Molecular Properties of Enzymes

Tavakoli

The Molecular Properties of Enzymes

Sean Tavakoli

March 8, 2010

Section: A020

Abstract:

In this experiment, the effects of boiling and inhibitors on enzyme activity were studied. Methods used to carry out this study were the use of an indicator substance, methylene blue, and qualitative measurements of the changes in color that occur. Structurally similar substances were used as substrates and inhibitors in order to allow competitive inhibition to occur. A pH buffer was used in order to keep the pH constant for each sample observed. All of the test tubes were exposed to the same temperature so that all enzymes have the same rate of reaction, however for another set of experiments I changed the temperatures around to determine if heat had an effect on enzyme efficiency. The variables in this experiment were whether the enzyme was fresh or boiled and the presences of substrate and inhibitor. I hypothesized that the presence of an inhibitor hinders an enzymes ability to react with substrate. Also, that after boiling, an enzyme is unable to catalyze a reaction (Frankel 2009 et al pg 32 and 53)

Introduction

One of the most essential inferences to existence is credited to the rate and efficiency of chemical reactions that occur within an individual cell. In order for many of these reactions to be successful, they need a unique type of globular protein recognized as an enzyme. These macromolecules act as a biological catalyst and can expedite reactions without being used up in the process. They also possess the ability to lower the amount of energy the reactants need to absorb in order for a reaction to commence (Campbell et al 2008 pg 153).

The reactant to which an enzyme acts on is called a substrate. Substrates bind to the region of an enzyme called an active site, which forms an enzyme-substrate complex. While the enzyme and substrate are joined together, a catalytic reaction occurs and converts the substrate to a product. The product is usually a broken down version of the substrate/reactant. An example of this would be hydrolysis. The reversible reaction, if the correct enzyme is used, is called dehydration. Enzymes are very specific to the type of molecule that they are reacting with. The active site will only bind with one very specific type of substrate, similar to that of a lock and key. This means that for our body to function we would need thousands of different types of enzymes to speed up the reactions that go on in our body (Campbell et al 2008 pg 154-156).

The thousands of different types of enzymes in our body not only specialize in one specific type of substrate, but also work in only certain types of pH levels and temperatures. Each enzyme is most active at high temperatures as well as high concentrations of substrates. However, they only work within a narrow pH range in which each type of enzyme has their own distinct range. If an enzyme is outside of their range, then they will denature and become utterly ineffective (Campbell et al 2008 pg 155-156). Also, the temperature that an enzyme in has a huge effect. Cold temperatures can slow down the speed of reaction for enzymes, while high temperatures can increase it. Unfortunately, if the temperature becomes too high, it possesses the power to damage these macromolecule proteins. A clear cut example of this is boiling enzymes can kill, or denature them (Frankel 2009 et al pg 32).

Enzymes possess many extraordinary properties and with the help of experimentation we can better understand their characteristics. Without the help of enzymes, life would cease to exist.

Hypothesis 1: Heating an enzyme up to a certain point, can increase the rate of reaction.

Hypothesis 2: Despite enzymes working ideally at high temperatures, boiling them will destroy and denature them.

Hypothesis 3: Competitive inhibition can slow down the rate of a reaction for enzymes.

Hypothesis 4: Enzymes posses the ability to accelerate chemical reactions, however in order for them to function, they need substrates to react with.

Materials and Methods

Hypothesis ...

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Enzymes possess many extraordinary properties and with the help of experimentation we can better understand their characteristics. Without the help of enzymes, life would cease to exist.

Hypothesis 1: Heating an enzyme up to a certain point, can increase the rate of reaction.

Hypothesis 2: Despite enzymes working ideally at high temperatures, boiling them will destroy and denature them.

Hypothesis 3: Competitive inhibition can slow down the rate of a reaction for enzymes.

Hypothesis 4: Enzymes posses the ability to accelerate chemical reactions, however in order for them to function, they need substrates to react with.

Materials and Methods

Hypothesis I:

The experiment I used to test this hypothesis was how temperature effects the hydrolysis of a protein. Raw liver was prepared by the teaching assistant to use in lab. I put 1 ml of it, the size of a small breath mint, into test tubes that I labeled 10 and 11. I then measured out 6 ml of casein, which will be used as the substrate. After the substrate was added, test tube 10 was placed in an incubator that had a temperature of 25⁰C and test tube 11 was placed in an incubator that had a temperature of 37⁰C. I let the test tubes sit in the incubators for ten minutes each and let protein hydrolysis occur. After the ten minutes expired I took the test tubes out and added two drops of Calcium Chloride salt solution into each tube. The Calcium Chloride salt was used to test for the presence of protein. If a protein is present the solution will turn to a milky white color with precipitates at the bottom of the tube. The higher the concentration of precipitates that appear at the bottom of the test tube means the more proteins got hydrolyzed (Frankel 2009 et al pg 53).

Hypothesis 2:

Similar to the procedure used in hypothesis one, I tested the effectiveness of enzymes when they are boiled. The teaching assistant prepared boiled liver to use in the lab. I labeled a test tube with the number 12 to identify it from the other test tubes in different experiments. Then I added 1 ml of the boiled liver to it (about the size of a small mint or a corn kernel). Next, I added 6 ml of casein, which is used as the substrate in the reaction known as protein hydrolysis. After both were added into test tube 12, I let it incubate for 10 minutes in a 37⁰C water bath. After ten minutes I removed the test tube from the bath and added two drops of CaCl₂. This was to determine if the boiled enzymes had any reactivity to casein. If the boiled pancreatin reacted, then the solution should turn milky white. If not, it should appear in the same way that it looked like before being added to the incubator (Frankel 2009 et al pg 53).

Hypothesis 3:

Place a raw piece of heart meat the size of a corn kernel into a test tube labeled 3 for distinguishing purposes. The sample of raw meat should contain enzymes known as succinic dehydrogenase. Next, I added an assortment of substances to the test tubes in different portions. These included malonate, succinate acid, Phosphate buffer, water, and methylene blue. The malonic acid acted as a competitive inhibitor that is used to measure the effect on enzyme-substrate complex interactions. It is structurally alike to sucinnic acid and acts as an extra substrate in the reaction. The sucinnic acid was the substrate which was supposed to interact with the enzymes found in the meat. The phosphate buffer was added to the solution in order to maintain a pH of 7.4. This is so that the shifting pH could not be related to enzyme activity. Once all the components are mixed together, add three drops of methylene blue. The methylene blue can accept hydrogen atoms during the oxidation-reduction reaction and exhibit this absorption in the form of a color alteration. Once the methylene blue has been added, I used paraffin oil to prevent reoxidation from occurring. I then placed the tube in a water bath at 37°C for 30 minutes. After the time expired, I removed the test tube, mixed it around, and placed it back into the water bath for an additional 10 minutes. The reason for putting the test tube into the warm bath was to increase the activity of the enzymes by putting it in my ideal environment for reaction. After the 10 minutes was over, I removed the test tube and made observations based on the color of the mixtures inside the test tubes (Frankel 2009 et al pg 32)

Hypothesis 4:

The procedure for this lab is very similar to that of hypothesis 3. The only difference is a switch in the components being used. Place a small, fresh piece of meat (about the size of a corn kernel) into a test tube labeled 1. Add 12 ml of a phosphate buffer to regulate the pH of the reaction and 10 ml of water to equalize the volume of the test tube to that of the volumes of the other test tubes in my previous hypotheses. After these components have been added, mix well and add three drops of methyelene blue to the solution. Seal the test tube with paraffin oil, which keeps the atmospheric oxygen from getting in and prevents reoxidation. Next, place the tube in the water bath at 37°C for 30 minutes. After the time runs up, remove the test tube, mix it around, and place it back into the water bath for an additional 5-10 minutes. Finally, record any observations that happened in the experiment (Frankel 2009 et al pg 32)

Results:

After completing each of my experiments, I recorded my observations into a table. For the experiments I did to test my first and second hypotheses on enzymes, I looked for a milky color change as well as if precipitates formed at the bottom of the test tube. This is shown in table 1-1. For the experiments that tested my third and fourth hypotheses I looked for a translucent color change. These results are summarized in figure 1-2.

Figure 1-1

Figure 1-2

In figure 1-1 a precipitate reaction occurred in test tubes 10 and 11, while no reaction occurred in test tube 12. The precipitate reaction can easily be recognized by the fact that precipitates (solids) were forming at the bottom of the tubes. Originally, test tubes 10, 11, and 12 all were the same clear color before any reaction occurred. This shows that test tube 12, which remained the same clear color even after being placed in the incubator, had no precipitates forming. One can concur from the result of test tube 12 that no reaction occurred at all. The information shown in Figure 1-2 shows that an oxidation reaction occurred in test tube 3, while no oxidation reaction occurred in test tube 1. The fact that an oxidation reaction occurred can be acknowledged by whether a color change occurred in the test tube. Originally both test tubes had a blue color and if an oxidation/reduction reaction occurred, the color of the test tube will become a lighter shade of blue.

Discussion:

In test tube 1 and 12, no reaction occurred based on the observation of the test tube colors. In test tube 1, no reaction occurred, because there was no substrate present for the enzyme to react with. Based on that, I can conclude that in order for an enzyme to work, substrate molecules must be present. In test tube 12, no reaction occurred when there was a substrate present. However, the enzyme was boiled prior to the start of the experiment. Therefore, I can conclude that enzymes become denatured and are unable to catalyze reactions when boiled at very high temperatures (Frankel 2009 et al pg 32)). These results showed that my hypothesis of boiling an enzyme will not allow it to function properly was correct.

In test tube 3, a partial color change occurred. The shade of blue lightened noticeably. This means that an oxidation-reduction reaction did occur, but there were still many substrates that were unable to fully react. Based on the information that I have found, I concluded that in the presence of an inhibitor, when an enzyme and substrate are both present, the reaction is slow or does not proceed to equilibrium (Frankel 2009 et al pg 32). My hypothesis, that the presence of an inhibitor reduces an enzyme’s effectiveness is true.

In test tube 11 and 12, a reaction did occur, based on the color properties that occurred over the time that it was in incubation. Test tube 12 however, had more precipitate at the bottom than did test tube 11. Additionally, while watching both test tubes incubate, I noticed that test tube 12 reacted much faster to form a milky white color than test tube 11 did. Both of these test tubes had the same amount of substrates, time of incubation, and other components, the only difference being that test tube 12 was placed in a warmer temperature environment to incubate. This goes hand in hand with my theory that most enzymes, to an extent, work more efficiently in warmer temperature environments than they do in colder ones.

The possible sources of error in the lab could have been the amount of enzyme used. There was not an exact amount, because they teaching assistant simply said to grab a small pinch of the liver meat. This means that there could have been more enzymes than inhibitors in test tube 3. The reaction would still proceed if that was the case, but at a slower rate. To fix this possible source of error, a set mass of meat that would be weighed out with an accurate and precise scale could be added to each test tube, rather than just a visually similar amount.

Acknowledgments:

I would like to thank my teaching assistant, Dean Able, for helping explain to me each of the lab procedures. My lab partner, Scott Hoover, was also a great help to me. Together we were able to tackle the labs and understand very important enzyme concepts. Also if there was something I did not know and my teaching assistant was busy, Scott would help answer my problem.

References:

Campbell, N and Reece, J. (2009). Biology Eighth Edition. Pearson Education, Inc., publishing as Benjamin Cummings, San Francisco, CA.

Frankel, Williams, Irish, and Stefaniak (2009). Principles of Biology I Laboratory Manual Sixth Edition. Pearson Custom Publishing, Boston, MA.

Introduction to Enzymes. Retrieved February 20, 2007 from http://www.worthington-biochem.com/introBiochem/introEnzymes.html