Jun 30, 2020
NADH-linked microtiter plate-based assay for measuring Rubisco activity & activation state – PK-LDH
- Cristina Rodrigues Gabriel Sales1,
- Anabela Silva2,
- Elizabete Carmo-Silva1
- 1Lancaster Environment Centre, Lancaster University, Library Avenue, Lancaster, LA1 4YQ, UK;
- 2Biosystems & Integrative Sciences Institute (BioISI), Science Faculty of Lisbon University, Lisbon, 1749-016, Portugal
Protocol Citation: Cristina Rodrigues Gabriel Sales, Anabela Silva, Elizabete Carmo-Silva 2020. NADH-linked microtiter plate-based assay for measuring Rubisco activity & activation state – PK-LDH. protocols.io https://dx.doi.org/10.17504/protocols.io.bf9rjr56
Manuscript citation:
Sales CRG, Silva AB, Carmo-Silva E. 2020. Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays. Journal of Experimental Botany, https://doi.org/10.1093/jxb/eraa289
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 11, 2020
Last Modified: June 30, 2020
Protocol Integer ID: 36881
Keywords: Enzyme activity assay , Rubisco, Crop improvement , NADH-linked assay, PK-LDH, Plant phenotyping, Microtiter plate,
Abstract
This protocol uses five reactions to couple RuBP carboxylation and 3-PGA formation to NADH oxidation to measure Rubisco activity, based on Scales et al. (2014).
Note
During the NADH-linked assays, some Rubisco active sites might become carbamylated as the leaf extract is exposed to high CO2 and Mg2+ in the assay buffer. Therefore, these assays are not suitable for measuring Rubisco initial activity and/or activation state at different levels in the canopy or in conditions in which low intercellular CO2 is promoted (e.g., low light, drought stress, cold stress); the 30 second 14CO2-based assay is recommended in such situations (protocol available in this collection).
Guidelines
- Check the "Materials" tab for a list of all the chemicals used in this protocol.
- In the "Steps" tab, there is a brief description of the materials and equipment necessary for the protocol execution.
- In the "Steps" tab, there is information on preparation of solutions, procedures for determining Rubisco initial and total activities, and notes to take into consideration to ensure reliable results.
- The references cited are at the end of the "Materials" tab.
Materials
MATERIALS
BicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #B3876
Magnesium chloride hexahydrate (MgCl2.6H2O)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M2393
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #E1644
BenzamidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #B6506
ε-Aminocaproic acid Merck MilliporeSigma (Sigma-Aldrich)Catalog #A2504
Sodium hydroxide (NaOH)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5881
2-Mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
DL-Dithiothreitol (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #43819
Phenylmethanesulfonyl fluoride (PMSF)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7626
Protease inhibitor cocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9599
D-Ribulose 1.5-bisphosphate sodium salt hydrate (RuBP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #83895
Sodium bicarbonate (NaHCO3)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Potassium chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
2.3-Diphospho-D-glyceric acid pentasodium salt (2.3-dPGA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D5764
Adenosine 5′-diphosphate sodium salt (ADP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A2754
β-Nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH)Merck MilliporeSigma (Sigma-Aldrich)Catalog #N8129
Enolase from bakers yeast (S. cerevisiae)Merck MilliporeSigma (Sigma-Aldrich)Catalog #E6126
Pyruvate kinase/Lactic dehydrogenase enzymes from rabbit muscle (PK/LDH)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P0294
2.3-dPGA-dependent phosphoglycerate mutase (dPGM)Home-made
Ethanol absolute 99.8 %Fisher ScientificCatalog #10437341
CITATION
CITATION
CITATION
CITATION
CITATION
CITATION
Safety warnings
Before using the protocol always check the Safety Data Sheet (SDS) for each chemical.
Before start
MATERIAL & EQUIPMENTS (for list of chemicals check "Materials" tab)
- Leaf sample frozen in -80°C
- Centrifuge for microtubes (speed 14000 g, 4 °C; VWR, Mega Star 600R)
- Microtiter plate reader (BMG Labtec, SpectroStarNano)
- 96-well microtiter plate with clear flat bottom (Thermo Scientific, 442404)
- Pipette set
- Mortar and pestle
- 1.5 mL microtubes
REAGENTS & SOLUTIONS
REAGENTS & SOLUTIONS
REAGENTS & SOLUTIONS TO PREPARE BEFOREHAND
Note
- Powder chemical stocks stored at -20°C: let warm up to room temperature on desiccant before opening container.
- Expensive chemicals purchased in very small amounts (mg), for which concentration in assay is not critical (e.g. in excess): trust quantity stated by the supplier and add ultrapure H2O / solvent to container for final concentration (e.g. 2,3-dPGA).
- Protein and substrate solutions should typically be stored at -80°C.
- Freeze proteins in LN2 before storing at -80°C. Store in small aliquots to prevent multiple freeze-thaw cycles. If using in consecutive days, protein solutions can be kept at 4°C.
- Buffers and solutions will last longer if filtered through 0.22 µm membrane.
Basic extraction buffer (1x)
50 millimolar (mM) Bicine-NaOH 8.2
20 millimolar (mM) MgCl2.6H2O
1 millimolar (mM) EDTA
2 millimolar (mM) Benzamidine
5 millimolar (mM) ε-Aminocaproic acid
- Dissolve in ultrapure H2O; adjust pH to 8.2 with NaOH; degas the solution bubbling with nitrogen (5 min/100 mL), then add:
50 millimolar (mM) 2-Mercaptoethanol
- Adjust for the final volume; it can be dispensed in aliquots (e.g. 50 mL Falcon tubes).
-20 °C (storage)
1 Molarity (M) DTT
- Dissolve in ultrapure H2O. 4 °C (storage)
100 millimolar (mM) PMSF
- Dissolve in ethanol 99%. 4 °C (storage)
Plant protease inhibitor cocktail
-20 °C (storage)
20 millimolar (mM) RuBP
-20 °C (storage)
Note
High purity RuBP (≥99%) is required to avoid interference in measurable activity due to the presence of RuBP-analogs that inhibit carboxylation (Kane et al., 1998; Sharwood et al., 2016). It is available commercially or it can be produced enzymatically from AMP-5’ monohydrate and ATP disodium salt (Wong, 1980).
STOCK COMPONENTS FOR THE ASSAY BUFFER
1 Molarity (M) Bicine-NaOH 8
- Dissolve in ultrapure H2O; adjust pH to 8.2 with NaOH; filter through 0.22 µm membrane for long shelf life. 4 °C (storage)
1 Molarity (M) MgCl2.6H2O
- Dissolve in ultrapure H2O; filter through 0.22 µm membrane for long shelf life. 4 °C (storage)
0.5 Molarity (M) NaHCO3
- Dissolve in ultrapure H2O; filter through 0.22 µm membrane for long shelf life. 4 °C (storage)
3 Molarity (M) KCl
- Dissolve in ultrapure H2O; filter through 0.22 µm membrane for long life shelf. 4 °C (storage)
0.1 Molarity (M) 2,3-dPGA
- Dissolve all solid by adding ultrapure H2O to the container; aliquot. -80 °C (storage)
0.5 Molarity (M) ADP
- Add powder to a tube, add some ultrapure H2O to dissolve, add 4 Molarity (M) NaOH (~10 µL/mL) to approximately 7 ; add ultrapure H2O to the final volume; aliquot. -80 °C (storage)
Note
To test the pH use universal pH paper, add 1 µL of the solution and check colour.
14 millimolar (mM) NADH
- Dissolve in 100 Molarity (M) Bicine-NaOH 8 ; aliquot. -80 °C (storage)
Note
Protect from light as it is light sensitive.
Please, check notes in section 2.1 to a more efficient way of aliquoting NADH.
36 U/ml Enolase
- Dissolve all solid by adding 100 Molarity (M) Bicine-NaOH 8 to the container; aliquot. -80 °C (storage)
1 KU/ml PK + 1.4 KU/ml LDH (PK/LDH)
-20 °C (storage)
3 KU/ml d-PGM
- Home-made (for more detail, please see the protocol “Purification of 2,3-bisphosphate-dependent phosphoglycerate mutase (d-PGM)"). -80 °C (storage)
Complete assay buffer for Rubisco activity
- The basic assay buffer (table below) can be prepared the day before the assays and kept at 4 °C , or prepared in advance (e.g. at the start of an experiment), snap-frozen in aliquots and kept at -80 °C .
- Each assay buffer aliquot should only be thawed once, as repeated freeze thawing can result in degradation of the coupling enzymes; thus, it is important to aliquot adequate volumes for use in a day.
- NADH is prepared separately, snap-frozen in aliquots and kept at -80 °C , and added to the assay buffer just before the assays.
Note
We recommend using the same assay buffer for all the samples of the same experiment.
Note
Stock solutions and the assay buffer should thaw On ice . The assay buffer should be kept in a tube wrapped in aluminium foil On ice during the assays, as NADH is light sensitive.
SOLUTIONS TO PREPARE JUST BEFORE USE
- Prepared with reagents/solutions described in step 1.
Complete extraction buffer
1x Basic extraction buffer (from step 1.1)
10 millimolar (mM) DTT (from step 1.2)
1 millimolar (mM) PMSF (from step 1.3)
1 % (v/v) Plant protease inhibitor cocktail (from step 1.4)
- Prepare the volume considering the number of extractions to be performed throughout the day plus two extras (to have a little excess). Mix all together. On ice
Note
The volume of extraction buffer will depend on the size of the leaf sample and the protein content, therefore it is species dependent and should be tested beforehand. Rubisco concentration in the assays should be approx. 15 µg mL-1 for purified enzyme and between 10-40 µg mL-1 for non-purified enzyme. Rubisco amounts above these values may limit the sensitivity of the NADH-linked assays.
Note
To test if the assay is giving reliable results (i.e, none of the chemicals are limiting the reactions) it is important to always perform a test when the plant species and/or growth conditions change. Perform the assay with different extract concentration (e.g. 1/2 the amount, 1/5 of the amount, etc) and check if the activity expressed by protein content (TSP or Rubisco) is mantained.
Complete assay buffer
- Thaw On ice the assay buffer and NADH aliquots (prepared according to step 2.1) to be used in the day.
- Mix the correspondent volume of both solutions together. Keep On ice , wrapped in aluminium foil.
Note
Example of how to prepare the complete assay buffer: e.g. 20 samples in a day
- 20 samples x (1 Blank + 3xTotal + 3xInitial activity assays) = 140 assays/wells
- Assay buffer without NADH per well = 62.3, for 140 wells = 62.3 x 140 = 8722 µL
- NADH per well = 5.71, for 140 wells = 5.71 x 140 = 799.4 µL
- Mix both together (8722 + 799.4 µL)
PROCEDURE
PROCEDURE
START
- Thaw the frozen solutions that will be used in the day.
- Prepare CO2-free ultrapure H2O by bubbling with nitrogen (5 min/100 mL).
- Turn on the microplate reader and set up for the temperature that Rubisco activity will be performed, select kinetic protocol at 340 nm.
Note
The temperature to be used for the Rubisco activity measurement depends on the experiment goals. Typical measurement temperatures are 25 °C (standard) and 30 °C , depending on the species. Assays can be performed at a range of temperatures, however high temperatures might lead to evaporation of the assay mix and, since rates will be faster, the assay might become less sensitive.
- Turn on the centrifuge and set to 4 °C .
- Collect samples from -80 °C into liquid nitrogen container.
- Prepare the complete extraction buffer (from step 3.1) and the complete assay buffer (from step 3.2) and keep it On ice .
EXTRACTIONS & RUBISCO ASSAYS
- Before starting the extraction, pipette to the 96-well microtiter plate 127 µL CO2-free ultrapure H2O for the blank (singlet) and 121 µL for the samples into each well (6 wells, i.e., triplicates for initial activities and triplicates for total activities), followed by 68 µL of complete assay buffer (from step 3.2).
- Gently mix components by pipetting up and down 5 times whilst stirring. Add 6 µL of 20 mM RuBP (from step 1.5) to the wells for measuring initial Rubisco activity (see table below). Cover to protect from light. Proceed to extraction.
Extraction
- Add the complete extraction buffer (from step 3.1) to an ice-cold mortar.
- Take a sample from the liquid nitrogen container and add to the mortar.
- Grind the sample thoroughly for 00:00:30 to maximum of 00:01:00 .
- Collect the homogenate into an ice-cold 1.5 mL microtube and centrifuge 14000 x g, 4°C, 00:01:00 .
Note
To prevent Rubisco deactivation (or even denaturation) the extraction should not take more than 1 min and it should be done in a ice-cold mortar, keeping the sample cold at all times. In our hands, with the extraction buffer described (containing protease inhibitors, mercaptoethanol and DTT, which keeps the enzyme reduced) 1 min centrifugation does not impact Rubisco activity. However, this should be tested for each species and extraction buffer used.
- When centrifugation stops, take the extract supernatant into another ice-cold 1.5 mL microtube.
- Proceed to the Rubisco assays straight away.
- Add 5 µL of sample supernatant to the wells for total activity first, followed by those for initial activity, mixing well by pipetting up and down 10 times whilst stirring. Place microplate in the reader and start monitoring the change in absorbance at 340 nm immediately. The addition of the extract initiates the reaction for the initial activity assays, which is measured while incubating Rubisco with CO2 and MgCl2 in the absence of RuBP (total activity) for 00:03:00 at 30 °C to enable carbamylation of the enzyme. The absorbance value should start decreasing in the wells for the initial activity assay (containing RuBP).
- Pause the reading in order to add 6 µL of 20 mM RuBP (from step 1.5) to the wells for measurement of total Rubisco activity 00:03:00 after addition of sample supernatant.
- Place the microplate in the reader and continue monitoring the change in absorbance.
- The reading can be stopped once the reaction reaches a plateau.
Note
Considering that the protein concentration in the extract is as suggested in step 3.1, 5 µL of sample supernatant will give a good NADH consumption rate. The rate can be adjusted by adding more or less supernatant, but note that the amount of CO2-free ultrapure H2O added in the wells will change (as the final volume needs to be 200 µL).
Note
The Initial activity assays start with extract addition, while total activity assays start with addition of RuBP after 3 min of extract incubation with CO2 and Mg2+ to allow for Rubisco carbamylation.
Note
This protocol can be adapted for measuring Rubisco activity with purified enzyme. In this case, Rubisco is frequently pre-activated and initial activity assays are performed.
- Below is a pipetting scheme for the microplate assay
Note
Conducting measurements at 30°C provides fast rates and reliable slopes, but the temperature can be adjusted according to the experimental aims and plant species used.
Note
It is important to ensure that air bubbles are not introduced in the wells during the pipetting steps, as these will interfere with the absorbance measurements.
CALCULATIONS
CALCULATIONS
- The activity of Rubisco is inferred from the consumption of RuBP (μmol s-1) measured by absorbance change per second at 340 nm due to NADH oxidation, using an extinction coefficient of 6220 M-1cm-1 or 6.22 μmol-1 mL cm-1.
- The carboxylation of one molecule of RuBP results in two molecules of 3-PGA, thus requiring two NADH in the final step. The rate of RuBP consumption (μmol s-1) in the assay volume, is therefore calculated by:
where the Slope represents the change in absorbance per second in the linear part of the absorbance trace change, Volume is the final volume per well in mL (0.2), 6.22 is the extinction coefficient of NADH in μmol-1 mL cm-1 and the factor 2 is used to account for the two molecules of NADH which are oxidized per molecule of RuBP. The Pathlength of the assay mix contained in each well is measured in cm.
- Rubisco initial (Vi) and total (Vt) activity expressed on a leaf area basis (µmol m-2 s-1) is then calculated by:
where the Extraction is the volume of buffer in mL used for leaf extraction, leaf area is in m2, and Aliquot is the volume of leaf extract supernatant used in the assay in mL.
- Rubisco activity can also be expressed on a Rubisco or total soluble protein (TSP) content basis (μmol min-1mg-1):
where 60 is to convert seconds to minutes, Protein is the Rubisco or TSP content in mg mL-1, and Aliquot is the volume of leaf extract supernatant used in the assay in mL.
- From the Rubisco activity calculations above for initial (Vi) and total activity (Vt), the Rubisco activation state (AS, %) can be calculated:
Note
Measured absorbance values in a microtiter plate need to be normalized to a 1 cm pathlength, which would be found in a typical cuvette used in spectrophotometers. Measurements are corrected using Lambert-Beer’s Law and considering both the volume in each well and the specific well dimensions for each type of microtiter plate. Modern microtiter plate readers frequently include a pathlength correction option, but this feature normally does not consider the properties of the solution. It is important to use the respective assay mix in determining the pathlength correction factor as the meniscus will affect the pathlength and absorbance reading in the microtiter plate. The pathlength can be determined according to Lampinen et al., (2012). Please, check the SI information in the publication linked to this protocol for more details.
Citations
Sales CRG, Degen GE, Silva AB, Carmo-Silva E. Spectrophotometric determination of Rubisco activity and activation state in leaf extracts
https://doi.org/10.1007/978-1-4939-7786-4_14Sharwood RE, Sonawane BV, Ghannoum O, Whitney SM. Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry
https://doi.org/10.1093/jxb/erw154Lampinen J, Raitio M, Perälä A, Oranen H, Harinen R. Microplate based pathlength correction method for photometric DNA quantification assay
https://assets.thermofisher.com/TFS-Assets/LCD/Application-Notes/AN-SkanIT-Microplate-Based-Pathlength-Correction-Technical-NoteWong C-H. Practical enzymatic syntheses of ribulose 1,5-bisphosphate and ribose 5-phosphate
https://doi.org/10.1021/ja00547a023Kane HJ, Wilkin JM, Portis AR, Andrews TJ. Potent inhibition of ribulose-bisphosphate carboxylase by an oxidized impurity in ribulose-1,5-bisphosphate
https://doi.org/10.1104/pp.117.3.1059Scales JC, Parry MA, Salvucci ME. A non-radioactive method for measuring Rubisco activase activity in the presence of variable ATP: ADP ratios, including modifications for measuring the activity and activation state of Rubisco.
https://doi.org/10.1007/s11120-013-9964-5