Investigation of Hill Reaction Efficiency in Chloroplasts Isolated with Three Buffer Systems Under Dark and Cold Conditions

Abstract

Photosynthesis consists of both light-dependent and light-independent reactions. The light reactions occur within the thylakoid membranes of chloroplasts, where light energy drives the splitting of water and the transfer of electrons through the electron transport chain. The Hill Reaction demonstrates this process in a lab setting. Allowing for the demonstration of photosynthesis in a lab setting, however, it is notorious for being unsuccessful. The purpose of this research is to develop an experimental method for the reaction that will yield successful results by preserving chloroplast integrity to maximize photosynthetic activity. Chloroplasts were isolated from a common source, spinach leaves. Variables such as implementing a three-buffer system, centrifugation time and force, spectrophotometer wavelength, and maintaining dark and cold conditions were explored. Photosynthetic activity was measured spectrophotometrically at 620 nm via a colorimetric approach using DCPIP, an artificial electron acceptor that changes from blue to colorless when reduced. DCMU, a photosystem II inhibitor, was added to some samples to determine the effects of inhibition of electron flow during photosynthesis. Successful graphs will show a negative linear trend when the indicator DCPIP is present, indicating a decrease in absorption due to a reduction. Samples containing DCMU were expected to show little or no change. This research can be applied in agriculture to enhance crop yield by maximizing photosynthetic activity and understanding the effects of different pesticides.