department of Science and Technology (EMR/2016/005375; http://www.serb.gov.in/home.php), Government of India. AB is the recipient of a Research Fellowship of the Department of Biotechnology, New Delhi, India (http://dbtindia.gov.in/).

The protocol is derived from the activity assays previously described for E. coli.-purified proteins (Herrou et al., 2016). This protocol for NAD(P)H:quinone oxidoreductase activity in C. glabrata cell extracts is described in our recent publication (Battu et al., 2021).

Competing interests

The authors declare that they have no conflict of interests.

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