TITLE: Carbonic Anhydrase Activity Assay
AUTHORS: Jack Koch, Virginia Weis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Carbonic anhydrase (CA) is an enzyme which catalyzes the interconversion of bicarbonate ions (HCO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span>) and carbon dioxide (CO</span><span style = "vertical-align:sub;">2</span><span>). The activity levels of CA can be measured by taking advantage of the chemical reaction that it catalyzes. When CO</span><span style = "vertical-align:sub;">2</span><span> is added to a tissue homogenate, the CA present converts the CO</span><span style = "vertical-align:sub;">2</span><span> into HCO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span> and a hydrogen ion (H</span><span style = "vertical-align:super;">+</span><span>), which results in a decrease in pH. The rate at which the pH drops can be measured and converted into a CA activity mesaure. If there is no CA activity, the pH level decreases slowly and if there is a high CA activity, the pH decreases quickly.</span></div></div>

[STEPS]
?. [Mixing Carbonic Anhydrase (CA) Buffers]
Stock 50mM barbital buffer
?. [Mixing Carbonic Anhydrase (CA) Buffers]
25 mM Veronal buffer (Buffer A)Mix 250mL of 50mM barbital buffer with 250mL of deionized waterAdjust pH of Buffer A to 8.2 with 1M HCl
?. [Mixing Carbonic Anhydrase (CA) Buffers]
Extraction bufferMix 250mL 50mM barbital buffer with 250mL of deionized waterAdd 0.93g EDTAAdd 1.23g MgSO4Stir solution until EDTA and MgSO4 are completely dissolvedAdjust pH of Extraction buffer to 8.2 with 1M NaOH
?. [Preparing your workspace]
First, take out three new 1 ml pipette tips. At the top edge of the tip, mark one with an “A” to use with Buffer A only, one with “E” to use with Extraction Buffer only, and the third with “C” to use with  CO2-saturated seawater only. Reuse these pipette tips on every sample you run, and be very careful that they don’t get contaminated by accidentally using them on other solutions. You will need to use new pipette tips for each anemone native and denatured samples.
?. [Prepare CO2-Saturated DI Water]
Prepare two flasks of CO2 saturated DI water in case the pH in one is not low enough when you are ready to begin assays. The pH of your water needs to be below 4.00. (go to steps 7-11 or 12-15)
?. [Preparing your workspace]
Calibrate the pH probe daily according to manufacturers specifications.
?. [Prepare CO2-Satured DI Water with CO2 Tank (Preferred Method)]
Bubble CO2 using an air stone into a 500 mL flask of 300 mL of chilled DI water for 15 minutes
?. [Prepare CO2-Satured DI Water with CO2 Tank (Preferred Method)]
Twist in the rubber stopper into the flask and let the CO2 equilibrate for 15 minutes.
?. [Prepare CO2-Satured DI Water with CO2 Tank (Preferred Method)]
Measure and record the pH of the water.
?. [Prepare CO2-Satured DI Water with CO2 Tank (Preferred Method)]
Keep your CO2 flask on ice (or the CO2 will start coming out of solution).
?. [Prepare CO2-Satured DI Water with CO2 Tank (Preferred Method)]
Once the necessary pH has been reached, keep the stopper tight on the flask to reduce CO2 offgassing.
?. [Prepare CO2-Satured DI Water with Dry Ice]
If a CO2 tank is unavailable, you can add dry ice to flask. Drop 4 or 5 half-dollar sized chunks of dry ice into the flask and place the rubber stopper on top loosely to allow excess pressure to escape the flask.
?. [Prepare CO2-Satured DI Water with Dry Ice]
Once the dry ice has dissolved completely, measure the pH of the water.
?. [Prepare CO2-Satured DI Water with Dry Ice]
If the pH is not low enough, add more dry ice and repeat.
?. [Prepare CO2-Satured DI Water with Dry Ice]
Once the necessary pH has been reached, keep the stopper tight on the flask to reduce CO2 offgassing.
?. [Run a Negative Control Daily, Before You Run Your Samples]
Run the assays in a 6-12 mL sample vial/shell vial (Thomas Scientific 60975L-3, 224744, 224745, 60965D-1, or 60965D-3) (referred to as "beaker" below) equipped with little stir bars (either custom made from pasteur pipet and piece of staple or tiny bars from science supplier). The beaker should be in ice during measurement to keep the reaction cold.
?. [Run a Negative Control Daily, Before You Run Your Samples]
Rinse out the beaker with 1 mL “Buffer A.” Dump into waste flask and do not lose the stir bar! If you lose the stir bar, wash it with DI water and repeat the Buffer A wash step.
?. [Run a Negative Control Daily, Before You Run Your Samples]
Add 2 mL “vernol Buffer A” to the beaker (using the “A” pipette tip).
?. [Run a Negative Control Daily, Before You Run Your Samples]
Place the beaker on the stir plate. Insert the pH probe. Ensure that the probe is submerged and that the stir bar is not hitting the probe.
?. [Run a Negative Control Daily, Before You Run Your Samples]
Hit “read/log” then quickly add 1 mL CO2-saturated seawater to the beaker, and watch the pH slowly drop as the CO2 offgasses.
?. [Run a Negative Control Daily, Before You Run Your Samples]
Wait at least 120 seconds before stopping the reading to ensure that the probe is reading ~-0.01 pH/second (normal CO2 offgassing).
?. [Run a Negative Control Daily, Before You Run Your Samples]
Rinse the beaker with DI water and empty into the waste beaker. Be carfeul not to lose the stir bar.
?. [Run a Negative Control Daily, Before You Run Your Samples]
Transfer the data onto flash drive or onto the computer log.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
Label two 15 mL tubes (“Anemone ID” + native or denatured) and two 1.5 mL tube (PROT or ALGAE + “Anemone ID”)
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
For A. elegantissima remove between a half and an eighth of the anemone for the assay (a half for very small anemones and an eighth for very large anemones). The minimum amount of material used for this assay should be about 0.2 grams frozen material. If you are concerned that you do not have enough material, try a few practice samples using the amount of material you plan to use for real samples.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
From the piece of anemone that will not be used for the assay, make a diagonal cut from the middle of the oral cavity to the outer edge of the anemone and place the slice into the algae 1.5 ml snap tube. Place this tube into the freezer for analysis later.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
Place the rest of the anemone back into the freezer.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
Finely mince anemone with a razor blade, then place the anemone in a glass-teflon tissue homogenizer with 3 mL of “extraction buffer.”
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
Keeping the grinder in ice, grind the anemone for 1-1.5 minutes (there should be no large tissue chunks). With the pestle still in the homogenizer, pour the homogenized anemone into the Anemone ID labeled 15 mL tube. Keep this tube on ice.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
Rinse the tissue grinder with 4 mL “extraction buffer”, swish, grind a few more times, and add to the anemone homogenate. You should have 7 mL of homogenate.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
Vortex the homogenate briefly. Centrifuge the homogenate at 4˚C at 4300 RCF for 10 minutes to pellet algae and large cellular debris. Don’t forget to balance the centrifuge.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
Carefully pour the supernatant into the Native 15 mL tube. You may want to freeze the cellular debris/algae pellet for future analysis.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
Use a P1000 pipettor to add 3 mL of the supernatant to the 15 mL Denatured tube.
?. [EXTRACT ANEMONE CA FROM ANEMONES (This can be applied to other organisms as well).]
You need 3 mL of homogenate in the Native tube. Remove between 200 µL and 1 mL of the homogenate and add to the “PROT” labeled 1.5 mL tube. Place the 1.5 mL tube in the designated box in the freezer for later protein analysis.
?. [PREPARE DENATURED SAMPLES]
Fill a 1000 mL beaker with ~900 mL of DI water and place on a hot plate. Let the water reach a boil. The high temperature denatures proteins, and as a result, inactivates enzymes (including CA).
?. [PREPARE DENATURED SAMPLES]
Take the tubes labeled “denatured” and loosen the cap very slightly to allow the pressure to equilibrate.
?. [PREPARE DENATURED SAMPLES]
Insert the tube into a hole on a float, and let the float sit on top of the beaker of boiling water. Make sure the homogenate is submerged in the water.
?. [PREPARE DENATURED SAMPLES]
Keep the denatured tube in the boiling water for five minutes.
?. [PREPARE DENATURED SAMPLES]
Remove the tube from the water and allow the tubes to cool on ice for five minutes.
?. [RUN NATIVE SAMPLES]
Rinse out the beaker with 1 mL “Buffer A.” Dump into waste flask and do not lose the stir bar!
?. [RUN NATIVE SAMPLES]
Add 1 mL “vernol Buffer A” to the beaker (using the “A” pipette tip).
?. [RUN NATIVE SAMPLES]
Briefly vortex/pipette mix your native sample homogenate. Add 1 mL of your native sample homogenate to the beaker.
?. [RUN NATIVE SAMPLES]
Place the beaker on the stir plate. Insert the pH probe. Ensure that the probe is submerged and that the stir bar is not hitting the probe.
?. [RUN NATIVE SAMPLES]
Hit “read” then quickly add 1 mL CO2-saturated seawater to the beaker, and watch the pH slowly drop as the CO2 is converted to HCO3- and H+ ions.
?. [RUN NATIVE SAMPLES]
Rinse the beaker with DI water and empty into the waste beaker. Be carfeul not to lose the stir bar. Repeat this three times.
?. [RUN NATIVE SAMPLES]
Repeat steps 40-45 two more times for the same sample so that you end up with three replicates from the native sample.
?. [RUN DENATURED SAMPLES]
Rinse out the beaker with 1 mL “Buffer A.” Dump into waste flask and do not lose the stir bar!
?. [RUN DENATURED SAMPLES]
Add 1 mL “vernol Buffer A” to the beaker (using the “A” pipette tip).
?. [RUN DENATURED SAMPLES]
Briefly vortex/pipette mix your denatured sample homogenate. Add 1 mL of your denatured sample homogenate to the beaker.
?. [RUN DENATURED SAMPLES]
Place the beaker on the stir plate. Insert the pH probe. Ensure that the probe is submerged and that the stir bar is not hitting the probe.
?. [RUN DENATURED SAMPLES]
Hit “read” then quickly add 1 mL CO2-saturated seawater to the beaker, and watch the pH slowly drop as the CO2 is converted to HCO3- and H+ ions.
?. [RUN DENATURED SAMPLES]
Rinse the beaker with DI water and empty into the waste beaker. Be carfeul not to lose the stir bar. Repeat this three times.
?. [RUN DENATURED SAMPLES]
Repeat steps 47-52 two more times for the same sample so that you end up with three replicates from the native sample.
?. [DATA ANALYSIS]
The data you will produce will be numerous pH values and a time stamp.
?. [DATA ANALYSIS]
You will be calculating the slope of each run and taking the steepest slope between two data points. Then you will average the slopes of your triplicate replicates. See my example below:RAW DATA  ABCDEFG1  Time (s)    Native 1    Native 2    Native 3    Denatured 1    Denatured 2    Denatured 3  2  0    8.00    8.1    8.00    8.00    8.1    8.0  3  1    7.5    7.5    7.5    7.9    7.9    7.9  4  2    7.2    7.0    7.2    7.8    7.8    7.8  5  3    7.1    6.9    7.1    7.7    7.6    7.7  6  4    7.0    6.8    7.0    7.6    7.5    7.6  7  5    6.9    6.7    6.9    7.5    7.4    7.5  8  6    6.8    6.6    6.8    7.4    7.3    7.4
ABCDEFG1  Time (s)    Native 1    Native 2    Native 3    Denatured 1    Denatured 2    Denatured 3  2  0    8.00    8.1    8.00    8.00    8.1    8.0  3  1    7.5    7.5    7.5    7.9    7.9    7.9  4  2    7.2    7.0    7.2    7.8    7.8    7.8  5  3    7.1    6.9    7.1    7.7    7.6    7.7  6  4    7.0    6.8    7.0    7.6    7.5    7.6  7  5    6.9    6.7    6.9    7.5    7.4    7.5  8  6    6.8    6.6    6.8    7.4    7.3    7.4
?. [DATA ANALYSIS]
Slope = (y2-y1)/(x2-x1)The x variable is time (in seconds)The y variable is pH
?. [DATA ANALYSIS]
Calculate the slope and find the largest change (min value since it it negative)  ABCDEFGHI1  Time
  (s)    Native
  1    Slope
  1     Native
  2    Slope
  2    Native
  3    Denatured
  1    Denatured
  2    Denatured
  3  2  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">0    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.00    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.00    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.00    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.0  3  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.3    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.9  4  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">2    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.2    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.0    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.2    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.8  5  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">3    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.7    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.7  6  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">4    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.0    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.0    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.6  7  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.7    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.4    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5  8  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-NA    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">NA    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.4    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.3    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.4  9  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">   10  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">MIN SLOPE    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">
ABCDEFGHI1  Time
  (s)    Native
  1    Slope
  1     Native
  2    Slope
  2    Native
  3    Denatured
  1    Denatured
  2    Denatured
  3  2  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">0    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.00    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.00    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.00    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">8.0  3  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.3    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.9  4  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">2    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.2    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.0    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.2    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.8  5  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">3    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.7    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.7  6  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">4    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.0    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.0    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.6  7  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.7    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.9    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.4    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.5  8  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-NA    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">NA    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">6.8    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.4    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.3    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">7.4  9  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">   10  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">MIN SLOPE    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">
?. [DATA ANALYSIS]
Average your replicates, calculate raw CA/second and convert to minutes. We convert to minutes since most literature reports CA activity in minutes; so, we can compare our values to other literature.  ABCDEFG1  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     Native
  1    Native
  2    Native
  3    Denatured
  1<span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">    Denatured
  2<span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">    Denatured
  3<span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">  2  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">MIN SLOPE    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.2    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1  3  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">AVERAGE SLOPE    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">Native    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.53    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">Denatured    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.13    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">   4  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">   5  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">RAW CA/SECOND    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">Native - Denature    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.4*-1 =     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">0.4    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">I convert to positive because it is more
  logical when graphing.  6  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">RAW CA/MINUTE    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">0.4 ∆pH/s * 60    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">24 ∆pH/minute    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">
ABCDEFG1  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     Native
  1    Native
  2    Native
  3    Denatured
  1<span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">    Denatured
  2<span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">    Denatured
  3<span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">  2  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">MIN SLOPE    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.6    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.5    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.2    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.1  3  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">AVERAGE SLOPE    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">Native    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.53    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">Denatured    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.13    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">   4  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">   5  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">RAW CA/SECOND    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">Native - Denature    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">-0.4*-1 =     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">0.4    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">I convert to positive because it is more
  logical when graphing.  6  <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">RAW CA/MINUTE    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">0.4 ∆pH/s * 60    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">24 ∆pH/minute    <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">     <span style="font-size:9.0pt;mso-bidi-font-size:12.0pt;
  font-family:&quot;Times New Roman&quot;,serif">
?. [DATA ANALYSIS]
LASTLY, MEASURE PROTEIN FROM YOUR “PROT” SAMPLE. We use a Bradford Protein Assay This should be converted to mg/mL. Then, divide RAW CA/MINUTE by PROTEIN (mg/mL).So in the example above, lets say that the protein was measured to be 10 mg/mL. 24/10 = 2.4 ∆pH/minute/mg animal protein = CA Activity