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ATP: Adenosine Triphosphate

ATP         ADP + Pi (Hydrolysis, energy release)

ADP + Pi         ATP (Condensation)

Properties of ATP:

-Small and soluble (easily transported in cells)

-Easily hydrolysed (instantaneous release)

-Remade easily (condensation, regeneration)

-Makes molecules reactive (phosphorylation)

-Cannot pass out of the cell

ATP used in active process e.g. active transport

Made up of adenine, ribose and 3 x phosphates

ATP is made by:

-Respiration in animals and plants

-Photosynthesis in plants

Structure of the chloroplast

Chloroplasts are double-membrane bound organelles found in plant cells. They are responsible for carrying out photosynthesis.

Thylakoid membranes: Contain important membrane proteins to carry out photosynthesis.

Thylakoid membranes: Form a stack called a granum, the plural of which is grana.

Integranal lamellae: Connect grana and provide a larger surface area for increased capture of light.

Chlorophyll: Small photosynthetic pigment which reflects green parts of the spectrum. Respond to light energy by releasing electrons.

Stroma: Aqueous medium which contains sugars, enzymes and some (organic) acids.

Photosystems: Protein machinery which couple light energy to the generation of an electrochemical gradient.

Cyclic photophosphorylation uses PSI only. No NADPH or O2 is produced, only small amounts of ATP (ATP synthase)

6CO2 + 6H2O                C6H12O6 + 6O2 (+ATP also made)

Photophosphorylation: Adding a phosphate group to a molecule using light.

Photolysis: Using light energy to split (lyse) a given entity e.g. H2O in the LDR.

Photoionisation: Light energy causes electrons to be raised to high energy level + released e.g. photoexcitation of chlorophyll in LDR.

Decarboxylation: Removal of CO2 (carboxyl group) from a given entity

Dehydrogenation: Removal of hydrogen from a given entity.

Light dependent reaction:

ATP

PSII

PSI

P680

P700

E-

ETC

H+

ADP + Pi

ATP Synthase

Light energy

Chlorophyll

H+

H+

H2O

2H+ + 0.5O2

Light dependent reaction explained:

-Light energy absorbed by PSII, causing a pair of electrons to become excited and move to a higher energy level, being released.

-Electrons move to the ETC (electron transport chain) where the movement of electrons drives H+ pumping into the thylakoid lumen.

-Electrons move to PSI, where they are passed to NADP to form reduced NADP (NADPH).

-H+ building up in lumen generates an electrochemical gradient (H+ gradient/proton gradient)

-H+ move down electrochemical gradient through ATP synthase, which couples proton movement to ATP synthesis (ADP + Pi = ATP)

-The photolysis of water replaces the electrons lost from the photoexcitation step.

Light independent reaction explained:

1. Carbon fixation- CO2 diffuses in through the stomata and joins RuBP (5 carbon compound). Catalysed by RuBisCo (enzyme).

2. 6C product is unstable and breaks down to form two 3C products known as G3P (Glycerate-3-phosphate)

3. G3P reduced to Triose Phosphate (TP) using 1xATP and 1xNADPH (from LDR). Note: This is per G3P molecule.

4. Most TP is recycled to form RuBP, with some TP molecules going on to form organic compounds for the plant.

5. Regenerating RuBP requires ATP (LDR)

*The calcin cycle needs six turns to make one hexose sugar. This is because 6x(2xTP, where each TP has 3C). Total carbon = 36C

6xRuBP regenerated (30C demand) leaving 6C for hexose.

Carbon dioxide

RuBP

RuBisCo

2 x G3P

2 x ATP

2 x ADP + Pi

2 x NADPH

TP

2 x NADP

ATP

ADP + Pi

The Calvin cycle:

Limiting factors for photosynthesis:

-Light: Plants absorb specific wavelengths of light. They reflect green light and so appear green (absorb red and blue)

-Temperature: Temperature affects kinetic energy of particles, stomata opening as well as enzyme action. If temperature is too high, enzyme active sites denature and lose complementarity to substrate (less ES-complexes formed). If temperature is too high, stomata close (water retention, less CO2 enters)

-Carbon dioxide: CO2 makes up a very small percentage of gas in the air. Increasing this concentration will increase the rate of photosynthesis, up to a point where stomata close (4%).

Investigating dehydrogenase activity in chloroplasts

-Cut and grind up leaves using pestle and mortar. Make sure this is done under ice (reduce enzyme activity).

-The isolation buffer should also contain sucrose, KCl and pH7 phosphate buffer.

-Transfer to centrifuge tubes and spin at high speed for 10 minutes. Chloroplasts form pellet, discard supernatant.

-Resuspend pellets in chilled isolation buffer and store on ice.

-Set up colorimeter using red filter and zero using cuvette with distilled water.

-Set up test tube rack and switch on light.

-Add volume of chloroplast extract and volume of DCPIP and mix.

-Immediately transfer small amount into cuvette and record reading of absorbance on the colorimeter.

*If dehydrogenase activity present, absorbance will decrease because DCPIP is reduced and the blue colour of the solution is lost. Plot absorbance against time for different variables (light intensity, temperature, distance from the lamp etc). The greater the decrease of absorption, the higher the dehydrogenase activity.

Thin Liquid Chromatography (TLC)

-Grind up leaves from plant and add anhydrous sodium sulphate, then a few drops of pronanone.

-Transfer to test tube, add ether (petroleum) then extract top layer (pigments). Transfer to second test tube and add a few more drops of ether.

-Draw line in pencil at bottom of TLC plate, create spots by adding small concentrated drops (point of origin) to the plate and ensuring they dry.

-Place plate into glass with a prepared solvent, place lip on top and allow time to develop.

-When solvent has nearly reached the top, remove the plate from the glass and mark the distance travelled by the solvent.

-Observe spots and calculate Rf values for each spot using the equation distance travelled by spot/distance travelled by solvent.

Practical knowledge:

Colorimeter: Measures how much light is absorbed by a solution when light is passed through it.

TLC: Allows determination of what pigments are present in the leaves

*Thin liquid chromatography involves a mobile phase (molecules can move- liquid solvent) and a stationary phase (molecules cannot move)

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