The purpose of this lab was to test if ethanol affects the reaction involving hydrogen peroxide and catalase. Tests were performed by putting chicken liver, ethanol solution (diluted ethanol solution for other trials) and hydrogen peroxide in a test tube with a side arm, and having a rubber tube lead the oxygen gas into a gas collection tube.
Results from the tests showed a negative correlation, this means that the more diluted the solution of 95% ethanol was, the less oxygen gas collected. Controlling a number of factors which include human error, temperature change, pH levels, substrate concentrations and ensuring a controlled experimental environment will work to increase the accuracy of the experiment. Purpose/Hypothesis
If the concentration of the 95% solution of ethanol was decreased, then the volume of oxygen gas collected through the reaction of hydrogen peroxide and catalase would decrease. Introduction The purpose of this lab is to see how various substrates such as ethanol affect reactions involving catalase. Enzymes are made up proteins; they function as a catalyst to accelerate chemical reactions without being consumed in the process. The enzymes lower the activation energy required and decrease the time needed for the reaction to take place.1 Enzymes are very specific to their substrates (reactant that the enzyme makes into a product).
They also have specific optimums, such as temperatures and pH levels. Catalase is a very common enzyme which is found in almost all living organisms that come in contact with oxygen, as it functions as a catalyst to decompose hydrogen peroxide (H2O2) into water (H2O) and oxygen gas (O2).3
Catalase is a very efficient enzyme for this process, as one molecule of catalase can convert 40 million molecules of hydrogen peroxide into water and oxygen per second. The effect that ethanol has on a functional liver is ultimately damaging it by causing inflammation and scarring through the production of toxins and reactive oxygen species (ROS) when consumed in excess.4 Ethanol cannot be removed and must be metabolized by the liver. The ethanol metabolism will eventually produce a toxic by-product such as acetaldehyde; it can eventually attack the liver and can also account for cirrhosis.
But the chicken liver that is used will not be able to metabolize the ethanol since it is non-functional. The liver contains cells called hepatocytes; the breakdown of ethanol requires multiple enzymatic processes in these cells, which requires the organism to be alive to activate these enzymes.6 Since the chicken liver is not a live sample, these enzymes are not able to perform its functions, therefore it is non-functional. Materials and Methods
6g of chicken liver was mixed with 6mL of distilled water and crushed up in a mortar and pestle. A 50mL gas collection tube was clamped onto a retort stand with a burette clamp, on top of a 1000mL beaker filled with water.
A rubber tube was attached to a test tube with a side arm, with a J-tube attached pointed into the gas collection tube. 5mL of 95% ethanol was measured with a syringe and poured into the test tube with a side arm. 1g of the crushed up chicken liver was weighed and put into the tube along with the ethanol. 3mL of 3% hydrogen peroxide was poured into the test tube with a side arm and capped immediately with a rubber stopper. The volume of oxygen gas was observed and recorded. The same steps were repeated by diluting the 5mL of 95% ethanol solution with 10mL of distilled water, this would make new concentration that is 32% ethanol (using the equation C1V1= C2V2). C1V1= C2V2
C1 = 95% ethanol = 0.95
V1 = 5mL
V2 = 5mL + 10mL = 15mL
(0.95) (5mL) = C2 (15mL)
0.3167 = C2
Therefore the new concentration is 31.67% ethanol.
The above calculation was used to calculate the new concentrations for 20mL, 30mL, 40mL and 50mL of water when added to 5mL of 95% ethanol.
After performing 6 trials, the results displayed a positive correlation, meaning that each time the ethanol concentration was decreased, the volume
of oxygen gas collected decreased. When diluting the ethanol with distilled water, the concentration of the ethanol decreased, and the volume of oxygen gas collected through the reaction of hydrogen peroxide and catalase decreased. (See figure 1)
1.The environmental factor that had an effect on this experiment would be altering the substrate concentrations. Substrate concentration means the number of substrate molecules in a solution, while enzyme concentration means the number of enzymes. For the substrate and enzyme reactions, only one enzyme can react with one substrate molecule at a time, this means that if you increase enzyme concentration, more substrate-enzyme reactions can occur (also vice-versa).
With more molecules floating around, there is a higher chance of the substrate molecules and the enzymes colliding into one another.8 2.There were many control groups in this experiment, one being the temperature of the solution. Keeping the temperature of the solution the same throughout the whole experiment would be important since increasing the temperature would cause the molecules of the solution to move faster as the molecules move faster within the solution, allowing higher chances of collisions. Increasing the temperature of the solution too much would cause the denaturing of the enzymes.
Another control group would be the mass of chicken liver used each time. Since the chicken liver contains the enzyme catalase, increasing the mass would increase enzyme concentration, allowing more reactions to occur. 3.Some things that have gone wrong during the experiment include pouring in the hydrogen peroxide into the test tube, and not being able to close the test tube fast enough, allowing some oxygen gas to escape instead of going through the rubber tube into the gas collection tube. Another thing would be while measuring the 1g of chicken liver on a scale and pouring it into a test tube, some of the chicken liver would get stuck onto either the plastic container holding the chicken liver, or it would get stuck on the side of the test tube.
This would not allow the chicken liver to fully react with the catalase enzyme. A big factor to the results of the lab would be the ethanol and the chicken liver itself. Ethanol contains alcohol, which cannot be removed and must be metabolized by the liver. As the liver breaks the alcohol down, many by-products such as acetaldehyde are formed, which can be extremely dangerous to the body than the alcohol itself. These by-products will eventually aggress onto the liver and cause cirrhosis, which is an abnormal liver condition that could ultimately stop liver function.10 However, since the liver that was used in this lab was an entirely non-functional chicken liver, the liver cannot metabolize the ethanol, therefore no toxic by-products can be produced to attack the liver.
This means that the ethanol diluted with distilled water only diluted the hydrogen peroxide in this reaction, decreasing the concentration of the hydrogen peroxide. 4.Ectothermic organisms are animals that cannot control its own body temperature; it is instead controlled by its environmental surroundings.11 We can use reptiles as an example, to stay warm, reptiles have to sit in the sun all day to keep warm, and during night time, they used stored heat to stay warm.
To lower their body temperatures, they must find a source of shade, for example under a rock. These changes in temperature will also affect the enzymes in ectothermic organisms; when increased, the enzymes will have a higher reaction rate, while decreasing in temperature below the optimal temperature will result in a lower reaction rate. Again, too much increase in temperature may lead to the denaturing of the enzymes, but through natural selection, animals today have evolved to have the enzymes in their bodies function at the range of temperatures that the animal experiences.13 Acknowledgements
I would like to thank Brian Vu, Kevin Huynh, Emre Koc, and Mr. Hedley for providing their assistance and support on this experiment/article.
1.Stephanie Strong. Enzyme Lab. . September 29, 2013.
2.Novozymes. What are Enzymes? . September 29, 2013.
3.Princeton. Catalase. . September 29, 2013.
4.July 2013. Drinkaware.co.uk. Alcohol and liver disease. . October 2, 2013. 5.November 20th, 2005. The Independent. What alcohol really does to your body. . September 29, 2013. 6.Carol C. Cunningham and Cynthia G. Van
Horn. October, 2004. NIH. Energy Availability and Alcohol-Related Liver Pathology. . October 2, 2013. 7.Chartgo. < http://goo.gl/UvfbW0>. October 2, 2013
8.wiseGEEK. What Is Substrate Concentration? < http://www.wisegeek.com/what-is-substrate-concentration.htm>. September 30, 2013. 9.Worthington. Temperature Effects. < http://www.worthington-biochem.com/introbiochem/tempeffects.html>. September 30, 2013. 10.November 26, 2009. MNT. What Is Cirrhosis? What Causes Cirrhosis?. . September 30, 2013. 11.Jennifer Kennedy. Marine Life. Ectothermic. . September 30, 2013. 12.Joseph Chiaro. November 14, 2006. Thermoregulation. . September 30, 2013. 13.Tesha. May, 2007. Ectoderm and Enzymes. . September 30, 2013.