Light Reactions of Photosynthesis - Windward · PDF file · 2016-07-07Light Reactions of Photosynthesis Laboratory 7. 1 Laboratory 7: ... is associated with certain proteins and...

Light Reactionsof

Photosynthesis

Laboratory

7

1 Laboratory 7: Light Reactions

OBJECTIVESAfter completing this lab you will be able to:1. Understand the cooperation between photosystems in plants

through electron flow (Hill Reactions)2. Use a redox dye to demonstrate electron flow during

photosynthesis between PSI and PSII3. Use a spectrophotometer to assess colorimetric changes4. Determine the absorption spectrum of chlorophyll5. Evaluate the effect of different wavelengths on excitation of

chlorophyll (fluorescence)

LAB PREPARATION1.Read Chapter 10 in Campbell (9th Ed.).

2.Bring your personal protective gear: lab coat, safety goggles,and safety gloves.

INTRODUCTION

Two Photosystems Cooperate. The characteristic feature of photosynthesis is the use of two lightreactions in series to drive electrons from water to NADP+, up thethermodynamic hill. This conception evolved over a period of twodecades from 1940 to 1960

How do the pigment molecules of the thylakoid membranestransduce absorbed light energy into chemical energy? The key toanswering this question came from the discovery made in 1937 byRobert Hill, a pioneer in photosynthesis research. He found thatwhen leaf extracts containing chloroplasts were supplemented with anonbiological hydrogen acceptor and then illuminated, evolution ofO2 and simultaneous reduction of the hydrogen acceptor took place,according to an equation now known as the Hill reaction:

2H2O + 2DCPIP ------> 2DCPIPH2 + O2

One of the nonbiological hydrogen acceptors used by Hill, the dye2,6-dichlorophenolindophenol (DCPIP), now called a Hill reagent.This reagent in its oxidized form is blue and in its reduced form iscolorless. When the leaf extract supplemented with the dye wasilluminated, the blue dye became colorless while O2 was produced.In the dark, neither O2 production nor dye reduction took place. Thiswas the first specific clue to how absorbed light energy is convertedinto chemical energy: it causes electrons to flow from H2O to anelectron acceptor. Moreover, Hill found that CO2 was not requiredfor this reaction, nor was it reduced to a stable form under theseconditions. He therefore concluded that O2 evolution can be

Laboratory 7: Light reactions 2

dissociated from CO2 reduction. Several years later it was found thatNADP+ is the biological electron acceptor in chloroplasts. Far-redlight (700 nm) does not drive the Hill reaction; it does, however,support the reduction of NADP+ with reduced DCPIP as the electrondonor:

Electron Acceptor/ Colorimetric Indicator: DCPIP DCPIP is one of many redox dyes. A redox dye is any coloredcompound that is easily inter-convertible between an oxidized formand a reduced form. The DCPIP inter-conversion is shown.

Note that during this process DCPIP accepts electrons in the form ofhydrogen atoms. Bonding to the hydrogen atoms results in fewerdouble bonds per dye molecule, producing a dramatic shift of itsabsorption maximum from the red end of the spectrum into theultraviolet. The UV absorbing form appears colorless, so the bluecolor disappears as the dye is reduced.

Figure 7-1. The “Z” scheme in photosynthesis. In the Hill reaction studied in today’s experiment, wewill focus on the region between plastiquinone (Pq) and plastocyanin (Pc).

NCl

OHCl

O

Oxidized (blue)

NCl

OHCl

HOH

Reduced(colorless)


In the Hill reaction, light is absorbed by molecules of chlorophyll II(P680) within the chloroplast membrane. The excited chlorophyll IImolecule gives up a pair of electrons to “Q” the primary electronacceptor of Photosystem II. “Q” in turn reduces plastoquinone (Pq).When DCPIP is present in the chloroplast support medium, Pq passesthe electrons to DCPIP rather than cytochrome b and c. Thechlorophyll II molecule recaptures two electrons from a manganese-containing enzyme (Z). This manganese-containing enzymeultimately obtains its electrons by splitting water into oxygen gas andhydrogen ions.

Electron inhibitor: DCMU The inhibitor 3-(3,4-dichlorophenyl)-1,1 di-methyl-urea (DCMU)blocks the passage of electrons from the primary electron acceptor“Q” to plastoquinone (Pq) as shown in Figure 7-1. It thereforeinterrupts both the Hill reaction and the normal transfer of electronsfrom Photosystem II to Photosystem I. All the molecules of “Q”quickly become reduced, and chlorophyll II (P680) can no longerdonate electrons to “Q.” The splitting of water ceases. ChlorophyllII will fluoresce under these circumstances, because fluorescence isthe simplest remaining pathway for the disposal of the absorbedenergy.

Light Absorption and Fluoescence by Photosynthetic Pigments When pigments absorb light, electrons are boosted to a higher energylevel, with three possible consequences: (1) the energy may bedissipated as heat; (2) the energy may be captured in a chemicalbond, as it is in photosynthesis; or (3) it may be re-emitted almostinstantaneously as light energy of longer wavelength, a phenomenonknown as fluorescence (when it is re-emitted as light at a later time, itis known as phosphorescence).

Chlorophyll can convert light energy to chemical energy only when itis associated with certain proteins and embedded in a specialized,thylakoid membrane. In the thylakoid membrane, pigments thatabsorb light rapidly pass on this energy, either by transfer to anotherpigment molecule, or by loss of electrons. Chlorophyll moleculesabsorb light most strongly at two extremes of the visible spectrum,the red and blue. Chlorophyll a, for example, has absorbancemaxima at 430 nm and 670 nm. Chlorophyll appears green becauseit reflects light in the green portion of the spectrum. The solarspectrum, however, has its maximum intensity in the middle of thevisible spectrum, near 530 nm (yellow light). Chlorophyll does notabsorb in this region, but accessory pigments such as carotenoids do.Carotenoid pigments normally pass their energy along to chlorophyllby electron transfer or electron loss.

Laboratory 7: Light reactions 4

If chlorophyll molecules are dissolved out of the thylakoidmembrane and light is permitted to strike them, the molecules absorblight energy, and their electrons are momentarily raised to a higherenergy level before falling back again to a lower one. However, noneof the light absorbed by the isolated chlorophyll molecules isconverted to any form of energy useful to living systems. Instead, asthe electrons fall back to a lower energy level, much of their energyis released as light, i.e., the molecules fluoresce. Because someenergy is dissipated as heat in the acts of absorption and release, thewavelength of the light released in fluorescence is generally longer(i.e., has less energy) than that originally absorbed.


LABORATORY EXPERIMENTS

Experiment 1: The Hill Reaction

Isolated chloroplasts are unstable!! Keep all tubes containingchloroplasts on ice and in the dark except to illuminate them and takea spectrophotometer reading.

PROCEDURE1.Turn on the spectrophotometer set the wavelength to 620 nm

(the absorption peak of DCPIP).

2.Place an empty test tube rack inside a clear plastic box and thenfill the box with ice.

A. Set up your experiment3.Prepare 5 spectrophotometer tubes according to Table 7-1. Add

the solutions to your tubes in the following order:

NOTE: Do not add DCPIP to any of the tubes until the lights inthe lab have been turned OUT.

a) Buffer b) Distilled water c) DCMU (add to tubes 3, 4, and 5)d) Chloroplastse) DCPIP (add to tubes 2,3, 4, and 5—in the dark)

Why should the DCPIP be added last?

4.After adding DCPIP to tubes 2, 3, 4, and 5, mix by covering thetube with parafilm and quickly inverting three times. Returnall tubes to the test tube rack in the box of ice and immediatelyplace the box in your dark bench storage area.

B. Time “zero” reading (no electron flow)

PROCEDUREKeep all tubes on ice and in the dark

1.Zero and blank the spectrophotometer using tube 1 (no DCPIP,no DCMU).

Laboratory 7``: Light reactions 6

NOTE: thoroughly dry the outside of each spectrophotometertube with a dry kimwipe before inserting it into thespectrophotometer and taking a reading.

2.For each tube: 2, 3, 4, 5, read the absorbance and record thisreading in your data table. Return the tube to the ice tray beingkept in the dark. They should read between 0.9 and 0.5. This isyour time zero reading.

C. First illumination of isolated chloroplastPROCEDURE

1.While the lab is still dark place the tubes in the ice bucket 20cm away from the lamp on your bench top (refer to the tape onyour bench) but take care that none of the tubes are shaded byothers.

2.Turn on the lamp to illuminate the tubes for 30 seconds, thenimmediately place the tubes and ice bucket back into yourdrawer.

3.Take out tube #1 and re-blank your spectrophotometer.Return the tube to your drawer and keep on ice.

4.Take out the remaining tubes one by one (replacing each inthe drawer when you’re through) and read the absorbance andrecord ii in your table.

5.After measuring the last tube repeat the above procedure for atotal illumination time of the tubes of five (5) minutes, i.e., tentimes. If tube #5 is not “clear” after the five minutes, continuethe experiment until it is, or consult your Teaching Assistant.Remember that the “clear” tube will still be green due to thechloroplasts. We are only concerned with the reduction ofDCPIP (blue to non-blue).

NOTE: Use your personal protective gear (safety goggles, gloves,and lab coat) for all procedures using acetone in Experiments 2and 3.

Experiment 2: Determining the Absorption Spectrum of Chlorophyll


PROCEDURE1.Fill a clean spectrophotometer tube with 5 mL of spinach leaf

pigment extract, cover with a square of parafilm, and place thetube in a small beaker with some ice.

2.Fill a second spectrophotometer tube with 5 mL of acetone,cover it with parafilm, and place it in the beaker containing icealong with the extract tube.

3.Set your spectrophotometer to 400 nm.

Dry the tube containing acetone using a kimwipe and zero andthen blank your spectrophotometer.

4.Dry the extract tube with a kimwipe, insert the tube into thespectrophotometer and record the absorbance in your table.Return the tube to the beaker of ice.

5.Re-adjust the spectrophotometer to 425 nm.

Blank the spectrophotometer with the acetone tube.

6.Now insert the extract tube and record the absorbance. 7.Increase the wavelength by 25 nm increments, each time

blanking the spectrophotometer using the acetone tube andreading and recording the absorbance of the extract until youreach 700 nm.

8.Dispose of the spinach extract in the special disposal bottle foracetone waste. However, KEEP your tube of acetone; you willuse it in the next experiment.

Experiment 3: Fluorescence of Chlorophyll Extract in AcetonePROCEDURE

1. Near the slide projector in the back of the room is a tube ofconcentrated spinach leaf extract (principally chlorophyll a andb) in acetone. Take this tube of extracted pigments and hold itdirectly in a beam of white light from the projector, along withthe tube of acetone from the previous experiment. Look at eachtube from the side (i.e., perpendicular to the projector beam)and compare their color. The fluorescence is visible becauseyou are observing at a right angle to the projector beam. Recordthe color and intensity (i.e., absent, dim, moderate, bright, verybright) of the fluorescence.

2.Repeat your observations with projector beams of blue, green,yellow, red (660 nm), and far-red (730 nm) light. With whichwavelengths do you expect to see fluorescence?

Laboratory 7: Light Reactions 8

LAB SUMMARY

Please include the following in your lab summary. 1. Descriptive title 2. Introduction describing purpose and objectives of this lab activity. Also very

briefly describe the general approach taken to achieve these objectives. 3. For the first experiment (The Hill Reaction) include the following: ÿ Working graphs used to determine the rate of DCPIP reduction (measured as

the reduction in absorbance per unit time) line slopes for each treatment (no DCMU and three different concentrations of DCMU). These do not need to be formatted for formal presentation, but do need to be clearly labeled (e.g., title, axes, reaction conditions, trend lines with slopes, etc.)

ÿ Formal production graph illustrating how rate of DCPIP reduction changes as a function of DCMU concentration.

ÿ A paragraph describing the trends observed. Be sure to include answers to the questions posed at the end of the previous lab description. Also include comment whether or not the results made sense and why. Comment on any sources of error.

4. For the second experiment (Absorption Spectrum of Chlorophyll) include the

following: ÿ Formal production graph that illustrates the absorption of light of different

wavelengths by a chlorophyll extract. This graph will be one of the few times when you “connect the dots”. If you make this graph using Excel you may use the graph format that connects the points with a smooth line.

ÿ A paragraph describing the trends observed. What is the significance of absorbance maxima observed in this graph?

5. For the third experiment (Fluorescence of Chlorophyll Extract) present your

observations in the form of a table . Provide a paragraph that explains these observations.

6. Conclusion that summarizes the experiments and interprets the significance of

the results.

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9 Laborotory 7: Light Reactions

Light Reactions of Photosynthesis - Windward · PDF file · 2016-07-07Light Reactions of Photosynthesis Laboratory 7. 1 Laboratory 7: ... is associated with certain proteins and...

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