Jun 30, 2020

Public workspace14CO2-based assay for measuring Rubisco activity & activation state

DOI
dx.doi.org/10.17504/protocols.io.bf8cjrsw
  • 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
Cristina Rodrigues Gabriel Sales
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DOI: dx.doi.org/10.17504/protocols.io.bf8cjrsw
Protocol Citation: Cristina Rodrigues Gabriel Sales, Anabela Silva, Elizabete Carmo-Silva 2020. 14CO2-based assay for measuring Rubisco activity & activation state. protocols.io https://dx.doi.org/10.17504/protocols.io.bf8cjrsw
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: 36836
Keywords: Enzyme activity assay, Rubisco, Radiometric assay, Crop improvement
Abstract
The Rubisco activity 14CO2-based assay measures the incorporation of 14CO2 into the acid-stable product 3-phosphoglycerate (3-PGA). This protocol is based on Parry et al. (1997).


Guidelines
  1. Check the "Materials" tab for a list of all the chemicals used in this protocol.
  2. In the "Steps" tab, there is a brief description of the materials and equipment necessary for the protocol execution.
  3. 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.
  4. The references cited are at the end of the "Materials" tab.
Materials
MATERIALS
ReagentBicineMerck MilliporeSigma (Sigma-Aldrich)Catalog #B3876
ReagentMagnesium chloride hexahydrate (MgCl2.6H2O)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M2393
ReagentEthylenediaminetetraacetic acid disodium salt dihydrate (EDTA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #E1644
ReagentBenzamidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #B6506
Reagentε-Aminocaproic acid Merck MilliporeSigma (Sigma-Aldrich)Catalog #A2504
ReagentSodium hydroxide (NaOH)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5881
Reagent2-Mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentDL-Dithiothreitol (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #43819
ReagentPhenylmethanesulfonyl fluoride (PMSF)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7626
ReagentProtease inhibitor cocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9599
ReagentD-Ribulose 1.5-bisphosphate sodium salt hydrate (RuBP)Merck MilliporeSigma (Sigma-Aldrich)Catalog #83895
ReagentSodium bicarbonate [14C] (NaH14CO3)Perkin ElmerCatalog #NEC086H005MC
ReagentPotassium hydroxide (KOH)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5958
ReagentFormic acidHoneywell FlukaCatalog #F0507
ReagentGold star quanta scintillation cocktail MeridianCatalog #QSQ1
ReagentEthanol absolute 99.8 %Fisher ScientificCatalog #10437341

CITATION
Carmo-Silva E, Andralojc PJ, Scales JC, Driever SM, Mead A, Lawson T, Raines CA, Parry MAJ (2017). Phenotyping of field-grown wheat in the UK highlights contribution of light response of photosynthesis and flag leaf longevity to grain yield. Journal of Experimental Botany 68: 3473-3486.
LINK
https://doi.org/10.1093/jxb/erx169

CITATION
Kane HJ, Wilkin JM, Portis AR, Andrews TJ (1998). Potent inhibition of ribulose-bisphosphate carboxylase by an oxidized impurity in ribulose-1,5-bisphosphate. Plant Physiology 117: 1059-1069.
LINK
10.1104/pp.117.3.1059

CITATION
Parry MAJ, Andralojc PJ, Parmar S, Keys AJ, Habash D, Paul MJ, Alred R, Quick WP, Servaites JC (1997). Regulation of Rubisco by inhibitors in the light. Plant, Cell & Environment 20: 528-534.
LINK
https://doi.org/10.1046/j.1365-3040.1997.d01-85.x

CITATION
Sharwood RE, Sonawane BV, Ghannoum O, Whitney SM (2016). Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry. Journal of Experimental Botany 67: 3137-3148.
LINK
https://doi.org/10.1093/jxb/erw154

CITATION
Wong C-H (1980). Practical enzymatic syntheses of ribulose 1,5-bisphosphate and ribose 5-phosphate. Journal of the American Chemical Society 102: 7938-7939.
LINK
https://doi.org/10.1021/ja00547a023







Safety warnings
Work with radiation should follow local safety procedures.
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)
  • Dry block heating system (Grant Instruments QBD4)
  • Vortex
  • Chronometer
  • Fume hood
  • Pipette set
  • Mortar and pestle
  • Glass vials for liquid scintillation counting (Perkin Elmer 6000167, 7 mL)
  • 1.5 mL microtubes
REAGENTS & SOLUTIONS
REAGENTS & SOLUTIONS

REAGENTS & SOLUTIONS TO PREPARE BEFOREHAND


Basic extraction buffer (1x)
Concentration50 millimolar (mM) Bicine-NaOH Ph8.2
Concentration20 millimolar (mM) MgCl2.6H2O
Concentration1 millimolar (mM) EDTA
Concentration2 millimolar (mM) Benzamidine
Concentration5 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:
Concentration50 millimolar (mM) 2-Mercaptoethanol
  • Adjust for the final volume; it can be dispensed in aliquots (e.g. 50 mL Falcon tubes).
Temperature-20 °C (storage)

Concentration1 Molarity (M) DTT
  • Dissolve in ultrapure H2O. Temperature4 °C (storage)

Concentration100 millimolar (mM) PMSF
  • Dissolve in ethanol 99%. Temperature4 °C (storage)
Plant protease inhibitor cocktail
Temperature-20 °C (storage)
Basic assay buffer (2x)
Concentration200 millimolar (mM) Bicine-NaOH
Concentration40 millimolar (mM) MgCl2.6H2O
  • Dissolve in ultrapure H2O; adjust pH to 8.2 with NaOH; adjust for the final volume; degas the solution bubbling with nitrogen (5 min/100 mL). It can be dispensed in aliquots (e.g. 50 mL Falcon tubes).
Temperature-20 °C (storage)

Concentration30 millimolar (mM) RuBP
Temperature-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).


Concentration0.1 Molarity (M) NaH14CO3 (0.5 Ci/mol)
Temperature-20 °C (storage)
Concentration0.3 Molarity (M) KOH
TemperatureRoom temperature (storage)

Concentration10 Molarity (M) Formic acid
TemperatureRoom temperature (storage)
Gold Star Quanta scintillation cocktail
TemperatureRoom temperature (storage)
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)
Concentration10 millimolar (mM) DTT (from step 1.2)
Concentration1 millimolar (mM) PMSF (from step 1.3)
Concentration1 % (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. TemperatureOn 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. It is important to ensure that the Rubisco concentration in the assays does not compromise the sensitivity of the assays (e.g. too much would consume all the substrate too quickly).


Complete assay buffer (volume per vial or reaction)

Amount250 µL 2x Basic assay buffer (from step 1.5) - final concentration in the solution = 1x
Amount50 µL 100 mM NaH14CO3 (from step 1.7) - final concentration in the solution = 10 mM
Amount165 µL ultrapure H2O
  • Prepare the volume considering that the activities are measured in duplicates (2 technical replicates).
  • If the goal is to assay Rubisco initial and total activities, for each sample extract, it is necessary to have the volume for 4 vials (2 initials + 2 totals).
  • In addition, account for additional volume for at least two more vials for Blanks (background counts, in the asbence of RuBP), plus one extra (to have a little excess).
Note
Example for 10 extractions: 10x (2xInitial + 2xTotal activity assays) + 2xBlanks + extra = volume for 43 vials.

Note
Concentration2 millimolar (mM) KH2PO4 can be added in the complete assay buffer for field samples to maximise the measurable Rubisco activity (Carmo-Silva et al., 2017).

PROCEDURE
PROCEDURE
START

  • Thaw the frozen solutions that will be used in the day.
  • Turn on the heat block and set to the temperature to be used for Rubisco activity measurements.
Note
The temperature to be used for the Rubisco activity measurement depends on the experiment goals. Typical measurement temperatures are Temperature25 °C (standard) and Temperature30 °C , depending on the species. Assays can be performed at a range of temperatures, however special care should be taken at high temperatures (e.g. Temperature50 °C ), as this can lead to evaporation of the assay mix; rates will be faster, i.e. the assay might become less sensitive; and may cause difficulty in handling hot vials.


  • Turn on the centrifuge and set to Temperature4 °C .
  • Collect samples from Temperature-80 °C into liquid nitrogen.
  • Prepare the complete extraction buffer (step 2.1) and the complete assay buffer (step 2.2) and keep it TemperatureOn ice .

EXTRACTIONS & RUBISCO ASSAYS
  • Immediately before starting the extraction, place 4 vials in the heat block (for 2 initial and 2 total activity assays).
  • Add Amount465 µL of complete assay buffer (from step 2.2.) to each of the 4 vials.
  • Add Amount10 µL of 30 mM RuBP (from step 1.6) to the 2 vials for initials.

Extraction

  • Add the complete extraction buffer to an ice-cold mortar.
  • Take a sample from the liquid nitrogen container and add to the mortar.
  • Grind the sample thoroughly for Duration00:00:30 to maximum of Duration00:01:00 .
  • Collect the homogenate into an ice-cold 1.5 mL microtube and centrifuge Centrifigation14000 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 with the Rubisco assays straight away.
  • Add Amount25 µL of sample extract consecutively to each of 4 vials, with 15 s intervals (Total1, Total2, Initial1, Initial2). All additions are completed within 1.5 min of finishing centrifugation.

Note
The Initial activity assays start with extract addition, while the 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.

  • Quench the assays after 30s by adding Amount100 µL 10 M formic acid (from step 1.9).
  • A possible time-line for the assays would be:


  • In the interval between quenching initial activities and initiating the reaction for total activities, it is possible to prepare the vials for the next assays (steps 1 and 2 in this table).
  • Repeat for all the extractions of the day.
  • Blanks can be prepared at the start or end of the day, by adding to each vial Amount465 µL of complete assay buffer (from step 2.2.) and Amount100 µL 10M formic acid (from step 1.9).
  • In addition, it is useful to prepare at least two background checks per experiment by adding to each vial Amount465 µL of complete assay buffer (from step 2.2.) , Amount25 µL sample extract, and 3 minutes later Amount100 µL 10M formic acid (from step 1.9) to test for background levels of carboxylation due to RuBP that may be present in the leaf extracts. In our hands, this tends to be negligible.
Note
We typically do a simple test to verify the total amount of radioactivity present in the complete assay buffer, prepared just before starting the extractions. This serves to verify that the amount of radioactivity in the solution is reliable and comparable accross days of assays. For this,
Add Amount490 µL 0.3 M KOH (from step 1.8) plus Amount10 µL complete assay buffer (from step 2.2). Add Amount3.6 mL Gold Star Quanta scintillation cocktail (from step 1.10). Close the vial as soon as possible and mix well.


  • Dry all the vials at Temperature100 °C in the heat block (it takes approximately Duration01:00:00 ).
  • Let vials cool, then add Amount400 µL ultrapure H2O to each vial to re-hydrate acid stable compounds. Wait Duration00:05:00
  • Add Amount3.6 mL of Gold Star Quanta scintillation cocktail (from step 1.10). Close the vials and vortex/mix well.
  • Determine 14C radioactivity using a scintillation counter.


CALCULATIONS
CALCULATIONS
Assumptions:
  • NaH14CO3: 0.5 Ci/mol CO2 = 0.5 µCi/µmol CO2
  • 1 µCi = 2220000 disintegration per minute (dpm);

Example:
Blank: 89.03 dpm
Vial 1: 23378.90 dpm

  1. correct vial DPM value by subtracting background counts (Blank): dpm
  2. convert dpm to µCi: µCi
  3. convert to CO2 concentration: μmol CO2

Rubisco activity is then converted to the unit of interest by accounting for the reaction time, the volume of sample extract used and the corresponding sample leaf area (µmol CO2 m-2 s-1) or protein content (µmol CO2 min-1 mg-1).

From the Rubisco activity calculations above for initial (Vi) and total activity (Vt), the Rubisco activation state (AS, %) can be calculated:

Citations
Kane HJ, Wilkin JM, Portis AR, Andrews TJ. Potent inhibition of ribulose-bisphosphate carboxylase by an oxidized impurity in ribulose-1,5-bisphosphate
10.1104/pp.117.3.1059
Sharwood 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/erw154
Wong C-H. Practical enzymatic syntheses of ribulose 1,5-bisphosphate and ribose 5-phosphate
https://doi.org/10.1021/ja00547a023
Carmo-Silva E, Andralojc PJ, Scales JC, Driever SM, Mead A, Lawson T, Raines CA, Parry MAJ. Phenotyping of field-grown wheat in the UK highlights contribution of light response of photosynthesis and flag leaf longevity to grain yield
https://doi.org/10.1093/jxb/erx169
Parry MAJ, Andralojc PJ, Parmar S, Keys AJ, Habash D, Paul MJ, Alred R, Quick WP, Servaites JC. Regulation of Rubisco by inhibitors in the light
https://doi.org/10.1046/j.1365-3040.1997.d01-85.x