Questions
Protocol
Select several leaves for each type of leaf and remove the large veins. Weigh out ~5-10 grams of each deveined tissue. Isolate chloroplasts from the two tissues separately using the following protocol.
Chop the tissue into small pieces with a scissors and suspend them in ~50 ml of grinding buffer. Then disrupt the leaf cells using short bursts from a polytron tissue homogenizer while swirling the suspension. Do not over-homogenize or you will break the chloroplasts.
Filter the solution through double-layered cheesecloth, squeezing the tissue pulp to recover the suspension in a 50 ml centrifuge tube (if you have more than 50 ml of suspension you may discard the extra).
Centrifuge at 1000 rpm for 1 minute to pellet unbroken cells and fragments.
Decant the supernatant into a clean centrifuge tube and recentrifuge at 1500-2000 rpm for 7 minutes. The pellet formed during this centrifugation contains chloroplasts. Decant and discard the supernatant.
Resuspend the chloroplast pellet in 5.0 ml of cold suspension buffer. Use a glass stirring rod to gently disrupt the packed pellet. This is the chloroplast suspension for use in subsequent procedures. Store the suspension in the dark at 4˚C. Be sure the suspension is well mixed before removing samples from it.
Determine the concentration of chloroplasts (chloroplasts/ml of suspension buffer) using a hemocytometer. Record the results.
Determine the concentration of chlorophyll in your chloroplast solution. Use suspension buffer to blank the spectrophotometer at a wavelength of 652 nm. You may need to dilute the solution (often by a factor of 10) to get a reading in the linear range of the spectrophotometer (roughly 0.02-1.0 absorbance units).
The conversion factor is: (35 mg chlorophyll / ml) / (Absorbance652 unit). Be sure to account for any dilutions you performed.
Calculate the average amount of chlorophyll per chloroplast (the average chloroplast volume is 0.6 fl). Record your results on the board so the values for the entire class can be compiled.
Grinding solution: 0.33 M sorbitol, 10 mM sodium pyrophosphate (NaP2O7), 4 mM MgCl2, 2 mM ascorbic acid, pH 6.5
Suspension solution : 0.33 M sorbitol, 2 mM EDTA, 1 mM MgCl2, 50 mM HEPES, pH 7.6
Homework Questions
1. What would happen to the chloroplasts if you resuspended them in water instead of in suspension buffer?
2. Would the amount of chlorophyll be affected by resuspending the chloroplasts in water? If so, would the measured amount increase or decrease?
3. Which type of tissue contains more chlorophyll per gram of tissue (you can tell this just by looking at which leaf is darker green)?
4. We do not know (and cannot determine from this experiment) how many chloroplasts are in an individual cell in either tissue.
What are the two possible explanations for why one tissue has more chlorophyll than the other (hint- think about how many chloroplasts are present in each possibility)?
5. What was the concentration of chloroplasts in both of your suspensions?
6. What was the concentration of chlorophyll in both of your suspensions?
7. What was the average amount of chlorophyll per chloroplast in each of your suspensions? (also write these values on the board)
8. Using your results only, why do you think one of your samples was darker green than the other (choose one of the answers from question 4) ?
9. Using the results of the entire class perform a t-test to see if the amount of chlorophyll per chloroplast is different between the two tissue types. Report your answer in an appropriate format.
10. Which answer to question 4 is supported by this t-test?
11. How much sorbitol (182 g/mol) do you need to make 25 ml of grinding buffer?
12. EDTA (372 g/mol) comes as a 0.5 M stock solution. What volume do you need to make 5 ml of suspension buffer?
