Enzyme Lab

Reetika Kashyap
September 20th, 2012
Enzyme Lab What is an enzyme? Enzymes are specialized protein molecules simplifying most of the body's metabolic processes such as, supplying energy, digesting foods, purifying your blood, executing the body of waste products etc. Enzymes act as catalyst by speeding up the reactions that happen in our bodies and decreasing the amount of activation energy needed to break a complex down. A reactant is any given enzymatic reaction is called a substrate for that specific enzyme. The place where the substrate fits and where catalysis occurs is known as the active site. The reason a specific enzyme will combine only with one kind of substrate is that the fit among the two is very precise. In fact, the fit is so precise, that the blend between the two results in a slight change in the shape of both. This precise fit that adjusts both original molecules is known as an induced fit. There are three ways in which enzymes are regulated, competitive and non-competitive inhibition, allosteric regulation and feedback inhibition. Competitive inhibition involves chemical compounds that bind to the active sit of the enzyme and inhibit enzymatic reactions. The compound competes with the true substrate for access to the active site, which is only possible due to their similar shape and structure compared to the enzymes substrate. In non-competitive inhibition, an inhibitor molecule binds to the enzyme at a site known as the allosteric site, which prevents the substrate from binding to the active site. Molecules known as activators that promote the action of enzymes can also bind to the allosteric site. There are many activities of any enzyme that can change, depending on the number of the activators and inhibitors in its environment. This process of regulation is known as allosteric regulation. Most metabolic pathways are regulated by feedback inhibition. For example, the end products of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway thus regulating the amount of end product made by the pathways. Enzymes are important because they speed up the rate of the reaction which would otherwise be too slow to support life. The rate at which an enzyme works is influenced by several factors including the concentration of substrate, temperature, pH, salt concentration and the presence of inhibitors or activators. Activity decreases when an enzyme is exposed to conditions that are outside the optimal range. Temperature plays a common affect on enzymes because after a temperature optimum is reached, temperatures above 40-50°C can denature many enzymes a change in pH can also affect he ionization of the enzyme and disrupt normal interactions. At extreme pH concentrations weather it is highly acidic or alkaline the enzyme too will eventually denature. Most enzymes function best in the pH range of 6-8. The enzymes involved in human digestion are an exception because they only work at acidic pH levels. . Catalase is an enzyme present in the cells of plants, animals and aerobic (oxygen requiring) bacteria. It is located in a cell organelle called the Peroxisome. Peroxisomes in animal cells are involved in the oxidation of fatty acids, and the synthesis of cholesterol and bile acids. Hydrogen peroxide is a by-product of fatty acid oxidation. White blood cells produce hydrogen peroxide to kill bacteria. In both cases catalase prevents the hydrogen peroxide from harming the cell itself. Catalase is very important due to the fact that is breaks down H202 or hydrogen peroxide, which is in the cells of our living tissue. This enzyme prevents cellular damage and can prevent H202 from killing a cell in animal and human tissue. Purpose: The purpose of this investigation was to witness how the rate of enzyme activity changes due to pH levels and temperature. The influence of low, medium and high temperatures and low, neutral and high pH on enzymes. Materials: * 10 ml H202 * HCl solution * NaOH solution * Fresh human liver * 6 Test tubes * Measuring Pipette * Grauduated cylinder * Test tube holder * Test tube holders * Ice bath * Warm water bath * Boiling water bath
Procedure:
To begin the experiment, start by setting up 6 test tubes on a rack. Label each test tube from 1-6. Pour fresh human liver into each individual test-tube roughly about 2 ml. Have a solution of 10 ml hydrogen peroxide ready in a Graduated cylinder to begin the experiment. To study the effects of pH on enzyme activity there were certain steps that are needed to be followed. Let’s start with test tube number 1, Measure 5 drops of HCl which has a pH scale of 1 into the test tube. To see what type of effect pH=1 holds on the enzyme, add 2ml of substrate in the test tube, which in this case is the hydrogen peroxide (H2O2). After having done that task, shake or flick the test tube well. Flick until the reactants have been mixed thoroughly. As the product beings to fizz up, wait about 30 seconds to for the end result. After having done, record the height of the bubble column. For accuracy, Measure from the bottom to the top of the fizz and record. Following that task, move on to test tube number 2 which should be currently filled with fresh human liver. Add 5 drops of distilled water into test tube number 2. To see what type of effect pH=7 holds on the enzyme, add 2ml of hydrogen peroxide into the test tube. After having done that task, shake or flick the test tube well. Flick until the reactants have been mixed thoroughly. As the product beings to fizz up, wait about 30 seconds to for the end result. Measure and record the result. Moving on to test tube number 3, begin by adding 5 drops of the solution Sodium Hydroxide (NaOH) into the test tube filled with fresh human liver. To see what type of effect pH=12 holds on the enzyme, add 2ml of hydrogen peroxide into the test tube. After having done that task, shake or flick the test tube well. Flick until the reactants have been mixed thoroughly. As the product beings to fizz up, wait about 30 seconds to for the end result. Measure and record the result. To find the effect of temperature on the activity of an enzyme, the experiment deals with the steps as follows. Begin by taking test tube number 4 which is filled with fresh human liver and place it in a cup full of ice cold water. Wait 3 minutes for the test tube to fully absorb the cold temperature of the ice. Remove the test tube from the cup and place it back on the rack to further the experiment. To display what time of effect cold temperature may have on the enzymes, add 2ml of substrate in the test tube, which in this case is the hydrogen peroxide (H2O2). After having done that task, shake or flick the test tube well. Flick until the reactants have been mixed thoroughly. As the product beings to fizz up, wait about 30 seconds to for the end result. Measure and record the result. To continue the experiment, take test tube number 5 and gently cup it with a pair of hands. The reason for this step is to make certain that the temperature for the test tube number 5 remains at room temperature. To display what time of effect room temperature has on the enzymes, add 2ml of substrate in the test tube. After having done that task, shake or flick the test tube well. Flick until the reactants have been mixed thoroughly. As the product beings to fizz up, wait about 30 seconds to for the end result. Measure and record the result. Lastly, the final step is done to warm up the fresh human liver. take test tube number 6 and put it in a hot bath which should be approximately 80 C hot. To display what time of effect room temperature has on the enzymes, add 2ml of substrate in the test tube. After having done that task, shake or flick the test tube well. Flick until the reactants have been mixed thoroughly. As the product beings to fizz up, wait about 30 seconds to for the end result. After having done, record the height of the bubble column. For experimental accuracy, measure from the bottom to the top of the fizz and record the result. Observations:
Table A: Temperature (measurements recorded in mm) Group | Ice Water | Water | Hot Water | 1 | 136 | 123 | 27 | 2 | 121 | 145 | 1 | 3 | 142 | 115 | 30 | 4 | 135 | 87 | 13 | 5 | 110 | 115 | 75 | 6 | 105 | 119 | 10 | 7 | 103 | 41 | 37 |

Table B: pH (measurements recorded in mm) Group | pH=1 | pH=7 | pH=12 | 1 | 28 | 113 | 147 | 2 | 5 | 125 | 131 | 3 | 1 | 131 | 102 | 4 | 36 | 120 | 128 | 5 | 15 | 125 | 100 | 6 | 15 | 121 | 109 | 7 | 30 | 64 | 82 |

Graph:
Table A

Table B

Discussion: 1. Temperature affect on enzyme activity is very complex. Most enzymes function best between 35 and 45 C which is why enzymes at room temperature scored the most. At low temperatures the rate of enzyme response is incredibly slow. The molecules contain low kinetic energy and collisions among them are less frequent. A boost in temperature increases the enzyme activity since the molecules now have larger kinetic energy. But at 80C, the temperature of our hot bath, the rate of reaction starts to decrease; the enzyme molecules are gradually denatured causing the figure of the active site to change. 2. Catalase work best at temperature between 37C-39C in humans. At this temperature, the properties of this protein are stable and flexible at the optimum level. This means that the enzyme will continue to stay in its shape and work better. 3. The rate of reaction depends on the enzyme. Each enzyme has its own optimum pH. Some survive better in very acidic pH and some vice versa. In the case of catalyses, the results were predictable. It was observed that this enzyme will not withstand any acidic ph. It was able to survive and performs well in the pH of distilled water which has a neutral pH of 7. However, it had the same result when observed in the pH of 12. 4. Like most enzymes, catalase is observed working best in neutral pH. The optimum pH for catalase is around 7 (6.8 - 7.5 (pH 7) because they are proteins, which are easily damaged by very acid or alkaline conditions. There are exceptions of course. For instance any enzymes in the stomach will be able to work at very acidic conditions because of its surroundings 5. Errors in this experiment could have been some common human errors with inaccurate measurements and timing. Another one is the fact that the catalase not being frozen when received most likely affected the data. We didn’t measure the room temp. The ice water only cooled the test tube, and hardly got into the liver

CONCLUSIONS 1. The increase and decrease of a temperature setting had an effect on the rate of the enzyme-catalyzed reaction. 2. The lowest and highest level of pH had no effect on the rate of enzyme activity, but the levels between pH 4 and pH 10 caused the rate of reactions to increase. 3. The rate of enzyme activity was caused by the amount of substance put in an enzyme concentration, which had to be proportional to the available substrate. 4. The increase in substrate concentration had an effect on the velocity of the enzyme concentration.

Read more: http://wiki.answers.com/Q/Why_is_Catalase_important_in_living_systems#ixzz27cjqQ7PC

http://163.16.28.248/bio/activelearner/06/ch6c3.html http://www.all-science-fair-projects.com/science_fair_projects/105/901/69adbb37c02f0fb4f8b674fbae189d9f.html http://faculty.valenciacollege.edu/tklenk/labs/enzymelab.htm