PHOTOSYNTHESIS IS ARGUABLY THE MOST IMPORTANT REACTION to living things. It is the initial reaction that harnesses light and converts it to sugars which are filtered up the food chain. Since this reation is so important, it is looked at in great detail many different times. In these experiments, we look at the effect that inhibitors have on the rate of the Hill Reation. Photophosphorylation is the first process of photosynthesis and its rate is very important to the process as a whole. We also look at how leaf structure can help a plant survive in their environment. C3 plants are the most common types of plants and use the enzyme RubisCO to break down their organic molecules into simpler, 3-carbon molecules which can in turn be converted into sugars that can be used for many processes within the plant.
Materials and Methods
Islotation of chloroplasts
In these first steps of the experiment, we must obtain our chloroplasts. We accomplish this by using spinach leaves, diluting with ice-cold NaCl-buffer, and centrifuging.
1. Obtaining chloroplasts
We obtained our organic material from spinach leaves. 4.25 grams of leaves were removed and then cut with scissors into smaller pieces before being put into a chilled mortar. 15-mL of cold NaCl-buffer and some sand were added to the leaves and then grinded for 2 minutes. The suspension was then filtered through four layers of cheesecloth into a centrifuge tube.
2. Creating a cholorplast suspension
The centrifuge tube with spinich juice and buffer was then placed in a centrifuge for 1 minutes at 1.5 g. The supernatant was then decanted into a clean, chilled centrifuge tube and put back in the centrifuge at 4.5 g for 5 minutes. The resulting supernatant was decanted and discarded. 10 mL of cold NaCl-buffer was then added to the remaining pellet and resuspended using a Pasteur pipet and inverting several times. 4 mL of this newly suspended cholorplast suspension are put into a clean, chilled test tube and diluted with 6 mL of cold NaCl-buffer. This test tube was covered with Parafilm, inverted several times and places in an ice bath.
Effect of inhibitors of the Hill Reaction
A blank, a non-illuminated control, and three other tubes were prepared with the chloroplast solution and differing amounts of ammonia and DCMU. These tubes were put in light and kept at 20°C and were put in a spectrometer and measured for transmittance at 600 nm.
A 150 mL water bath was prepared in a 250 mL beaker. This water bath is kept at a temperature of 20°C by adding ice. The...