Benedique C. ValdezBS-Chemistry IIIFebruary 10, 2015UNIVERSITY OF SAN CARLOSDEPARTMENT OF CHEMISTRY

Name: Benedique C. ValdezDate: February 10, 2015Course: BS-Chemistry IIIApproved _____________________Exercise No. 3Enzymes

AbstractEnzymes are naturally occurring biomolecules in the bodies of both plants and animals. They are essential because they are said to be the biological catalysts in bodily reactions. Enzymes are found all-through-out the bodies of living things. Examples of these enzymes are amylases and catalases. Amylase is found in the saliva and is responsible for the breakdown of complex starch molecules to simpler glucose units. Catalase on the other hand is present in both animal and plant cells and is responsible for the fixation of toxic hydrogen peroxide in both. The difference lies in the optimal temperature of the enzymes depending on the source. Animal catalase from liver have a higher optimal temperature than that of the potato filtrate catalase which can be attributed to the normal temperature of the bodies of the sources. Not only temperature but pH can dictate the activity of an enzyme. Certain enzymes act optimally at neutral pH like catalase while others like pepsin will be optimally active at low pH. Another factor that dictate the activity of enzymes in biological systems is the presence of inhibitors which either compete with the substrate of interest or destroy the form the active site of the enzyme by binding in another part.IntroductionEnzymes are abundant in all living organisms as they are biological catalysts for bodily reactions. Due to its biological nature, these enzymes are constrained by certain factors. With the aid of this experiment, the factors that dictates the enzymatic activities can be represented with a series of results in support to the literature data available. Also, this experiment could lead to the discovery of side reactions and thus limiting factors to the usage of certain chemicals to demonstrate the factors affecting enzymatic activities.Experimental DetailsPart I: Activity of Enzymes AmylasePrior to collection of saliva, the mouth of the source was washed. Two milliliters of saliva was collected from the source in a 50 mL beaker. Ten milliliters of 1% starch solution was gathered in a test tube. Two test tubes were prepared labelled as Test tube 1 and Test tube two containing 1 mL of the starch solution with 2 drops of iodine solution and 1 mL of starch solution with 1 mL of Fehlings solution A and 1 mL of Fehlings solution B. Test tube 1 was set aside. Test tube 2 was then heated in a water bath for 5 minutes. Observation was gathered.The remaining (8 mL of starch solution) was mixed with 2 mL of collected saliva in another test tube. It was shaken well to ensure homogeneity. It was then maintained in a water bath at 37C for thirty minutes. After, the solution was then halved and tested with the same solutions (iodine and Fehlings solutions). Observation was gathered.CatalaseA small potato was pared and grated into fine pulp. The pulp was the mixed with 100 mL of ice water, left standing for 15 minutes, then filtered through a cheese cloth. The filtrate gathered was then separated into two parts in two test tubes. One of the half was boiled in a water bath. A few drops of 3% H2O2 was then added into each test tubes.Part II: Factors Affecting the Activity of EnzymesA. Effect of TemperatureSeparate test tubes containing 1 mL of pork liver extracts were prepared. Each test tubes were immersed in a water bath following the order: Test tube 1 (ice bath at 0C-5C), Test tube 2 (37C-40C), Test tube 3 (boiling water bath). After 15 minutes of exposure to the temperatures, 1 mL of hydrogen peroxide was added to each test tube without removing the vials from the baths. Avoid shaking the mixtures. The height of the foam was measured in centimeters after 5 minutes of standing. A graph was constructed with the y-axis as foam height and the x-axis as the temperature.

B. Effect of pHSeparate test tubes containing 1 mL of pork liver extracts were prepared. Each test tubes were treated as follows: Test tube 1 (1 mL of 1M HCl and 1mL of hydrogen peroxide), Test tube 2 (1 mL of hydrogen peroxide), Test tube 3 (1 mL of NaOH and 1mL of hydrogen peroxide). The foam height was then measured in centimeters after 15 minutes of standing. Avoid shaking the mixtures. A graph of pH versus foam height (x versus y) was then made. C. Effect of InhibitorSeparate test tubes containing 1 mL of pork liver extracts were prepared. Each test tubes were treated as follows: Test tube 1 (1 mL of 95% ethanol), Test tube 2 (1 mL of 0.1 M mercury(II) nitrate solution), Test tube 3 (1 mL of distilled water). All three test tubes were immersed in a water bath maintained at 37C for 5 minutes. Each of the test tubes were treated with 1 mL of hydrogen peroxide. The height of the foams generated was compared per test tubes.Results and DiscussionAmylase is an enzyme found in saliva specifically called salivary amylase. This enzyme catalyzes the breakdown of starch into smaller sugar units like glucose (-D-glucose). Due to this reason, the test tubes untreated with amylase showed a positive result with iodine in KI solution (indigo solution) and negative with Fehlings solutions (no brick red precipitate; no formation of CuO2). This means that starch is present in both solutions for starch will form a blue-violet starch-iodo complex. However, upon treating the solutions with salivary amylase fresh from the source and maintaining it at 37C for 30 minutes, the exact opposite results occurs. The solutions treated with amylase showed positive result for Fehlings solution upon heating (brick red precipitate formed; CuO2 present) which tells us that there is a reducing sugar present in solution. The other solution treated with iodine solution did not form any blue-violet color, signifying that there was no starch present in solution. Therefore, the enzyme salivary amylase did act on the starch present in solution which is the substrate, and formed glucose as a product.Another diverse type of enzyme present in both plants and animals is the catalase. It is present in humans primarily in the liver for fixing toxins. In plants, potatoes for example, this enzyme is used in fixing toxic metabolism side-products, specifically hydrogen peroxide. This enzyme also act the same in humans livers to that in plants. The reaction of the substrate, hydrogen peroxide, with the enzyme catalase goes:3H2O2 + catalase 2H2O+ O2 However, due the fact that enzymes themselves are proteins, they are very sensitive to temperature changes. Some of these enzymes have their optimum temperatures at low temperatures such as the potato catalase. Heating the enzyme solution would cause denaturation of the enzymes and thus destroying their secondary, tertiary, and quaternary structures. Without these structures, the enzymatic action of proteins are deactivated or removed and thus minimal or absolutely zero enzymatic activity on hydrogen peroxide can be observed. This is shown in the reaction of heated potato filtrate with hydrogen peroxide which yielded minimal bubbles (O2 (g)) as product.As mentioned earlier, enzymes are proteins themselves. As biological catalysts, they have their specific and optimum conditions for optimal activity towards their specific substrates in the biological systems. Pork liver extract like the humans liver, contains catalase which fixes the toxic metabolism hydrogen peroxide side-product. Temperatures are to be specific for each enzyme according to their optimal temperatures for optimal activity. Pork liver will be on its optimal activity when the normal body temperature of a pig is attained upon reaction with hydrogen peroxide, theoretically. This is supported by the data gathered from the experiment. The test tube maintained at 37C had the highest foam height among the three treatments for it is close to the normal body temperature of a pig at 38.739.8. The treatment maintained in an ice bath still had enzymatic activity lower than that of the optimally treated one. The test tube held in a boiling water bath had zero enzymatic activity (no bubbles present) because the catalase in the solution was already denatured due to intense heating.pH is another constraint for enzymatic activity. Experimentally, the result were as follows (in decreasing order of enzymatic activity): base treated, neutral solution, acid treated. According to literature, however, the optimal pH of catalase in pork liver is around pH 7 or neutral solution. This can be rationalize by focusing on the side reaction of hydrogen peroxide and sodium hydroxide which yields a gaseous product and thus entails a higher foam height than the neutral solution. The reaction of hydrogen peroxide and sodium hydroxide goes:2NaOH +H2O2Na2O2 (g)+2H2OStill, the neutral solution did have a relatively high enzymatic activity compared to the zero activity presented by the acidic solution. Theoretically the only solution that should have had foam present could have been the neutral solution.Unlike catalase, pepsin helps in the digestion of proteins in the stomach at pH 2. Therefore, the optimal pH for pepsin must be pH 2. Upon entering the intestinal part of the human body system, which has a pH of 8, the enzymatic activity of pepsin is hindered and thus digestion of proteins by pepsin is lowered or ultimately stopped.Another factor that limits the activity of enzymes are the presence of inhibitors. Inhibitors are substances that tend to lower the enzymatic activity towards the specific substrate of interest by blocking the active site of the enzyme disabling the substrate of interest to bind on the site. Ethanol and as well as heavy metals (mercury(II) nitrate solution) are inhibitors. They tend to bind with the enzyme themselves and denatures the enzyme disabling its enzymatic activity towards the substrate which is hydrogen peroxide. Heavy metals denature proteins in a higher degree than ethanol as what was observed in the experiment. This is rooted to the nature of denaturation of both substances. Heavy metals such as mercury denatures the protein by blocking the active site of the enzyme and or by binding itself in another site of the protein and consequently altering the form of the active site of the enzyme. The combined effects of these two results to a greater degree of denaturation caused by heavy metals. Ethanol, on the other hand, only disrupts the hydrogen bonding of the proteins which results to relatively lower denaturation effect to the protein and thus lower inhibiting effect than heavy metals.ConclusionEnzymes are biological catalysts and therefore are biomolecules that are very sensitive to the pH, temperature, and the presence of inhibitors. These enzymes are important in each and every living creatures as they hasten the reactions and bodily processes. Without these biological catalysts, life could be impossible. For example, without catalase, hydrogen peroxide would not be metabolized to water and oxygen in cells leading to poisoning in cells and ultimately death of the creature. These enzymes are under the category of proteins in the list of general biomolecules. Due to this nature, enzymes like any other proteins, are sensitive to pH, temperatures, and inhibitors. Most of these enzymes, are programmed by their source ,which is the thing from which they came from, to do their specific tasks at a specific set of conditions. Catalase of example is present in both animals and plants. The optimal temperatures of the catalases from both differ. Plants for example, have a generally lower optimal temperature for their catalases compared to animal catalases. This can be attributed to the differences of the body temperature of the sources. Plants have a relatively colder body temperature than animals so their enzymes are set to act optimally on a lower temperature than that of animals. pH also is another condition which can alter enzymatic activity. Pork liver extract contains catalase which fixes hydrogen peroxide optimally at pH 7. Due to the side reaction of sodium hydroxide and hydrogen peroxide, however, it showed the highest enzymatic activity which is a false positive as the activity was not fully attributed to the enzymatic activity of catalase to hydrogen peroxide but as a combination with the side reaction. Another factor limiting the activity of enzymes is the presence of inhibitors. These substances can inhibit by blocking the path of the substrate to the enzyme which deforms the active site such as heavy metals which greatly inhibits the enzymatic activity relative to the action of ethanol which only disrupts hydrogen bonding. All of these behaviours can be summarized and pointed out into one single cause, which is the nature of enzymes: enzymes are proteins.ReferencesCampbell, Mary and Farrell, Shawn. "Chapter 6: The Behaviour of Proteins: Enzymes"Biochemistry. 7th edition, International Edition ed. Pacific Grove, CA: Brooks/Cole Pub., 2012. N. pag. Print.Normal Rectal Temperatures, http://www.merckmanuals.com/vet/appendixes/reference_guides/normal_rectal_temperature_ranges.html