The Activity of Chloroplasts and Mitochondria Lab Report

Chloroplasts are organelles in a plant cell that are the site of photosynthetic reactions (Boardman, 1966). Mitochondria are organelles that are actively involved in energy generating reactions popularly known by many biochemists as the electron transport chain and oxidative phosphorylation. In these processes, electrons are carried through several acceptor molecules and molecular oxygen is the final electron acceptor. This transfer of electrons provides energy to proton pumps on the mitochondrial inner membrane, which pump protons setting up a proton gradient that powers ATP synthase causing the formation ATP, an energy-rich molecule (Bednarczyk, Kicinska, Kominkova, Ondrias, Dolowy and Szewczyk, 2004). Photosynthesis converts light energy into the chemical energy in sugars, whereas, respiration converts the chemical energy in sugars into the chemical energy stored in ATP molecules.

Isolation of mitochondria The outer 4 mm of cauliflower florets were shaved until there was 20g of tissue. The tissue was placed in a chilled mortar with 40mL of ice-cold mannitol grinding buffer (0. 3 M D-mannitol-0. 02 M phosphate buffer) and 5g of purified sand, and was then ground with a chilled pestle for 4 min. The suspension was filtered through four layers of cheesecloth into a chilled 50mL centrifuge tube, and then set on ice for 2 min to allow the sand and debris to settle. To run these reactions, each group added 0. 5mL of chloroplast/mitochondria suspension to tube 1 and used it to blank the spectrophotometer, so that each tube was set “blank” before reading. The spectrophotometer wavelength was set to 600nm. In each tube, 0.

5mL chloroplast/mitochondria suspension was then added to one tube at a time and immediately read to get the initial absorbance. 5mL buffer, 0. 5mL dH2O, 0. 5mL chloroplast Tube2- 0. 5mL 0. 4mM DCIP, 3. Three reactions were run containing buffer, DCIP, and azide. The three reactions were tested every 5 minutes for 30 minutes. One reaction had no succinate or malonate, one reaction had succinate, and the other reaction had succinate and malonate (0. 5 ml of 0. 2 M). As shown in figure 3, DCIP was not reduced during the succinate and malonate conditions. Discussion In this lab we performed reactions with chloroplast/mitochondria suspensions and tested the effect of inhibitors. The spectrophotometer was used to measure the amount of light that was absorbed by each test tube.

An artificial electron acceptor DCIP was used to “see” the reactions. Chloroplast and mitochondria both reduced DCIP. Ultimately, this experiment displayed the differences between mitochondria and chloroplasts through the DCIP redox reaction. The next experiment I would perform is to find out the enzymes that are interfered with inhibitors in the electron transport chains of the chloroplast and mitochondria. References Swier, K. Lab/Activity #6, Cell Biology; The Activity of Chloroplast and Mitochondria Bednarczyk, P. , Kicińska, A. [Ebook]. Retrieved from http://www. jbc. org/content/178/1/241. full. Ruuska, S. The Role of Chloroplast Electron Transport and Metabolites in Modulating Rubisco Activity in Tobacco. Insights from Transgenic Plants with Reduced Amounts of Cytochrome b/f Complex or Glyceraldehyde 3-Phosphate Dehydrogenase. Retrieved from https://www.

For the chloroplast, 2, 6dichlorophenolindophenol (DCIP) was used as the reagent to assay for the activity that occurs therein. Contrariwise, malonate was used as an inhibitor of the reactions that generate energy within the mitochondria (Bartels, Matsuda, Siegel and Weier, 1967) to assay for the same concept herein stated. Both malonate and DCIP are down-regulators of the reactions that occur in the mitochondria and in the chloroplast respectively and thus; it was set and done. Mitochondria are organelles that are actively involved in energy generating reactions popularly known by many biochemists as the electron transport chain and oxidative phosphorylation. In these processes, a hydrogen ion is carried catalytically through several acceptor molecules known as principal oxidants and molecular oxygen is the final electron acceptor resulting into the formation of ATP, an energy-rich molecule (Bednarczyk, Kicinska, Kominkova, Ondrias, Dolowy and Szewczyk, 2004).

The tubes, having been placed on the centrifuge,balancing was doneusing a pan balance before putting them into the centrifuge. They were centrifuged at 10,000rpm for half an hour at 4°C. The mitochondria were in the pellet into which frozen mannitol buffer amounting to 5. 0 ml was poured. The pellet was resuspended by scraping and pipetting mannitol assay media. 4ml, 0. 5mM DCIP and 0. 5 ml dH2O. To tube 5 were added all the contents of tubes 2 and 3 and 0. 2M succinate. Into twotubes labeled 5 were added all the contents of tubes 2 and 3 and 0. 2M succinate. To tube 6 was added 3. 0 ml buffer plus0. 5ml of0. There is a continual rugged trend in decrease and increase in the absorbance values recorded over the spectrophotometer as time increases and as the experimentation progresses.

This indicates that the substances in the reaction mixture reacted in such a way that the turbidity of the solution reduces with time. Table 1: Mitochondria Absolute change Time (min) Tube 2 Tube 3 Tube 4 Tube 5 no inhibitor (-) succinate (+) malonate (-) azide 0 0 0 0 0 5 0. 036 Figure 1: Table of values for the absolute change involving mitochondria. There occurred an observable reduction with succinate and increment with malonate in the absorbance with time in the mitochondria reaction illustrated by the experimental results in the above table. 041 Tube 4 Ambient 0. 812 Tube 5 (+) succinate 0. 776 Tube 6 (+) Azide 0. 546 Figure 3: A table of values for absorbance in various test tubes under varied lighting. Figure 4: Bar graph for chloroplast reaction Test tube # Variable Absolute Change Initial After 10 min Tube 1 Blank 0 0 0 Tube 2 No light 0. Generally, inhibitors are negative regulators of the activities of the chloroplasts and the mitochondria thus the immense decrease in absorbance observed.

Mitochondria reaction ± inhibitors. Figure 7: line graph on mitochondria reaction. Malonate is an inducer of cell death because it is capable of bringing about potential mitochondrial collapse because, although it acts like succinate in oxidative phosphorylation, it does not form fumarate and is therefore a toxic substance in regard to the functionality of the mitochondria (Brookes, Digerness, Parks and Darley-Usmar, 2002). This is why there is a deceleration in the curve which again begins to accelerate because the azide added promotes mitochondrial metabolic activity (energy generating activity). Azide acts as a substance that speed up the conversion of malonate derivatives to less harmful forms because succinate does not form fumarate as it happens to it in the citric acid cycle (Ritenour, Joy, Bunning and Hageman,, 1967).