BIO 121 LAB INSTRUCTIONS - Wilkes University



Thylakoid extraction

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Chloroplast preparation Use Percoll instead of sucrose and test steps ahead of time (most banded at 1.8/1.3 M sucrose interface).!

Chloroplasts are relatively large and dense, so they sediment rapidly upon centrifugation. Consequently, chloroplasts can be separated from smaller organelles after breaking open leaf cells by giving the brei a short spin at low speeds. Chloroplasts will be in the pellet, while most of the other organelles will be in the supernatant. For many purposes, this is adequate purification. However, nuclei, unbroken cells and other debris will also be in the pellet, so often further purification is needed. Fortunately, these all have different densities from chloroplasts, so we can separate chloroplasts from these contaminants by density gradient centrifugation. There are three ways to do this: 1) you can load your sample suspended in low-density buffer on the top of a tube filled with medium which becomes increasingly dense progressing down the tube. Upon centrifugation your organelles will sediment down the tube until they reach a point where the density of the surrounding medium is equal to their own. 2) you can prepare your sample in high-density medium, then layer media of increasingly lower density on top of your sample. Upon centrifugation your organelles float up until they reach a point where the density of the surrounding medium is equal to their own. 3) you can prepare your sample in a medium which forms a density gradient upon centrifugation. Cesium chloride does this upon ultracentrifugation, and is therefore frequently used for purifying DNA (as you will recall from the Meselson-Stahl experiment). Silica sols such as percoll will also form density gradients upon centrifugation. In this case you prepare your samples in such a medium then spin them long and hard enough for the medium to form a density gradient. Once the contents of the tube have reached equilibrium you collect the portion of the gradient containing chloroplasts.

Unfortunately, there is no best method for preparing chloroplasts. It all depends upon what you plan to do with them. For many purposes chloroplasts prepared by differential centrifugation are adequate, whereas to obtain the purest fractions density gradients are the method of choice. Unfortunately, they also take much longer, and during that time chloroplast integrity is often impaired such that light- and CO2 -dependent photosynthetic activity may be lost.

Measuring photosynthetic activity of isolated chloroplasts

There are three basic approaches to measuring photosynthesis. The first is to measure incorporation of CO2 into sugar. This can be done either by measuring the removal of CO2 from the atmosphere using an instrument called an IRGA (Infra-Red Gas Analyzer) or by measuring the incorporation of radioactive CO2 into sugar. The second approach is to measure light-dependent oxygen evolution. If very high-quality chloroplasts are obtained one can measure light- and CO2-dependent oxygen evolution. Frequently, chloroplast envelopes are disrupted during isolation, allowing critical stromal components such as Calvin cycle enzymes or NADP to leak out. In this case, it is no longer possible to measure CO2-dependent oxygen evolution. However, in such cases it is still often possible to measure light-dependent oxygen evolution provided that another acceptor for the electrons removed from water is provided, and numerous electron acceptors which substitute for various steps in photosynthetic electron transport have been identified. Today we will provide our chloroplasts with ferricyanide as an alternative electron acceptor. If all goes well they will reduce ferricyanide Fe(III)(CN)63- to ferrocyanide Fe(II)(CN)64-.

Photosynthetic electron transport (the Hill reaction)

In 1937 Robin Hill demonstrated that isolated chloroplasts could transfer electrons to an artificial electron acceptor and evolve oxygen in the process when they were illuminated. This showed that electron transport and CO2 fixation (i.e. the light and light-independent reactions) are separate reactions. We now know that what happened was that Photosystem II used light energy to remove electrons from water and donate them to the artificial electron acceptor. The general equation for the Hill reaction is:

light

H2O + A ----------------------> AH2 + 1/2 O2

chloroplasts

where A is an artificial electron acceptor. The specific reaction which we will measure today is:

light

H2O + 2 Fe(CN)63- ----------------------> 2 Fe(CN)64- + 1/2 O2

chloroplasts

THIS WEEK’S ACTIVITIES

This week we will prepare spinach chloroplasts using differential and density-gradient centrifugation. We will grind spinach in a buffer which stabilizes and protects them, then remove large debris by filtering them through cheese-cloth. Next we will separate them from smaller organelles by giving the brei a short spin at low speeds. Chloroplasts will be in the pellet, while most of the other organelles will be in the supernatant. Unfortunately, nuclei and unbroken cells will also be in the pellet. Therefore we will use flotation gradients to purify the chloroplasts. Next we will recover the band containing chloroplasts and use them to measure factors affecting photosynthetic electron transport.

First we will learn how to measure the Hill reaction, and examine two chemicals known to affect it.

Recall that ordinarily electron transport is used to pump protons across the membrane to create a proton gradient , which, in turn, is used to generate ATP. Chemicals which allow this proton gradient to be dissipated without generating ATP are known as uncouplers, and an example of an uncoupler is ammonia. Therefore, you will treat one of your samples with ammonia to see how this affects the rate of the Hill reaction. Another inhibitor which you will test is DCMU ( 3-(3-4-dichlorophenyl)-1,1-diethylurea). DCMU interrupts electron transport on the reducing side of PSII by inactivating one of the electron carriers.

PREPARING CHLOROPLASTS

1. Obtain 5 g of fresh spinach leaves from which the major veins have been removed.

2. Cut the leaves into small pieces with a razor blade,

• divide into 2 equal batches

• grind each batch in a chilled mortar and pestle for 2 minutes in 10 mls ice-cold grinding buffer.

3. Prechill and dampen four layers of cheesecloth and two layers of miracloth by pouring 10 mls ice cold grinding buffer through it, then squeeze the brei through this filter into a 50 ml test-tube kept on ice.

4. Centrifuge 2 minutes at 2,000 x g.

5. Discard the supernatant, and resuspend the pellet in 5 mls 60% Percoll in assay medium with a paint brush.

6. Carefully layer on 10 mls 40% Percoll, then 10 mls 20 % Percoll then 10 mls assay medium

7. Centrifuge 15 minutes at 10,000 x g.

8. Carefully remove the green band at the interface between the 40% and 20% layers using a pasteur pipet.

9. Transfer to a second centrifuge tube and dilute with 25 mls assay buffer.

10. Centrifuge 2 minutes at 2,000 x g.

11. Discard the supernatant, and resuspend the pellet in 1 ml assay buffer with a paint brush.

12. Determine the chlorophyll concentration by mixing 50 µl of your chloroplasts with 5 mls 80% acetone, filtering, then measuring the absorbance at 652 nm.

mg chlorophyll/ml = A652 x dilution factor

34.5

(before starting check that the spectrophotometer is warmed up, set to 652 nm and adjusted to 0% transmittance. Use 80% acetone as your blank)

13. Dilute your chloroplasts to 0.2 mg/ml chlorophyll using assay buffer, then keep the suspension in the dark and on ice.

14. Boil a 0.6 ml aliquot of chloroplasts for 5 minutes.

MEASURING THE HILL REACTION

1. Turn off the stirrer and remove the water, then rinse with assay buffer.

2. Add the contents of tube 1 prepared as described below.

• First, add the correct volume of buffer.

• Next add the correct volume of water.

• Third, add the potassium ferricyanide.

• Finally, add the chloroplasts.

3. Carefully insert the oxygen electrode. Locate the groove made in the plunger and tilt the chamber so that the groove is on the high side. Carefully push down the plunger squeezing the bubble through the groove so that no bubbles are trapped beneath the plunger.

4. Turn on the stirrer, waterbath and floodlamp, then once the needle has settled down read the % oxygen saturation at 30” intervals for 5 minutes, entering the data in Table I.

5. Remove the contents of the cuvette and rinse out with assay buffer.

6. Add the contents of tube 2, then wrap the cuvette with aluminum foil and turn off the light.

7. Repeat steps 3-5.

8. Add the contents of tube 3. Note that the boiled chloroplasts are added to this cuvette.

9. Repeat steps 3-5.

10. Add the contents of tube 4. Note that you leave out the K3Fe(CN)6

11. Repeat steps 3-5.

12. Add the contents of tube 5, then turn on the stirrer, waterbath and lights. Once you the needle settles to a steady rate measure the % saturation at 30” intervals for two minutes, entering the data in Table I.

13. Inject 0.5 mls of ammonium hydroxide, then once the needle settles to a steady rate measure the % saturation at 30” intervals for three minutes, entering the data in Table I.

14. Remove the contents of the cuvette and rinse out with assay buffer.

15. Add the contents of tube 6, then turn on the stirrer, waterbath and lights. Once you the needle settles to a steady rate measure the % saturation at 30” intervals for two minutes, entering the data in Table I.

16. Inject 0.5 mls of DCMU, then once the needle settles to a steady rate measure the % saturation at 30” intervals for three minutes, entering the data in Table I.

|Cuvette |Buffer |water |chloroplasts |K3Fe(CN)6 |

|1 |1.0 ml |2.5 ml |0.5 ml |1.0 ml |

|2 |1.0 ml |2.5 ml |0.5 ml |1.0 ml |

|3 |1.0 ml |2.5 ml |0.5 ml* |1.0 ml |

|4 |1.0 ml |3.5 ml |0.5 ml |- |

|5 |1.0 ml |2.5 ml |0.5 ml |1.0 ml |

|6 |1.0 ml |2.5 ml |0.5 ml |1.0 ml |

* these are the boiled chloroplasts

17. Report your findings on your datasheet. Your introduction should explain what the Hill reaction is, why it can be measured using an artificial electron acceptor, and what you specifically set out to do. Results should include a table showing the raw data, a graph in which the % saturation is plotted as a function of time for tubes 1-4, and a few sentences explaining why you performed the experiment, where the data are presented, and describing the major trends observed. Be sure to include separate graphs for tubes 5 and 6, and state the amount the rate changed upon adding ammonia and DCMU. Your discussion should explain the results obtained with each tube. Be sure to explain the effects of DCMU and ammonia in your discussion.

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