Investigation 13B

Introduction
On Friday, September 4th, a lab group consisting of Vinay Ayyappan, Shreyan Jain, Vikram Vasan, and myself were tasked with observing and tracking the rate of reaction for the enzyme peroxidase breaking down the harmful toxin hydrogen peroxide in differing conditions of pH and temperature. Hydrogen peroxide (composed of 2 hydrogen and 2 oxygen atoms) is a naturally occurring toxin that will break down in sunlight, but very slowly. Because it is harmful to quite a few organisms, many have developed ways to break it down with the use of enzymes such as peroxidase. Humans have this natural defense mechanism, but we took our peroxidase from ground-up turnips because turnips are readily available and people don't take too kindly to being ground up. To measure the rate of the reaction between the enzyme and hydrogen peroxide, we decided to use the alcohol guiacol as an indicator because, in the presence of oxygen gas (which is a product of the reactions between peroxidase and hydrogen peroxide), molecules of guiacol bind with three others to form tetraguiacol. Whereas hydrogen peroxide, peroxidase and guiacol are all clear in solution, tetraguiacol is dark brown in solution, so as the reaction progresses, more tetraguiacol will be produced, thus turning the solution a darker shade of brown.

The problem with simply using tetraguiacol as an indicator and watching as the solution turns a darker brown is that this is only a qualitative set of data. It is impossible to write down that the solution is "dark brown" because dark brown could be any number of shades of brown for different people. It is an inexact way of measuring data, so to change this qualitative data into quantitative data, we used a "colorimeter". This small device takes a sample of the solution in a specially crafted tube and shoots a certain wavelength of light through it, and then photoreceptors on the other side of the chamber record how much light made it through the solution and present this data as a percentage. Therefore, if the solution is darker, less light will find its way through and then we will find a decreasing numerical value as the percentage of light making its way through the chamber shrinks as the solution gets darker.

There were multiple purposes to this experiment. The first, and most obvious, being to measure the rate of reaction between peroxidase and hydrogen peroxide in normal conditions. This reaction would be our control reaction because the reaction would occur at a pH of 7 (normal, distilled water) and at room temperature. The other two purposes of this experiment were to observe the changes in the rate of reaction at different pH's (5, 6, and 8) and at different temperatures (0 degrees Celsius and 37 degrees Celsius). Because peroxidase is an enzyme within our own bodies, we, as a lab group, hypothesized that the rate of reaction would be fastest at a temperature of 37 degrees Celsius because the molecules would be hitting each other faster and there would be more energy to push the reaction along, and at a pH of 7 because humans are not particularly acidic or basic.

Procedure
Control

1. Prior to mixing the test tubes together, we filled one of the colorimeter tubes up with regular, distilled water and tested the calibration to make sure that it read 100% of the light got through for water.
2. We then took 2 test tubes and marked one "E" for enzyme and the other "S" for substrate.
3. For the substrate tube, we added 7mL of distilled water, 0.3mL of 0.1% hydrogen peroxide solution, and 0.2mL of guiacol for a total volume of 7.5mL and then upended it once to mix the ingredients, using Parafilm as a stopper.
4. For the enzyme tube, we added 6mL of distilled water and 1.5mL of peroxidase that we had gathered from turnips for a total volume of 7.5mL. We then upended it once, again using Parafilm as a stopper.
5. Then we combined the contents of both tubes, upended the combination twice using a stopper of Parafilm and then began timing the reaction.
6. We then moved a few milliliters into a new colorimeter tube and immediately put it into the colorimeter.
7. At 30 second intervals for 5 minutes we recorded the percentage of light getting through the solution in the colorimeter and took pictures of the test tubes as it gradually changed color.
8. After 5 minutes we discarded the tests tubes and moved onto testing pH's.

pH Change for pH 5, pH 6, and pH 8

1. Repeat steps 1 through 3 of the control procedure for testing the colorimeter and mixing the substrate tube.
2. For the enzyme tube, we added 6mL of a buffer solution with pH 5 to the test tube and then added 1.5mL of peroxidase for a total volume of 7.5mL. We then upended the solution once with a stopper of Parafilm to properly mix the contents.
3. Repeat steps 5 through 7 of the control procedure, using the newly created tubes.
4. We then repeated the procedure for the pH test tubes with a buffer solution of pH 6 and another buffer solution of pH 8.
5. After we were done we discarded these test tubes and moved on to testing the effects of temperature change on the reaction.

Temperature Change

1. Repeat steps 1-4 of the control procedure, producing the enzyme test tube and the substrate test tube.
2. Then dip the test tubes into an insulated cup filled with water of temperature 0 degrees Celsius.
3. After 3 minutes, combine the test tubes and upend twice, using Parafilm as a stopper.
4. Move a few milliliters to a new colorimeter tube and immediately put it into the colorimeter.
5. At 30 second intervals for the next 5 minutes we took pictures of the color change in the test tube and recorded the percentage of light getting through the solution as displayed by the colorimeter.
6. After 5 minutes, discard the test tubes and repeat the entire procedure, substituting 37 degree Celsius water instead of 0 degree Celsius water.

Data/Results

This is a data table of all the percentages as measured by the colorimeter for each experiment, including the time at which each measurement was taken:

Time/Experiment	Control	pH 5	pH 6	pH 8	0⁰C	37⁰C
0:30	51.3%	52.1%	31.0%	64.2%	29.1%	40.9%
1:00	43.7%	37.1%	24.0%	56.5%	23.4%	30.7%
1:30	37.5%	28.4%	18.9%	50.9%	21.9%	24.9%
2:00	32.9%	22.4%	13.5%	45.8%	21.6%	19.9%
2:30	29.1%	17.7%	11.0%	42.0%	21.4%	16.8%
3:00	26.0%	14.7%	9.3%	38.1%	23.2%	14.3%
3:30	23.5%	12.5%	7.9%	34.8%	23.0%	12.4%
4:00	21.3%	10.7%	6.8%	32.0%	23.0%	10.9%
4:30	19.7%	9.4%	6.1%	29.4%	22.9%	9.7%
5:00	18.2%	8.3%	5.4%	27.2%	22.7%	8.8%

Here is a graph of the visibility through the drakening solutions vs. time:

Here are the pictures that show the color change of the solution in the test tube for each trial:

Trial/Time	0:30	2:30	5:00
Control
pH 5
pH 6
pH 8
0⁰C
37⁰C

Conclusions
For the control reactions, in a mere 30 seconds, the percentage of light getting through the solution was 51.3%, by 2:30 it had reached 29.1% and by the end of the reaction the percentage of light getting through was only 18.2%. For a pH of 5, the reaction was even speedier, going from % at 30 seconds to 17.7% at 2:30. The final visibility was even less than the control, a minuscule 8.3%. The reaction was even faster at a pH of 6, hitting only 31% visibility after 30 seconds! Then, it dropped to tiny 11% visibility at 2 minutes and 30 seconds, ending at a measly 5.4% visibility after 5 minutes. It seems as though our initial prediction of the reaction working best at pH was wrong, that instead peroxidase works best at a slightly acidic pH. The pH cannot be too acidic though, because at only one pH point lower (which is 10 times more acidic), the reaction slowed quite a bit. The slightly basic pH 8 reaction was slower than the control, so that must mean that the enzyme hates basic conditions.

As for temperature, the reaction at 37 degrees Celsius was at 40.9% visibility after only 30 seconds, and then hit 16.8% visibility after another 2 minutes, finishing off at 8.8% visibility after 5 minutes total. The colder reaction started off incredibly fast, reducing visibility in the colorimeter to only 29.1% after 30 seconds, but then it slowed substantially, reaching 21.4% visibility after 2:30 and finishing off with a confusing 22.9% visibility after 5 minutes, higher than what it had previosuly been. We can safely say that the warmer reaction was much faster than the control, so peroxidase likes to be in higher temperature (but obviously not too hot otherwise the enzyme would break apart). For the 0 degree Celsius reaction, our results were very strange. At the 30 second mark, the visibility was much lower than any other reaction thus far, including the fastest, the pH 6 reaction. Then the visibility in the colorimeter went down very quickly and then rebounded a bit between the 2:30 and 3 minute marks. This could have been due to a variety of factors, such as equipment malfunction, or the more likely misreading of the device. This is proven by the photographs that we took which show that the color of the colder reaction is not as dark as the color of the warmer reaction after 30 seconds, so therefore there must be some error. But the final darkness for the colder reaction after 5 minutes was higher than both the control and the 37 degree trials, which makes sense because at lower temperatures, the molecules are moving slower and will bump into each other less for the reaction to occur. This is due to the fact that at higher temperatures, there is more energy in the solution, so the bonds will break much faster. Thus, our hypothesis about temperature was proven correct.

Our group decided to consider the lab a success because we were able to observe the effects of pH and temperature that we were looking for, seeing that these variables do affect the reaction of peroxidase and hydrogen peroxide. We discovered that the enzyme prefers to be in lower pH ranges yet still close to neutral 7, but can also function in more basic or extreme pH ranges, such as 8 and 6. Our hypothesis about the enzyme preferring a neutral pH was proven wrong, so that must mean that the area in which the enzyme is produced or used by the body must be somewhat acidic so that it functions at the most efficient rate possible to defend the body. We also learned that the enzyme peroxidase prefers higher temperatures over freezing temperatures because the reaction sped up at 37 degrees Celsius in comparison to both the control and our trial with the temperature at 0 degrees Celsius. This proved our hypothesis about temperature correct because we believed that the enzyme would work best near body temperature (which is 98.6 degrees Fahrenheit or 37 degrees Celsius) because it is an enzyme contained in the human body.