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2008-07554
BIO 120 S-5L
EXERCISE 6. The Hill Reaction
DISCUSSION
Experiment Rationales
During the preparation of the experiment, solutions and glass wares were
pre-cooled to maximize the rates of reactions as centrifuge rotors and the blender
generate heat which denature enzymes involved. A hypertonic solution, 0.35M NaCl,
was mixed when grinding the leaves and suspending the chloroplasts to cause
plasmolysis, wherein the cells shrivel and become easier to lyze or break
mechanically.
After centrifugation, Pellet 1, which contained the heavier organelles and lipid
fragments, was discarded. Centrifugation speed was only at 1400 x g for 15 minutes
as chloroplasts are relatively lightweight and will sediment at this RCF.
During spectrophotometric measurements, the blank contained chloroplasts
to eliminate absorbance of chloroplasts in the mixture, as only DPIP absorbance is of
interest. The chloroplast suspension was diluted to 0.05 mg chl/mL to control the
levels of DPIP reduction. If the suspension is too concentrated, all DPIP will be
consumed too quickly.
Two different blanks were used as heated and unheated chloroplasts absorb
light differently. The wavelength used when determining the amount of chlorophyll
per mL of the extract was 652 nm because chlorophyll absorbs red light maximally.
Red light has a wavelength of 652 nm. All the other mixtures that contained DPIP
maximally absorb light at 605 nm, the wavelength of orange light.
Effect of Light on Hill Reaction and the Role of DPIP
In the presence of light, a process known as photo-phosphorylation occurs
wherein ATP is synthesized from ADP and Pi. The requirement of light entails that
photosynthesis is part of a photoinduced electron transport system which results to
the production of chemical energy to be supplied to the light-independent or dark
reactions in which CO2 and H2O are converted to carbohydrates.
2,6-dichlorophenolindophenol (DPIP), when added to the chloroplast solution,
substitutes NADP+ as the final electron acceptor in the photosynthetic electron
transport chain as it has a higher affinity for electrons (ie, higher reduction
potential).
DPIP is initially blue in its oxidized form and turns colorless in its reduced
form. During this reaction the nitrogen atom joining the two benzyl groups of the
compound is reduced and changes its double bond to a single bond, forcing several
carbon bonds in the entire left benzyl ring to change confirmation. This makes the
molecule reflect light differently and ultimately absorbs and reflects a different
range of wavelength, accounting for the change in color of the solution.
Results
REFERENCES:
Giebek, P. E. (n. d.) Extraction of Chloroplasts from Plant Tissue and Their Use in
Demonstrating the Hill Reaction. Virginia Commonwealth University.
Richmond, Virginia, USA.
OCR (Oxford, Cambridge and RSA) pamphlet. 2014. The Hill Reaction Instructions
and answers for teachers. Retrieved on March 18, 2015 from
http://www.ocr.org.uk/Images/170050-the-hill-reaction-activity-teacherinstructions-.pdf