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Scientific Investigation of the Peroxidase Enzyme & Temperature Essay

Abstract: 

In this lab we tested the effect temperature has on the rate of enzyme activity. The way we figured this out was by taking four different temperatures and testing the different absorbance levels they produced every 20 seconds for two minutes straight using a spectrophotometer. The important part of this experiment was the temperature the enzyme concentration was made at. What we got from the experiment was at lower temperature we got very low numbers for the absorbance, which gave us a lower rate for the enzyme reaction to complete, and visa versa for higher temperatures. From our results we saw that the higher the temperature the enzyme concentration was held at the faster the reaction was produced. Therefore concluded that enzymes work faster at higher temperatures and slower at lower temperatures.

Introduction:

An enzyme is a substance produced by a living organism that acts as a catalyst to bring about a specific biochemical reaction. Enzymes are generated by our body, and are usually categorized as proteins. They speed up reactions and have a structure that is important to its function. It has an active site, which is opened until a substrate binds to it and activates it to play a specific function in a chemical reaction. In biology terms a substrate is the surface or medium on which an organism grows or is attached.

In addition the chemistry term for a substrate is simply the substance that is acted upon by an enzyme or the molecules at the beginning of the reaction. A great example of an enzyme and substrate reaction is curd formation, which is a reaction where rennin is added to milk. In this example the substrate would be milk and the enzyme would be rennin. At the end of the reaction a product is made. A good way to memorize what happens in these chemical reactions is by symbols, such like this:

Enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. In this experiment your enzyme extract will be the horseradish mixed with the citrate-phosphate buffer and the substrate will be H2O2. These will be kept separate until mixed in different temperature surroundings.

A lot of factors contribute to the way an enzyme works in a cell. For example temperature, PH, inhibitors, enzyme concentration, and substrate concentration all contribute to the way enzyme reactions occur. The main focus on this experiment is temperature. Just like us human’s enzymes have a desirable temperature they like to work under. Furthermore heat enhances the kinetic energy of the participant molecules, which results in more number of collisions between them. These collisions of molecules cause the reaction to happen better and quicker. So if he environment is cold instead of hot then the molecules move slower causing a less chance of these molecules colliding.

Meaning in hotter temperature the reaction will be faster than in colder environments. Even though this is true in extreme hot temperature the opposite occurs, which the enzyme becomes denatured and fails to carry out its function. Denature is when something unfolds and cannot follow up with its function due to its structure. A good temperature for enzyme reaction is our body temperature. Here we want to test this and make sure that with different temperature the warmer it is the more robust the reaction will be.

Scientist are constantly trying to catalyze reactions using enzymes. Catalyze is another way of saying accelerate a reaction. Recently, “A PLP-dependent aminotransferase PctV, encoded in the pactamycin biosynthetic gene cluster, was found to catalyze the formation of 3-aminobenzoate from 3-dehydroshikimate with L-glutamate as the amino donor. The PctV reaction comprises a transamination and two dehydration reactions. This is the first report of a simple 3-ABA synthase in nature” (Pub Med). In this lab you will experience a simple enzyme reaction using horseradish and temperature.

Material and Method:

When doing this lab we started off by making an enzyme extract using horseradish, citrate-phosphate buffer, and a blender. When cutting off a small piece of horseradish with a knife and measuring it to about one gram we placed it in the blender with about 100 mL of the buffer. Once both are placed in the blender we blended it for about 15-20 seconds. After this was blender we used a double layer of cheesecloth to pour what we blended into a beaker so any left over chunks of horseradish was left out.

This was labeled our enzyme extract. We use this throughout the lab experiment. Next we labeled another graduated cylinder “buffer” which contains citrate-phosphate ph 5 and then have two dispensers of H2O2 and Guaiacol solutions. Using all these solutions/buffer we filled nine test tubes with the following the chart below, remembering to make sure we use test tube number one as a blank for the spectrophotometer. The spectrophotometer is a machine used to calculate the Absorbance. Even though we are calculating the rate dependent on the temperature in its environment we can calculate rate using absorbance and the time. The formula for rate is:

Absorbance Final-Absorbance Initial
Time Final-Time Initial

In order for the experiment to work we first set the Spectrophotometer a 500 wavelength and blanked it using our blank test tube. In the lab room we had certain environments to make sure the enzymes reactions would occur. We had a bucked of ice, room temperature, body temperature, and boiling point. Each time before putting the test tubes in the spectrophotometer we placed the test tube in the ice then the next test tube we tested in the body temperature bucket etc. *Note – Once we mixed the test tubes one with the enzyme and one with the substrate we started the timer.

We got the absorbance for each mixed test tube for two minutes marking each absorbance 20 seconds. After getting all the numbers we need we can calculate the rates for each enzyme reactions that were in different temperature settings using the formula above. Here we can see if our hypothesis and predictions were correct or false. Remember we are testing for Rates in different temperature settings.

Cite:
Symbiosis, Biology Department, Middlesex County College, General Biology I, BIO 123

Results:
When completing the experiment we saw that as we went from freezing to boiling absorbance increases dramatically as you see in Figure 2 below. This means that the higher the absorbance the higher the rate as shown in Figure three.

As you see in the final results in figure 3 (Rate vs. temp) the fastest reaction would occur in a boiling environment and slowest in the freezing environment. However the Room Temperature would be the best fit for enzyme activity based on Figure 2 data and Chart 4. Reason being because in the Graph (Chart 4) the highest peak is at room temperature with a rate of .0042. 

Figure 3: Rates Vs Temp

Figure 4: Chart – Temperature vs. rates


This chart 4 also backs up with what I was trying to explain earlier in the Introduction. Even though I am stating that as temperature increases so does rate of enzyme activity at extreme temperature enzyme rate will decrease due to unfolding of the enzyme. You see this happening in Chart 4 (Temp vs. rate) where it reaches its max at Room temperature and stays high even at body temperature but decreases dramatically when it reaches boiling.

Discussion:

In this lab we were trying to see the affects different types of temperature have on an enzyme reaction. We predicted that the higher the temperature the faster the reaction meaning the darker the solution would be inside the test tube. We saw that each time the temperature increased the rate of reaction was faster. We could tell by the darker the reaction the faster it occurred. In the coldest temperature we saw that the enzyme reaction was a lighter color compared to the other three test tubes. Even though there was some human error involved it was very minor, which kind of messed up our results. This happened due to lack of preparation of the spectrophotometer before we stared mixing test tubes two and three, which threw off our time for a difference of about 10 seconds.

Even though we tried to adjust our time difference we noticed the test tubes color and numbers for absorbance were a bit off for the temperature it was put in, which was the freezing temperature. Even though this occurred our hypothesis and predictions still concurred with what we expected to happen based on simple knowledge. We already knew that at higher temperature the faster the molecules inside the solution move creating a higher chance of them colliding and creating a reaction, and for lower temperature the slower the molecules moves causing a slower reaction.

On pub Med I found something similar to the concept of our experiment. It stated, “The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme” (Pub Med). Here it shows at higher temperature this D503F mutants has a longer half life just like our test tubes have a faster reaction at higher temperatures.

References:

http://www.ncbi.nlm.nih.gov/pubmed/23624477

http://www.ncbi.nlm.nih.gov/pubmed/23744829

http://linus.chem.ku.edu/hewlett/Chem188/Enzyme/enzyme_background.htm

http://www.anyvitamins.com/enzymes-info.htm

Symbiosis, Biology Department, Middlesex County College, General Biology I, BIO 123

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