TITLE: NADH-linked microtiter plate-based assay for measuring Rubisco activity & activation state – GAPDH-GlyPDH
AUTHORS: Cristina Rodrigues Gabriel Sales, Anabela  Silva, Elizabete Carmo-Silva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol uses five reactions to couple RuBP carboxylation and 3-PGA formation to NADH oxidation to measure Rubisco activity, based on Kubien et al. (2011).</div><div class = "text-block"><div class = "justify" style = "text-align:center"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/6ebbf137347306d4f5b66038cf0224ede4e26ba63bb212101b7af5c54dad5bd6/b8g8bdagf.png" /></div></div>

[STEPS]
?. [REAGENTS & SOLUTIONS]
REAGENTS & SOLUTIONS TO PREPARE BEFOREHAND
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. PCK).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.
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Basic extraction buffer (1x) Bicine-NaOH  MgCl2.6H2O EDTA Benzamidine ε-Aminocaproic acidDissolve in ultrapure H2O; adjust pH to 8.2 with NaOH; degas the solution bubbling with nitrogen (5 min/10 mL), then add:      2-MercaptoethanolAdjust for the final volume; it can be dispensed in aliquots (e.g. 50 mL Falcon tubes). 2-Mercaptoethanol
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DTTDissolve in ultrapure H2O.
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PMSFDissolve in ethanol 99%.
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Plant protease inhibitor cocktail
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RuBP
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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).
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STOCK COMPONENTS FOR THE ASSAY BUFFER Bicine-NaOH Dissolve in ultrapure H2O; adjust pH to 8.2 with NaOH; filter through 0.22 µm membrane for long shelf life.  MgCl2.6H2ODissolve in ultrapure H2O; filter through 0.22 µm membrane for long shelf life.  NaHCO3Dissolve in ultrapure H2O; filter through 0.22 µm membrane for long shelf life.  PhosphocreatineDissolve in ultrapure H2O; aliquot;  ATPAdd powder to a tube, add some ultrapure H2O to dissolve, add NaOH (~10 µL/mL) to approximately ; add ultrapure H2O to the final volume; aliquot.   NADHDissolve in  Bicine-NaOH ; aliquot.  PCKDissolve all solid by adding   Bicine-NaOH  to the container; aliquot.  GAPDHDissolve all solid by adding   Bicine-NaOH  to the container; aliquot.  PGKStore at when in ammonium sulfate suspension.Before use, it is necessary to buffer exchange as ammonium sulfate can affect the assay.Pipette a portion, e.g.,100 µL.Proceed to a buffer exchange using Amicon molecular weight cut-off of 10000 (Sigma-Aldrich); substitute ammonium sulfate for   Bicine-NaOH   in the same volume centrifuged (in this example 100 µL).Assay for TSP to know the concentration; aliquot.         GlyPDH +    TPI (GlyPDH/TPI)Store at  when in ammonium sulfate suspension.Before use, it is necessary to buffer exchange as ammonium sulfate can affect the assay.Pipette a portion, e.g.,100 µL.Proceed to a buffer exchange using Amicon molecular weight cut-off of 10000 (Sigma-Aldrich); substitute ammonium sulfate for  Bicine-NaOH  in the same volume centrifuged (in this example 100 µL).Assay for TSP to know the concentration; aliquot.  GlyPDH +   TPI (GlyPDH/TPI)Store at when in ammonium sulfate suspension.Before use, it is necessary to buffer exchange as ammonium sulfate can affect the assay.Pipette a portion, e.g.,100 µL.Proceed to a buffer exchange using Amicon molecular weight cut-off of 10000 (Sigma-Aldrich); substitute ammonium sulfate for  Bicine-NaOH  in the same volume centrifuged (in this example 100 µL).Assay for TSP to know the concentration; aliquot.
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To test the pH use universal pH paper, add    of the solution and check colour.
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Protect from light as it is light sensitive. Please, check notes in section 2.1 to a more efficient way of aliquoting NADH.
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4 °C
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4 °C
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?. [REAGENTS & SOLUTIONS]
Complete assay buffer for Rubisco activityThe basic assay buffer (table below) can be prepared the day before the assays and kept at , or prepared in advance (e.g. at the start of an experiment), snap-frozen in aliquots and kept at .NADH is prepared separately, snap-frozen in aliquots and kept at , and added to the assay buffer just before the assays.
4 °C
-80 °C
-80 °C
*As PGK and GlyPDH/TPI need to be buffer exchanged, the stock concentration will change in each preparation, thus the volume added in 200 μL assay will change.
We recommend using the same assay buffer for all the samples of the same experiment.
Stock solutions and the assay buffer should thaw  . The assay buffer should be kept in a tube wrapped in aluminium foil   during the assays, as NADH is light sensitive.
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SOLUTIONS TO PREPARE JUST BEFORE USE
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Complete extraction buffer    1x Basic extraction buffer (from step 1.1) DTT (from step 1.2) PMSF (from step 1.3)  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
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.
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 per protein content (TSP or Rubisco) is mantained.
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Complete assay bufferThaw  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 , wrapped in aluminium foil.
on ice
on ice
Example of how to prepare the complete assay buffer: e.g. 20 samples in a day20 samples x (1 Blank + 3xTotal + 3xInitial activity assays) = 140 wellsAssay buffer without NADH per well = 38.8, for 140 wells = 38.8 x 140 = 5432µLNADH per well = 5.71, for 140 wells = 5.71 x 140 = 799.4 µLMix both together (5432 + 799.4 µL)
?. [PROCEDURE]
STARTThaw 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.Turn on the centrifuge and set to .Collect samples from  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  .
The temperature to be used for the Rubisco activity measurement depends on the experiment goals. Typical measurement temperatures are   (standard) and  , 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.
4 °C
-80 °C
on ice
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EXTRACTIONS & RUBISCO ASSAYS
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Before starting the extraction, pipette to the 96-well microtiter plate  CO2-free ultrapure H2O for the blank (singlet) and  for the samples into each well (6 wells, i.e., triplicates for initial activities and triplicates for total activities), followed by  of complete assay buffer (from step 3.2). Gently mix components by pipetting up and down 5 times whilst stirring. Add  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.
150.5 µl
144.5 µl
44.5 µl
6 µl
?. [PROCEDURE]
ExtractionAdd 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   to maximum of  .Collect the homogenate into an ice-cold 1.5 mL microtube and centrifuge  . When centrifugation stops, take the extract supernatant into another ice-cold 1.5 mL microtube.Proceed to the Rubisco assays straight away.Add   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   at  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   of 20 mM RuBP (from step 1.5)  to the wells for measurement of total Rubisco activity   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.   Below is a pipetting scheme for the microplate assayGrind the sample thoroughly for  to maximum of .Collect the homogenate into an ice-cold 1.5 mL microtube and centrifuge .When centrifugation stops, take the extract supernatant into another ice-cold 1.5 mL microtube.Proceed to the Rubisco assays straight away.Add   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   at  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   of 20 mM RuBP (from step 1.5)  to the wells for measurement of total Rubisco activity   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.   Below is a pipetting scheme for the microplate assayAdd  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  at 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  of 20 mM RuBP (from step 1.5)  to the wells for measurement of total Rubisco activity  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.   Below is a pipetting scheme for the microplate assay
Centrifuge: 14000 34, 1 min, 4 10
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.
5 µl
30 °C
6 µl
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).
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.
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.
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.
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]
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 four NADH in the subsequent coupling reactions. 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 4 is used to account for the four 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: RuBP consumption=(Slope×Volume)/(6.22×4×Pathlength)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 4 is used to account for the four molecules of NADH which are oxidized per molecule of RuBP. The Pathlength of the assay mix contained in each well is measured in cm. 
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							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: V_i  or V_t=(RuBP consumption×Extraction)/(Leaf area×Aliquot)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. 
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							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: V_i  or V_t=(RuBP consumption×60)/(Protein×Aliquot)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. 
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							AS=100×V_i/V_t
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.