BIO 120 Exer 6

RANELLE JANINE L. ASI 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

Figure 6.1. Spectrophotometric readings of three solutions consisting of heated and unheated chloroplasts, phosphate buffer, NaCl, and DPIP In the results of the experiment, the control setup had absorbance readings that constantly fluctuated (although no extreme deviation is noted) as the control contained heated chloroplasts. At high temperatures, enzymes in the chloroplasts are mostly deactivated; hence the control exhibited random spectrophotometric readings in the absence of photosynthetic activity. The absorbance trend of the light setup is decreasing and eventually flat lining. The extreme decrease in absorbance is attributed to the loss in color of the solution caused by the light-dependent reaction reducing the DPIP dye indicator. The flat lining of the graph indicates that all DPIP molecules have been used up by the photosynthetic electron transport chain. Lastly, the dark setup showed no increase or decrease in absorbance readings and stayed constantly near 0. There is no change in the bluish color of the solution. This is due to the absence of light energy supposedly powering the transfer of electrons across the electron transport chain to finally reduce the DPIP dye. This result confirms the requirement of light in the photosynthetic reactions within the chloroplast. Increasing absorbances may be attributed to impurities in the prepared solution leading to the oxidation of DPIP or interference during measuring. Other methods may be employed in the measurement of photosynthetic activity. The manometric technique (which is based on direct measurement of the pressure of CO2 or O2 in an isolated chamber with photosynthetic organisms), or the electrochemical sensor method (which is based on the use of O2 and pH electrochemical electrodes to measure the O2, CO2 or pH aqueous concentrations of the analyzed sample) may be implied but requires more sophisticated equipement.

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