Post on 03-Jan-2016
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Lab 7: The Light Reactions of Photosynthesis
• Study the Hill Reaction and the effects of DCMU on electron transport
• Determine absorption spectrum of chlorophyll
• Observe fluorescence in chlorophyll
Purpose of the lab exercises:
Properties of Light
Source of energy
• wave and particle (photons)
Wavelength of light: Peak to Peak
• Different wavelengths have different characteristics and energies
(wavelength)
The Electromagnetic Spectrum
Short wavelengths have high energies
Long wavelengths have lower energies
Visible portion between 380 and 750 nm Different wavelengths = different colors.
Today, you will examine the Hill Reaction
The chemical equation for photosynthesis is:
Photosynthesis
6CO2 + 6 H2O + ENERGY C6H12O6 + 6O2
2)The Dark Reactions
• Calvin CycleCombines H2O and CO2 to produce sugars in stroma
PhotosynthesisTwo sets of reactions: (1) The light reactions • Light energy trapped by
chlorophyll • (NADPH) and (ATP) are formed
in thylakoid membranes
Light Reactions of PhotosynthesisComplexes embedded in thylakoid membrane Organized cluster of chlorophyll and proteins• Harvest light energy, resonance transfer• Reaction centers = chlorophyll a + primary
electron acceptor
Two Photosystems: PSII and PSIContain chlorophyll a in reaction center• PSII chlorophyll a is P680 (Absorbs 680)• PSI chlorophyll a is P700 (Absorbs 700)
Light Reactions of Photosynthesis
Primary electron acceptors
• associated w/ chlorophyll a of reaction center
• traps high-energy electrons (excited)
• prevent return to ground state.
Light Reactions of Photosynthesis
Photosystem II (P680) Electrons lost to primary electron acceptor
How are they replaced?Splitting of water• Each water molecule:
provides 2 electrons• An atom of oxygen • Two atoms of oxygen
form O2
What happens to electrons at the primary electron acceptor?
Electrons move from PS II to PS I
Light Reactions of Photosynthesis
Lose energy Lower energy level Produce ATP
Plastoquinone (Pq) Complex of two cytochromes Plastocyanin (Pc)
Electron Transport Chain
As electrons move through electron transport chain
Hill Reaction
• Named after Robin Hill
• Chloroplast preparations can split water
Light Reactions of Photosynthesis
Colorimetric indicator (DCPIP)
• Intercepts electrons in electron transport chain
• Between Pq and cytochrome complex
• Reduced (gains electrons)
Study of Hill Reaction: DCPIP
• As DCPIP becomes reduced, gradually turns from blue to colorless
• Over the 30s intervals, drop in absorbance and readings in spectrophotometer
Study of Hill Reaction: DCPIP
Reduction of DCPIP by Isolated Chloroplast
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Time (seconds) exposed to light
A62
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Study of Hill Reaction: DCMU
DCMU inhibitor of electron transport
• Blocks passage of electrons from primary acceptor of PS II - plastoquinone
• Prevents DCPIP from being reduced
• Degree of inhibition depends on concentration
High concentrations of DCMU, electrons are almost completely blocked from passing to Pq very little reduction of DCPIP, little change in spec readings
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0 50 100 150 200 250 300 350
Time (seconds) exposed to light
A62
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High [DCMU]
No DCMU
Study of Hill Reaction: DCMU
• Lower concentrations of DCMU, electrons are only moderately inhibited from passing to Pq,
• DCPIP continues to be reduced
Interaction of Light with MatterLight can be
reflected transmitted absorbed
Color of objects due to reflected or transmitted light
Chlorophylls absorb red and blue light Reflects and transmits green light.
Pigments: Chlorophyll
Pigments Absorb visible light
Chlorophyll a and b:
• Two primary pigments in photosynthesis
• Differ slightly in chemical structure
Chlorophyll molecule
Absorption Spectrum
Graph of light absorbence vs. wavelength
Today you will create your own absorbance spectrum using isolated chlorophyll.
But what happens if chlorophyll is isolated from the intact structure of chloroplast, and then illuminated with light?
Isolated chlorophyll molecules
Fluorescence in Isolated Chlorophyll
Fluorescence!
• Electrons still boosted to higher energy levels
• No electron acceptor
• They quickly drop back down to ground state
• Energy released as light and heat.
Fluorescence in Isolated Chlorophyll
Why does it fluoresce red?
Fluorescence in Isolated Chlorophyll
• Looking at the spectrum, red is associated with lower energy
• In returning back to ground state, some energy is lost as heat
• Energy of fluorescing light is less than that which illuminated it
• Longer wavelengths have lower energy
Today you will illuminate isolated chlorophyll with different wavelengths (colors) from the visible portions of the spectrum
• Observe the INTENSITY and red fluorescence.
• Must use absorption spectrum.
Would you expect the intensity of fluorescence to be high, moderate, or low if chlorophyll was exposed to blue light?
Fluorescence in Isolated Chlorophyll
• Little energy from green light is absorbed• Most is reflected or transmitted • Few electrons boosted
Fluorescence in Isolated ChlorophyllChlorophyll absorbs most of the blue light Electrons boosted to higher orbitals fall back to ground state: “ High Fluorescence”
What about green light (@550 nm) on isolated chlorophyll, the intensity of fluorescence will be low, moderate, or high?
Low!
Experiment 1: The Hill Reaction• DCPIP (e- acceptor; blue to non-blue)• DCMU (e- inhibitor)
Experiment 2: Determining the Absorption Spectrum of Chlorophyll• Spinach leaf pigment extract
Experiment 3: Fluorescence of Chlorophyll Extract in Acetone
Time to
work!