Cell Fractionation: Preparation of Chloroplasts

BC2004 Lab Exercise 5 Spring 2005 Cell Fractionation: Preparation of Chloroplasts The green color of many plant organs (primarily leaves and stems) is due to the presence of the green pigments chlorophyll a and chlorophyll b in the subcellular organelles called chloroplasts. The remainder of a plant cell is often colorless. The chloroplasts are found in the cytoplasm of the cell, unattached to other cellular components such as the cell wall, the plasma membrane, the nucleus, and the mitochondria. When the cell wall is disrupted, the plasma membrane breaks, and the subcellular components are released as separate particles of various sizes and densities. In this laboratory, the cells of spinach leaves will be disrupted, freeing the untethered organelles, which can then be sorted out from each other by filtration and differential centrifugation as cell fractions. Filtration will remove large debris (e.g., cell walls) and unbroken cells, providing a filtrate that contains organelles (nuclei, chloroplasts, mitochondria, ribosomes), small membrane vesicles, and soluble components; most of these will not be visible under the compound light microscopes we use in this course. Low-speed centrifugation will sediment remaining large bodies from the filtrate, and moderatespeed centrifugation will sediment chloroplasts, leaving most of the mitochondria, ribosomes, and soluble components in the supernatant. (The mitochondrial fraction could be collected by highspeed centrifugation, and ribosomal and membrane vesicle fractions could be collected by ultrahigh-speed centrifugation.) Repeated rounds of differential centrifugation can be used to further purify the chloroplasts when highly purified preparations are required for experimentation, but one round of low-then-moderate centrifugation will suffice for the purposes of this exercise. In today s laboratory, you will prepare a crude suspension of chloroplasts and determine several characteristics of the preparation. You will measure the chlorophyll a and b content of the suspension, count the chloroplasts per unit volume of the suspension, and use those data to estimate the quantities of chlorophylls a and b and total chlorophyll per chloroplast BC 2004, Spring 2005, Lab exercise 5-1

Procedure (work in pairs during today s lab; if there is an odd number of students in your lab section, have one group of three) **Keep all tissue and fractions ice-cold throughout the procedure.** [When you prepare your protocol outline, don t forget that you need to allow the spectrophotometers to warm up for about 15min before using them.] A. Chloroplast separation by differential centrifugation 1. Obtain 8 grams of deveined leaf tissue. Rinse it in ice water, blot it dry (it does not need to be completely dry) using clean paper towels, and cut it into pieces about 1 cm square. Why do you think the veins might be removed from tissue used for a chloroplast preparation? 2. Place the leaf pieces in a prechilled blender cup containing 40ml of ice-cold 0.5 M sucrose (use a graduated cylinder to measure this amount, having the bottom of the meniscus at the 40mL mark). Blend for 15 sec at top speed, pause about 10 sec, then blend again for 10 sec. Why do you think it is best to keep everything well chilled? What is the advantage of having the cells broken open under cold conditions? 3. Remove (and discard) the ice from the 100-ml beaker, then squeeze the leaf homogenate through four layers of prechilled cheesecloth into the cold beaker by twisting the top corners of the cloth around each other. Use your gloved fingers to accomplish the twisting (it will be a bit messy, but you can throw the gloves away when you are finished). What are you filtering out during this step? 4. Pour 14 ml of the homogenate into each of two 15-ml centrifuge tubes and centrifuge at 200 x g for 5 min. (Don t forget to balance the centrifuge by having tubes of the same volume/weight directly across from each other.) What is being collected in the pellet during this low-speed spin? 5. Using a Pasteur pipet, transfer each supernatant (containing the chloroplasts) to a second, clean 15-ml centrifuge tube (be careful not to disturb the pellet; it is better to leave a little supernatant behind than to get pieces of the pellet), and centrifuge at 1,000 x g for 7 min. (Save the pipet on a piece of clean paper towel.) What is being collected in the pellet during this moderate-speed spin? How and why is it different than the materials collected during the low-speed spin? 6. Using the Pasteur pipet you saved from step 5, remove and discard the supernatants [what is in the supernatants?], being careful not to disturb the pellets. Pipet 2 ml (using a 5-mL graduated pipet) of phosphate buffer onto each pellet and gently resuspend the chloroplasts by moving the liquid up and down in the pipet leave some liquid in the pipet at all times to avoid the formation of bubbles. Combine the two suspensions into one of the tubes, and discard the empty tube. Why should you be gentle during resuspension? Why should you avoid bubbles? BC 2004, Spring 2005, Lab exercise 5-2

Name 7. Using a clean Pasteur pipet, add phosphate buffer to a total volume of 8 ml (using the markings on the side of the tube), and mix the diluted suspension by gently moving it up and down in the pipet. What do you predict is present in this tube? Why is it important to be gentle during your resuspension? This is your chloroplast suspension. Use this suspension for parts B and C. B. Estimation of chlorophyll a and b concentrations of the suspension. 1. Measure 4.75 ml of 80% acetone into a 13 x 100 mm tube. What will the acetone do to the chloroplast membranes and the pigments therein? Why don t you need to keep things cold after adding the acetone? 2. Add 0.25 ml of your chloroplast suspension and mix well. What is the dilution factor? Why don t you need to be gentle any more? 3. Using a vis-specrophotometer (the Spec 20, not the UV-spec), read the absorbance at 663 and 645nm, using a reference blank of (4.7 5ml acetone+ 0.25 ml phosphate buffer why use this particular blank?). Don t forget to zero the spec before using and to blank the spectrophotometer after changing the wavelength. A 663 = (Why read absorbance at 663nm?) A 645 = (Why read absorbance at 645nm? BC 2004, Spring 2005, Lab exercise 5-3

C. Determination of chloroplast concentration of the suspension. 1. Measure 4.75 ml of the phosphate buffer into a clean 13 x 100mm test tube, add 0.25 ml of chloroplast suspension, and mix well. What is your dilution factor? 2. Prepare the clean, dry hemacytometer with a cover slip in place supported by the frosted-glass shoulders of the chamber (your instructor and/or TA can help you with this). A hemacytometer was designed to perform blood counts (of different cellular components of blood). Why is it so useful for counting chloroplasts? 3. Making certain that the chloroplasts are evenly suspended (not settled or clumped), take up some of the suspension into a clean Pasteur pipet; let part of a droplet from the pipet tip flow under the cover slip of the chamber. When properly delivered, the liquid will fill the space between cover slip and etched surface of the chamber and will not overflow into the side troughs beneath the cover slip. (Helpful hint: have a KimWipe ready, folded to a point; if the chamber fills, but there is still a droplet standing at the side of the cover slip, pull it up gently into the point of the tissue, taking care not to pull any liquid from under the cover slip.) If overflow nevertheless occurs, re-clean and dry the chamber and cover slip, wiping finally with alcohol and a KimWipe, and begin again with another sample of suspension. 4. Using the 40x objective (what is the total magnification?), count the total number of chloroplasts in five sections of the large central square of the counting chamber the square that is bounded by a triple-line border and is itself subdivided into 25 sets of 16 very small squares. Count the chloroplasts in the five sections indicated in the diagram below: number of chloroplasts in 5 sections of the chamber = x 5 to estimate number of chloroplasts in counting chamber = BC 2004, Spring 2005, Lab exercise 5-4

5. Draw and label a large diagram of a chloroplast in the space below. You may wish to use to your textbook and/or recitation notes to help you with your drawing and labeling of the following: outer chloroplast membrane, inner chloroplast membrane, stroma, stroma thylakoid, grana, and grana thylakoid. Specifically where do the light-dependent reactions of photosynthesis occur? Specifically where do the light-independent reactions of photosynthesis occur? Even more specifically, where within the chloroplast are the chloroplast pigments located? 6. After counting the chloroplasts in your sample, make a note of the purity of your chloroplast suspension. Are there other organelles present in your suspension? It is unlikely that you ll be able to identify other organelles, but describe them using general terms. What color are they? Are they bigger or smaller than the chloroplasts? What are their approximate proportions in your sample (not their dimensions, but is your sample ~25% tiny yellow triangle shaped organelles?) BC 2004, Spring 2005, Lab exercise 5-5

Calculations Name B. Estimation of chlorophyll concentrations of the suspension. A 663nm of diluted chloroplast suspension: A 645nm of diluted chloroplast suspension: Reminder from Lab Exercise 1: The basis of this application of spectrophotometry is that the proportion of light of a given wavelength that is absorbed by a solution of a particular compound is a function of the concentration of the solute. This allows quantitative analysis of concentration of a substance from the Beer-Lambert relationship (below). A spectrophotometer directs light of a specific wavelength into the solution. This light is the incident light. The light that passes through the solution is the transmitted light. The Absorbance, A, of the solution is the logarithm of the ratio of these two intensities: A (Absorbance) = log 10 (Intensity of incident light / Intensity of transmitted light) The spectrophotometer will calculate and display the Absorbance value. From the Absorbance, the concentration of the solute can be calculated from the Beer-Lambert equation: where: A = E x C x L E (Extinction Coefficient) = Absorbance of a l mm solution of the substance measured through a l-cm light path. This is a constant for each substance at a given wavelength. C = molar concentration, in mmoles/liter. L = length of the light path through the solution, in cm. For the spectrophotometer you will be using, L is equal to 1 cm, so that C = A / E. The proportion of light of a given wavelength that is absorbed by a solution of a particular compound is a function of the concentration of the solute. Knowing the extinction coefficients for chlorophyll a and b, the path length of the spectrophotometer, and the absorbance of solutions of chlorophyll a and b, we could easily use the Beer-Lambert equation to calculate the concentrations of pigment in those solutions. The matter is complicated in this lab exercise by having in the solution two pigments with overlapping absorption spectra. Next week we will learn a method for physically separating the BC 2004, Spring 2005, Lab exercise 5-6

two pigments, but this week we can calculate the pigment concentrations by measuring the absorbance at two different wavelengths for which the extinction coefficients of each pigment are known. The maximum absorption for chlorophyll b occurs at about 645 nm and for chlorophyll a at about 663 nm. Total absorption (A) at a wavelength at which both pigments absorb will depend on the concentration (C) and extinction coefficient (E) of each pigment. In today s lab, we can calculate concentrations because the extinction coefficients are known. A 645 nm = [(E chl a/645 )(C chl a )(L)] + [(E chl b/645 )(C chl b )(L)] equation 6-1 Extinction coefficients at 645 nm: chl a = 16.75 ml/cm mg chl b = 45.6 ml/cm mg A 663nm = [(E chl a/663 )(C chl a )] + [(E chl b/663 )(C chl b )] equation 6-2 extinction coefficients at 663 nm: chl a = 82.04 ml/cm mg chl b = 9.27 ml/cm mg. We can use the given extinction coefficients and the path length of 1.0 for our spectrophotometers to rewrite these equations as follows: A 645 nm = (16.75) (C chl a ) + (45.6) (C chl b ) A 663nm = (82.04) (C chl a ) + (9.27) (C chl b ) equation 6-3 equation 6-4 Finally, these equations can be solved to allow us to calculate the concentration of each chlorophyll using the absorbance values measured in lab: C chl a = 0.0127A 663-0.00269A 645 C chl b = 0.0229A 645-0.00468A 663 equation 6-5 equation 6-6 Thus, the total chlorophyll in mg/ml = C chl a + C chl b = 0.0202A 645 + 0.00802A 663. equation 6-7 BC 2004, Spring 2005, Lab exercise 5-7

Name Using these equations, you can (and will!) calculate the concentrations of the pigments in your diluted chloroplast suspension: C chl a/diluted (mg/ml) = 0.0127A 663-0.00269A 645 equation 6-8 C chl b/diluted (mg/ml) = 0.0229A 645-0.00468A 663 equation 6-9 C total chl /diluted (mg/ml) = C chl a + C chl b = 0.0202A 645 + 0.00802A 663 equation 6-10 Next, using your calculated dilution factor (from B.2. on page 5-3) in an appropriate manner, calculate the concentrations of these pigments in the undiluted chloroplast suspension. (You should remember how to do these calculations from the first lab of the semester.) C Chla/undiluted (mg/ml)= C Chlb/undiluted (mg/ml)= C total chl /undiluted (mg/ml) = BC 2004, Spring 2005, Lab exercise 5-8

Name C. Determination of chloroplast concentration of the suspension. number of chloroplasts in chamber = (already multiplied by 5) The volume contained in the chamber over the large square in which you counted chloroplasts was 0.1 µl, or 10-4 ml. Therefore, you estimated the number of chloroplasts in 10-4 ml, and you calculated the dilution factor for this sample (is this the same dilution factor used directly above?). You can now calculate the concentration of chloroplasts/ml of undiluted suspension as: number of chloroplasts x 1/D.F. = number of chloroplasts/ml of undiluted suspension 10-4 ml equation 6-11 x 1/ = chloroplasts/ml of undiluted suspension 10-4 ml D. Combine calculations B and C to calculate the quantities of chlorophylls per chloroplast: mg chl/ml chloroplast suspension number of chloroplasts/ml suspension = mg chl/chloroplast equation 6-12 mg chl a/ml = mg chl a/chloroplast chloroplasts/ml mg chl b/ml = mg chl b/chloroplast chloroplasts/ml mg chl total/ml = mg chl total/chloroplast chloroplasts/ml BC 2004, Spring 2005, Lab exercise 5-9

Name Finally, convert mg to an appropriate unit of mass, a unit that will allow expression of the calculated value as a number between 0.1 and 100 (e.g., as 20pg or 5mg or 0.6g), using the following relationships as needed. 10 12 fg = 10 9 pg = 10 6 ng = 10 3 µg = 1 mg = 10-3 g = 10-6 kg mg chl a/chloroplast = chl a/chloroplast (unit) mg chl b/chloroplast = chl b/chloroplast (unit) mg chl total/chloroplast = chl total/chloroplast (unit) Is there more chlorophyll a or b in the chloroplasts? Keep your answer to this question in mind next week when you study the characteristics of the pigments in more detail. Remember that you will be responsible for understanding the techniques you used in lab this week and for performing calculations like these on your exams. BC 2004, Spring 2005, Lab exercise 5-10

BC2004 Materials and Laboratory Preparation Information Lab 5 There should be a supply of extra reagents available in the refrigerator in case of a problem (if a student spills something, she needs to be able to get more.) All reagents need to be clearly and legibly labeled, preferably using computer-printed labels. General supplies: gloves of various sizes, replenished throughout the week as needed. Table top centrifuge (with conversion from rpm to g if needed; students have instructions in g, so if centrifuge is only marked in rpm, they need to be able to convert). KimWipes (one box per group of 4), ethanol (one small squirt bottle per group of 4), Parafilm (preferably cut into strips or with scissors available for students to cut their own). Spectronic 20 (as many as we have functioning). Per pair of students: crushed ice in ice bucket ON ICE: 1 100-ml beaker containing crushed ice 8g deveined leaf tissue rinsed in ice water, blotted, and cut into pieces about 1 cm square students can do the rinsing, blotting, and cutting into small pieces, but leaves should be pre-weighed and de-veined (in plastic wrap or small baggies) 45ml 0.5 M sucrose, ice-cold 15 ml phosphate buffer, 0.1 M, ph 6.5, ice-cold 100 ml water, ice-cold four-layered cheesecloth, ~5 inches square, prechilled (wrapped in plastic wrap and placed on ice) 4 15-ml centrifuge tubes, conical, graduated (yes, these should be on ice); must be able to withstand 1000 x g centrifugal force. do not need to be on ice, but needed per PAIR of students: 10 ml acetone, 80 % (acetone will eat the labels off of tubes, so be careful with labeling), does not need to be chilled 4 test tubes, 13 x 100 mm, (as cuvettes) (plus a generous supply at the front of the lab) 4 Pasteur pipets, 4 bulbs (plus a generous supply at the front of the lab) 2 5-ml pipets (plus a generous supply at the front of the lab) and large pump 2 1-ml pipets (plus a generous supply at the front of the lab) and small pump 1 Sharpie pen 1 pair of scissors 1 50-mL graduated cylinder We won t have enough for all pairs of students: blender cup and blender: the cup needs to be prechilled. We will not have enough of these for each pair of students to have their own. Please put out all available blenders and small blender cups (more might be able to be borrowed from elsewhere in the department). hemacytometer, cover slips (we ll have enough regular cover slips, but probably not enough hemacytometers, please distribute evenly between sections) BC 2004, Spring 2004, Lab exercise 5-11