TITLE: 14CO2-based assay for measuring Rubisco activity & activation state
AUTHORS: Cristina Rodrigues Gabriel Sales, Anabela  Silva, Elizabete Carmo-Silva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The Rubisco activity </span><span style = "vertical-align:super;">14</span><span>CO</span><span style = "vertical-align:sub;">2</span><span>-based assay measures the incorporation of </span><span style = "vertical-align:super;">14</span><span>CO</span><span style = "vertical-align:sub;">2</span><span> into the acid-stable product 3-phosphoglycerate (3-PGA). This protocol is based on Parry et al.</span><span style = "font-style:italic;"> </span><span>(1997).</span></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/b8avbdagf.png" /></div></div>

[STEPS]
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REAGENTS & SOLUTIONS TO PREPARE BEFOREHAND
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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/100 mL), then add:      2-MercaptoethanolAdjust for the final volume; it can be dispensed in aliquots (e.g. 50 mL Falcon tubes). 2-Mercaptoethanol
[(storage)]
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PMSFDissolve in ethanol 99%.
[(storage)]
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Plant protease inhibitor cocktail
[(storage)]
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Basic assay buffer (2x) Bicine-NaOH MgCl2.6H2O
[(storage)]
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RuBP
[(storage)]
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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NaH14CO3 (0.5 Ci/mol)
[(storage)]
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KOH
[(storage)]
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Formic acid
[(storage)]
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Gold Star Quanta scintillation cocktail
[(storage)]
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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. 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).
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DTTDissolve in ultrapure H2O.
[(storage)]
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Complete assay buffer  (volume per vial or reaction)  2x Basic assay buffer (from step 1.5) - final concentration in the solution = 1x 100 mM NaH14CO3 (from step 1.7) - final concentration in the solution = 10 mM ultrapure H2OPrepare 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).
250 µl
50 µl
165 µl
Example for 10 extractions: 10x (2xInitial + 2xTotal activity assays) + 2xBlanks + extra = volume for 43 vials.
KH2PO4 can be added in the complete assay buffer for field samples to maximise the measurable Rubisco activity (Carmo-Silva et al., 2017).
?. [PROCEDURE]
STARTThaw 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.Turn on the centrifuge and set to .Collect samples from  into liquid nitrogen.Prepare the complete extraction buffer (step 2.1) and the complete assay buffer (step 2.2) 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 special care should be taken at high temperatures (e.g.  ), 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.
4 °C
-80 °C
on ice
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EXTRACTIONS & RUBISCO ASSAYS
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Add  of complete assay buffer (from step 2.2.) to each of the 4 vials.Add  of 30 mM RuBP (from step 1.6) to the 2 vials for initials.
465 µl
10 µl
?. [PROCEDURE]
ExtractionAdd 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   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 with the Rubisco assays straight away.Add    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.  Quench the assays after 30s by adding   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   of complete assay buffer (from step 2.2.) and   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   of complete assay buffer (from step 2.2.) ,   sample extract, and 3 minutes later   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. Dry all the vials at   in the heat block (it takes approximately  ).Let vials cool, then add   ultrapure H2O to each vial to re-hydrate acid stable compounds. Wait  Add   of Gold Star Quanta scintillation cocktail (from step 1.10). Close the vials and vortex/mix well.Determine 14C radioactivity using a scintillation counter.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 with the Rubisco assays straight away.Add    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.  Quench the assays after 30s by adding   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   of complete assay buffer (from step 2.2.) and   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   of complete assay buffer (from step 2.2.) ,   sample extract, and 3 minutes later   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. Dry all the vials at   in the heat block (it takes approximately  ).Let vials cool, then add   ultrapure H2O to each vial to re-hydrate acid stable compounds. Wait  Add   of Gold Star Quanta scintillation cocktail (from step 1.10). Close the vials and vortex/mix well.Determine 14C radioactivity using a scintillation counter.Add   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.Quench the assays after 30s by adding  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   of complete assay buffer (from step 2.2.) and   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   of complete assay buffer (from step 2.2.) ,   sample extract, and 3 minutes later   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. Dry all the vials at   in the heat block (it takes approximately  ).Let vials cool, then add   ultrapure H2O to each vial to re-hydrate acid stable compounds. Wait  Add   of Gold Star Quanta scintillation cocktail (from step 1.10). Close the vials and vortex/mix well.Determine 14C radioactivity using a scintillation counter.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   of complete assay buffer (from step 2.2.) and   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   of complete assay buffer (from step 2.2.) ,   sample extract, and 3 minutes later   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. Dry all the vials at   in the heat block (it takes approximately  ).Let vials cool, then add   ultrapure H2O to each vial to re-hydrate acid stable compounds. Wait  Add   of Gold Star Quanta scintillation cocktail (from step 1.10). Close the vials and vortex/mix well.Determine 14C radioactivity using a scintillation counter.Blanks can be prepared at the start or end of the day, by adding to each vial  of complete assay buffer (from step 2.2.) and  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  of complete assay buffer (from step 2.2.) ,  sample extract, and 3 minutes later  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.Dry all the vials at  in the heat block (it takes approximately ).Let vials cool, then add  ultrapure H2O to each vial to re-hydrate acid stable compounds. Wait Add  of Gold Star Quanta scintillation cocktail (from step 1.10). Close the vials and vortex/mix well.
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.
25 µl
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.
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.
100 µl
465 µl
100 µl
465 µl
25 µl
100 µl
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   0.3 M KOH (from step 1.8) plus   complete assay buffer (from step 2.2). Add   Gold Star Quanta scintillation cocktail (from step 1.10). Close the vial as soon as possible and mix well.
100 °C
400 µl
3.6 mL
?. [CALCULATIONS]
Assumptions:NaH14CO3: 0.5 Ci/mol CO2 = 0.5 µCi/µmol CO21 µCi = 2220000 disintegration per minute (dpm); Example:Blank: 89.03 dpmVial 1: 23378.90 dpmcorrect vial DPM value by subtracting background counts (Blank):  23378.9 — 89.03=23289.87 dpmconvert dpm to µCi: 23289.87/2220000=0.010491 µCiconvert to CO2 concentration: 0.010491/0.5=0.0210 μmol CO2Rubisco 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: AS=100×V_i/V_t