Dr. Denise Marie Ratterman Archives

Abstract

Photosynthesis in plants is affected by the intensity of the light the plant is exposed to. For this experiment, DCPIP was added to cuvettes with spinach chloroplasts, which were exposed to an incandescent light at different distances for different intervals of time. After each exposure, the cuvettes were placed in a spectrophotometer set at 621 nm and the absorbance of DCPIP was measured. At 37 mm away from the light, the absorbance decreased at 0.033 absorbances/minute. At 27 mm away from the light, the absorbance decreased at 0.043 absorbances/minute. At 5 mm away from the light, the absorbance decreased at 0.050 absorbances/minute. As the distance from the light increased, the absorbance readings on the spectrophotometer went down at a faster rate. This means that the amount of DCPIP in the chloroplasts decreased quicker as the intensity of light was higher, and that photosynthesis occurs more quickly or slowly depending on the intensity of the light.

Introduction

Photosynthesis is the process that allows plants to survive (Chiras, 1993). It provides ATP (which can be used for energy), starch, cellulose, fats, and nucleic acids among other large molecules, and it consumes CO₂ to produce O₂ (Chiras, 1993). The plant chloroplast cell consists of an inner and outer membrane (Thorpe, 1984). Inside of the cell consists of chlorophyll and stroma. The stroma is a fluid substance comparable to cytosol in animal cells. Chlorophyll are particles that absorb light and pass the energy obtained onto thylakoid disks (Thorpe, 1984). Situated in the membrane of the thylakoid membrane is the electron transport chain. Along the electron transport chain, two electrons from H₂O ­are excited by light in Photosystems I and II to reach a higher energy level. This energy is used to convert NADP+ to NADPH and to drive electrons into to thylakoid space, which creates a gradient. This gradient fuels ATP Synthase, which converts ADP + Pi to ATP. ATP is used for energy in many processes in plant cells. In the experiments performed, DCPIP was added to chloroplast cells, which replaced the NADP+ along the electron transport chain. Using a spectrophotometer, the amount of DCPIP present was able to be determined and tests using variable intensities of light were performed on spinach chloroplast cells.

Materials and Methods

First, a room was insulated from light and the lights were turned off. A green light was used in order to see. Six cuvettes were then obtained and labeled with the numbers 1 through 6. Tube 1 consisted of 0.5 mL chloroplast, 3.0 mL cold buffer, 1.5 mL cold distilled water, and 0.0 mL of DCPIP, which were all dispensed into the tube using a micropipettor. This tube was used as a blank for the spectrophotometer which was set to 621 nm. Tube 2 was used as a control and was covered completely with tin foil in order to insulate it from light. Tube 2 and 3 were filled with 0.5 mL chloroplast, 3.0 mL cold buffer, 0.5 mL cold distilled water, and last with 1.0 mL DCPIP. Immediately after being filled with the DCPIP and agitated, the tubes were both placed in the spectrophotometer and their absorbances were recorded. Tube 2 was first removed from its foil before being put in the spectrophotometer, and it was put back on after the reading. They were than placed 11.5 mm away from an incandescent light for 3 minutes. Their absorbances were again recorded and this was repeated until the control tube came to a constant reading while tube 3 gradually went down. Tubes 4 through 6 were also filled with 0.5 mL chloroplast, 3.0 mL cold buffer, 0.5 mL cold distilled water, and 1.0 mL DCPIP right before being subject to light. Tube tubes were placed 37 mm, 27 mm, and 5 mm away, respectively. Their initial absorbances were recorded and following absorbances were recorded every 60 seconds for 6 minutes was exposed to the light.

Results

Table I:

Table II:

Tubes 2 and 3 were used to prove that it was indeed the light causing the absorbance to go down, and not the heat from the light. Tube 2 was covered with foil to prevent it from being exposed to light and its absorbance stayed constant, while the absorbance from tube 3 which was uncovered went down. As the tubes were placed closer to the light, their absorbances went down quicker, which was expected. The absorbance of tube 6 went all the way down to 0.16, which seemed very low (Table I). Test tube 5’s absorbance went low also, going down to 0.21 (Table I). Test tube 4 had very sporadic readings (Table I). This may be attributed to it not being thoroughly mixed enough.

Discussion

The absorbances readings recorded measured the amount of DCPIP_oxidized in the cells. As photosynthesis occurred, electrons were donated to the DCPIP_oxidized, forming DCPIPH_{2 reduced}. DCPIP_oxidized is able to absorb light from the spectrophotometer at 621 nm, hence that it why it was calibrated at 621 nm. As photosynthesis took place, there was less and less DCPIP_oxidized available for absorbance. That is why the absorbance readings went down over time.

Bidwell (1979) reported that light absorption is not really affected by temperature. The results gained from the experiment were consistent with his findings. When tube 2, which was covered in tin foil, was exposed to the light, its absorbance stayed constant. Though light was not affecting the tube, it could still be heated up. Because its absorbance did not move, this showed that the heat did not affect any of the absorbance readings for any of the test tubes.

Tubes 3 through 6 were all exposed to the light and their absorbances went down over time. The rate at which their absorbances went down increased as their distance from the light source decreased. The intensity of light directly affects the rate of photosynthesis (Bidwell, 1979). Graphs show that the higher the intensity, the higher rate of photosynthesis. The intensity in this experiment was increased by moving the tube closer to the light.

Lastly, the reason a green light was used in order to see was because chlorophyll absorbs all colors of light except for green and yellow (Chiras, 1993). Additional experiments using different colors of light or different light bulbs would be interesting, as the chlorophyll trap different kinds of lights at different rates. This could show what range of the color spectrum chlorophyll accept best.

Literature Cited

Bidwell, R. G. S. 1979. Plant Physiology. (MacMillian Publishing Co., NY, NY) 726 p.

Chiras, Daniel D. 1993. Biology: The Web of Life. (West Publishing Co., St. Paul, Mn) 896 p.

Thorpe, N. O. 1984. Cell Biology. (John Wiley & Sons, NY, NY) 719 p.

This study determined whether or not temperature had an affect on CO₂ production between yeast and different sugars. For this experiment, 2 mL of 10% fructose solution, 10% glucose solution, and distilled water were each combined with 2 mL of baker’s yeast. Each of these three solutions was measured for CO₂ production at three different temperatures: 3 ºC, 22 ºC (room temperature), and 42 ºC. None of the solutions produced CO₂ when they were at 3 ºC. At room temperature, the fructose and yeast solution produced 0.37 mL of CO₂ in 60 minutes, while the glucose and yeast solution produced 0.45 mL of CO₂ in 60 minutes. The control sample did not produce any CO₂. At 42 ºC, the fructose and yeast solution produced 1.28 mL of CO₂ in 45 minutes, while the glucose and yeast solution produced 1.33 mL of CO₂ in 45 minutes. Again, the control group did not produce any CO₂. Temperature had a great impact of the production of CO₂. When chilled, the solutions did not even produce any CO₂ at all, but when heated, the solutions produced CO₂ almost four times as fast as normal.

1.33= pipet exhausted all of the yeast

*1 ml. bubble appeared in the pipet

Abstract

Enzymes are catalysts which lower the activation of chemical reactions, thus making them happen more rapidly. In this experiment, different amount of enzyme and substrate were put in a test tube, then were observed using a spectrophotometer to see how fast the reacted to produce product. It was found that as the concentration of enzyme was increased, the speed of reaction increased. Likewise, it was found that as the concentration of substrate was increased, the speed of reaction increased. As there is more enzyme, it is able to react with more substrate at once, therefore increasing the rate of reaction. When there is more substrate, there will be just as much enzyme, also making the rate of the reaction increase. Thus, higher levels of enzyme or substrate mean there will be a higher turnover rate of product.

Introduction

A catalyst is a substance that lowers the activation energy of a chemical reaction (Zubay et al., 1995). In order words, this means that a catalyst makes chemical reactions happen faster than they normally do. Enzymes are catalysts that speed up reactions in living cells. For example, the enzyme carbonic anhydrase makes the chemical reaction CO₂ + H₂O —> H₂CO₃ happen 10⁷ times faster than it normally does. In this example, the enzyme reacted with CO₂. The substance which an enzyme catalyzes is called a substrate. Enzymes bind to substrates in order to speed the reaction in turning the substrate to a product. When there is a little amount of substrate, there will be a small amount of enzyme, but as the level of substrate increases, the level of enzyme increases. In this experiment, the speed at which enzymes and substrates react was observed.

Materials and Methods

For the first experiment, different enzyme concentrations were tested to see how fast they reacted. A Spec20 spectrophotometer was first turned on and set to a wavelength of 340 nm. A reaction mixture consisting of 0.850 ml distilled water, 1.000 ml buffer stock, 0.100 ml 15 mM NAD+, and 1.000 ml ethanol was then put into the test tube using a micropipettor. This mixture was agitated to mix it together, and the spectrophotometer was then calibrated using this test tube. Once calibrated, 0.050 ml ADH enzyme was added to the test tube mixture and agitated. The test tube was then quickly put into the spectrophotometer and its absorbance readings were recorded. The readings were recorded every 15 seconds for 3 minutes. This process was repeated for 2 more reaction mixtures with adjustments to the amount of distilled water and AHD enzyme included. The second reaction mixture had 0.100 ml ADH enzyme and 0.800 ml distilled water, and the third one had 0.025 ml ADH enzyme and 0.875 ml distilled water.

For the second experiment, different substrate concentrations were tested to see their reaction rates. Once again, a Spec20 spectrophotometer was turned on and set to a wavelength of 340 nm. A reaction mixture consisting of 1.85 ml distilled water, 1.0 ml buffer stock, 100 ul NAD+, and 0 ul ethanol stock was put into a test tube using a micropipettor. The mixture was agitated, then put into the spectrophotometer and the spectrophotometer was calibrated. Once 50 ul of ADH was added to the reaction mixture, the test tube was agitated and quickly put into the spectrophotometer. Absorbance readings were then recorded every 15 seconds for 2 minutes. Another reaction mixture with the same amounts of each substance was made, observed, and recorded. This process was repeated for five more reaction mixtures with differences in the amount of distilled water and ethanol stock used. The amounts of distilled water and ethanol stock for these reaction mixtures were 1.80 ml distilled water and 50 ul ethanol stock, 1.75 ml distilled water and 100 ul ethanol stock, 1.65 ml distilled water and 200 ul ethanol stock, 1.35 ml distilled water and 500 ul ethanol stock, and 0.85 ml distilled water and ethanol stock.

Results

Table I

Table II

The concentration of enzyme had an effect of the reaction rate. As the concentration of the enzyme went up, the velocity went up (Table II, Figure 2). The initial slope also became steeper as the concentration of the enzyme increased (Table II, Figure 1).

The concentration of substrate affected the rate of reaction. As the concentration of substrate went up, the velocity increased steeply then evened out (Figure 4). From Figure 4 (v vs. [S]), the estimated Vmax is 0.0620 umol/min and the estimated Km is 0.0140 mM. From Figure 3 (1/v vs. 1/[S]), the estimated Vmax is 0.0568 umol/min and the estimated Km is 10 mM. The actual Vmax and Km recorded from SigmaPlot were 0.06566 umol/min and 0.01522 mM. The Vmax obtained from each graph are fairly close, but the Km are not very close at all. The Vmax and Km from the v vs. [S] graph are very similar to the actual Vmax and Km obtained from SigmaPlot.

Discussion

Shono and co-workers (1995) observed the rate of reaction versus the concentration of a substrate different from the one used in this experiment. Their graphs resulting from their experiment are very similar to the graph resulting from this experiment. The “velocity vs. substrate concentration” graphs follow the almost exact same curve, but the levels of concentration were higher in Shono’s experiment, resulting in higher rates of reaction. In the “1/v vs. 1/[S]” graphs, Shono’s line seems to have about the same slope, but it crosses the x-axis at a much lower value than it did in the graphs for this experiment.

This can be attributed to error in procedure of the experiment, which caused outlier values the line to be skewed. It was not known these outlier values should have been disregarded until computing calculations for Km and Vmax using the “1/v vs. 1/[S]” graph and comparing to other similar graphs. The Vmax recorded from Figure 3 was fairly close to the Vmax found from Figure 4. The Km from Figure 3, 10 mM, is not very close, however. That value is way larger than the Km found from Figure 4, 0.0140 mM, and the actual Km calculated by SigmaPlot, 0.01522 mM.

From the experiment, the results showed that with a higher concentration of enzyme, the higher rate of reaction. When catalyzing a reaction, the enzyme binds to the substrate (Bolsover et al., 1997). If there is a higher concentration of enzyme, this means that there will be more enzyme to bind to substrate at once, therefore making the turnover rate of substrate to product higher. This is why a higher concentration of enzyme produces a higher turnover rate. The turnover rate begins to slow down and stop as the amount of substrate runs out, and that is why the absorbance rates began to even out in Figure 1. There was a limited amount of substrate that could be converted into product.

As there was a higher concentration of substrate, the rate of reaction increased and then leveled off, as shown in Figure 4. The Michaelis-Menten graph assumes that the enzyme and substrate are in equilibrium (Zubay et al., 1995). Therefore, as there is a higher concentration of substrate, there will be an equally high concentration of enzyme to react with the substrate. As there is a higher concentration of each, the rate of reaction increases. The curve evens off, however. This is because each substrate has a maximum velocity at which it can convert from substrate to product. The enzyme can not catalyze the substrate to turnover faster than this.

The affinity for the enzyme and substrate in this experiment was fairly high. The Km, 0.01522 mM, is a low number. This means the enzyme and substrate reacted very quickly to produce product.

Literature Cited

Bolsover, S.R., J.S. Hyams, S. Jones, E.A. Shepard, and H.A. White. 1997. From Genes to Cells. (Wiley-Liss, NY). 424 p.

Shono, M., M. Wada, T. Fujii, 1995. Partial Purification of a Na+ -ATPase from the Plasma Membrane of the Marine Alga Heterosigma akashiwo. Plant Physiol 108: 1615-1615.

Zubay, Geoffrey, William M. Parson, and Dennis E. Vance. 1995. Principles of Biochemistry. (Wm. C. Brown, Dubuque, Iowa) 863 p.

Abstract

Sugars catabolize through the process of glycolysis. Glycolysis causes the sugar to undergo phosphorylation and ferment, which yields CO₂. In this experiment, different sugar solutions were mixed with a yeast solution. The yeast solution caused the sugar solutions to undergo glycolysis and produce CO₂. Glucose, fructose, and mannose all produced CO₂, yet galactose did not. Mannose and fructose followed very similar curves of time versus the production of CO₂, while glucose followed a different curve. The ability for the sugar to undergo glycolysis was dependent on its ability to accept a phosphate group during phosphorylation. Therefore the sugar molecules most easily able to accept a phosphate group produced the most CO₂.

Introduction

Everyone knows that yeast makes dough rise, but exactly why does this happen? Sugars are broken down through the process of glycolysis (Black, 1999). In order to begin this catabolizing (breaking down) process, the sugar must first gain a phosphate group, which is called phosphorylation. Often the phosphate group is gained from ATP. Once the sugar has undergone these processes, it will begin to ferment and yield a byproduct of CO₂. Buchner (1897) performed experiments using a yeast “juice” and different sugars such as can sugar, glucose, fructose, and maltose. When these two ingredients were combined, they reacted to produce CO₂ for days. When the yeast juice alone was heated, it would also produce CO₂. He also noticed that some sugars did not undergo the fermentation process with the yeast juice to produce CO₂. In this study, different sugar solutions were combined with a yeast solution in order to see how fast and if they react to produce CO₂.

Materials and Methods

A yeast solution along with three sugar solutions and laboratory instruments and supplies were distributed. The three sugar solutions included mannose, fructose, and glucose. A fourth sugar solution had to be concocted by measuring 1 g of raw galactose on a top loading scale and mixing it with 10 ml of distilled water measured in a graduated cylinder. The galactose and water were mixed in a test tube. All of the sugar solutions were 10% solutions.

Using a micropipettor, 2 ml of yeast solution and 2 ml of the first sugar solution, mannose, were measured and mixed together in a test tube. After being combined, a Pasteur pipette was filled with 1 ml of the new solution. “Play-Doh” was put on the tip of the pipette so that CO₂ would not be able to escape and also to create vacuum so the solution would not fall out of the pipette. The pipette was then placed upside down in the test tube with the remaining solution of yeast and mannose. The pipette was then observed every 5 minutes to see if any CO₂ had accumulated at the top. Any progress was recorded. This process was repeated with the rest of the sugar solutions. A control sample was also tested, using distilled water instead of a sugar solution. All of this was done at a room temperature of about 20º C.

Results

Table I:

Graph I:

Out of the samples that did produce CO₂, the mannose sample took the longest to begin producing CO₂ (Figure 1). It took 25 minutes until it started. Its biggest jumps in CO₂ production were between the 20 minute and 25 minute, and 25 minute and 30 minute marks. This was when right when it began producing CO₂. Its CO₂ production increased steadily until the 65 minute mark when production started to even out.

The fructose sample took 20 minutes to begin producing CO₂, which was second fastest out of the samples that did produce CO₂. The fructose had its highest CO₂ production in the first three time intervals it began producing CO₂. After that it steadily increased and only seemed to slightly begin to even out production of CO₂. It produced the most CO₂ out of all the samples.

The glucose sample started producing CO₂ the earliest out of all the samples, at the 15 minute mark. It increased production of CO₂ steadily until the 45 minute mark when production suddenly spiked in production for 15 minutes. The CO₂ production looked like it may have been starting to even out when recording stopped.

Galactose and the control sample did not produce any CO₂ at all. They remained at 0 mL of CO₂ production throughout the whole experiment.

Discussion

Prescott and co-workers (1999) took a look at the chemical reactions between microorganisms and carbohydrates. They noted that the sugars glucose, fructose, and mannose are all catabolized, or broken down, through the process of phosphorylation, which is process of adding a phosphate group to a molecule (Black, 1999). Most commonly ATP adds the phosphate group to the sugar molecule, and then it is able to enter glycolysis, which is the process of breaking down a sugar. Once in the process of glycolysis, the sugar will begin to ferment. One common byproduct of fermentation is CO₂.

In this experiment, glucose, fructose, and mannose were the only sugar solutions that produced CO₂. As described by Prescott and co-workers (1999), those are the only sugars that are able to be broken down through phosphorylation. The yeast must have had ATP in it, which would have added a phosphate to the sugar molecules. After gaining the phosphate, the sugars began to break down, ferment, and produce CO₂.

Prescott and co-workers (1999) also noted that in order for galactose to be catabolized, it must first go through a three-step process to be converted into a type of glucose. In the experiment, the galactose solution did not react with the yeast to produce any CO₂. This is because the galactose was not converted, so therefore it could not gain a phosphate and begin the process of phosphorylation to break down, undergo glycolysis, ferment, and produce CO_2.

Lastly, the control group did not produce any CO₂ either. That is because there was nothing for it to react with and it was at room temperature. If the yeast was heated, it would have given off CO₂ (Buchner, 1897).

Literature Cited

Buchner, E. 1897. “Alcoholic Fermentation without Yeast Cells”.

Prescott et al., 1999. “Catabolism (breakdown) of Carbohydrates”.

Black, 1999. “Glycolysis and Fermentation”.

Table I

Table II

Table III

Table IV

As each egg size got bigger, its measurements did indeed get bigger (Table I). For example, none of the measurements for the large eggs were smaller than the measurements for the medium eggs (Table I). The two eggs measured for each size were each about the same length, width, and mass. Their measurements only differed by at most 1.65 mm in length, 0.74 mm in width, and 2.5 g in mass (Table II). The greatest factor is separating the different eggs sizes was mass. The average masses for the two eggs measured for each size differed the most between egg sizes than their lengths or widths (Table IV).

A half dozen eggs were distributed to each team. The six eggs consisted of two medium sized eggs, two large sized eggs, and two extra large sized eggs. Each egg was labeled with a number and letter in order to keep track of it. Upon being distributed, the eggs were measured with a digital caliper to find their lengths and widths to the nearest hundreth millimeter. The lengths and widths recorded were the largest lengths and widths found. After dimensions were taken, the eggs were then weighed on a digital top loading scale. The weights were recorded in grams to the nearest tenth. The class’ data was then entered into a spreadsheet on the computer and the lab was completed.