Three Characteristics of Enzymes - Lab Report Example

Effects of Temperature and pH on Enzyme Activity INTRODUCTION Life consists of a dynamic process that involves constant changes in chemical composition. These changes have to take place in specific manner thus are regulated by catalytic reactions. Enzymes are biocatalysts, protein in nature that catalyze and regulate metabolic reactions that sustain life. The composition of enzyme is primarily a complex protein molecule and function by catalyzing specific biochemical changes within the bodies of animals and plants such as digestion of food in animals and photosynthesis in plants. Enzymes acts on substrates and converts them into different products. As stated by Onteh et al (2005), most biochemical reactions would be very slow without enzymes since enzymes increase the rate of biochemical reactions by thousands times. It is therefore important to study the environmental factors: pH and temperature and how they would affect specific enzymes. There are three characteristics of enzymes that make them function as catalysts. First enzymes increase the rate of biochemical reaction, second, they are specific to a substrate and thirdly they can be regulated to increase or reduce rate of biochemical reaction. Studies have confirmed that enzymatic activities are affected by certain factors such as enzyme concentration, pH, and temperature as well as substrate concentration. According to (), all these factors except substrate concentration have the effect of modifying the specificity nature of the enzyme there by making it incompatible with the substrate. Moreover enzymes act as catalysts because they exhibit three dimensional protein structures. As stated by Onteh et al (2005), this structure is particularly sensitive to changes in salts, pH and temperature thus a slight changes in temperatures of reaction can significantly change the rte of reaction while extreme temperatures can irreversibly alter the both the three dimensional structure of the enzyme thus making it impossible to catalyze a reaction. The enzymatic activity is based on the fact that enzymes are made up of certain active sites that are specific to the substrate’s active sites. As stated by Onteh et al (2005), it is on this active sites that enzymes bind to a given substrate in a geometrical fashion after which substrate undergo its inherent reaction at a much faster rate. This mode of enzymatic action is referred to as lock and key. It is important to note that enzyme does not actually react with a substrate, but brings and aligns the substrate in order for it to react with other substances. Enzymes therefore have a best fit configuration to the protein that enables it align to the substrate that increase reaction rate. It therefore follows that with substrate concentration held constant, when enzymes are in their ideal configuration, reaction rates would be maximum. Factors that affect enzyme activity do so by affecting these active sites, substrate-enzyme contact or both. We began this project by stating that enzymes are found in both animal and plant cells. Potato tuber is a living system that contains many molecules used for metabolism. Enzymes form part of the biological molecules found in potato tubers. Potato tubers contain Catalase enzymes which is responsible for the conversion of hydrogen peroxide into water and oxygen according to the reaction below. As you can see in the reaction below, the products are oxygen and water. Hydrogen peroxide Water + Oxygen Catalase H2O H2O + O2 Catalase This study investigated the effect of temperature and ph on catalase enzyme activity and how the release of carbon dioxide gas in humans affects the pH of the surrounding environment. METHODS Three different experiments were conducted in this study. One was to investigate the effect of temperature on the catalase enzyme activity, effect of pH on catalase enzyme activity and effect of release of carbon dioxide gas during gas exchange in humans. Preparation of the Enzyme Source Potato was cut into potato slices equaling to half centimeter each thick. Circular borer was then used to punch uniform, small equal cylinders of potato tissue and placed in a dish filled with distilled water until they were ready to be used. Potato cylinders were made equal hence t was assumed that the amounts of catalase enzyme available within the potato cylinders are the same. Each of the teams participating in this experiment were then provided with 10 ml test tubes cocks with small holes in each, hydrogen peroxide, 150 mil beakers and a supply of fleshly-made potato tissue cylinder. Procedure for Effect of Temperature on Catalase Enzyme Four clean test tubes were taken and labeled 1, 2, 3 and 4 before filling each with hydrogen peroxide. The tubes were pre-incubated by placing tube 1 in ice at (0oC) bath, tube 2 in room temperature bath, tube 3 in human body temperature at (370C) bath and tube 4 was left at the work station for a while. A raw potato cylinder was then placed in tubes 1, 2 and three after five minutes. The tubes were then firmly corked and inverted in water bath in which they had been pre-incubated. Time for which each tube was incubated was recorded in table 1. A fourth boiled potato cylinder was placed in test tube 4, firmly corked, inverted in a beaker at the workstation and time recorded upon inversion. The tubes were then left to form gas and after exactly five minutes, each tube was brought back to the workstation and the length of gas bubble formed measured and recorded. After recording the gas length, the contents of the test tubes were poured into a sink and test tubes cleaned. When all teams were done with this, experiment, average values from other group members were calculated, compiled and recorded in table 2. Procedure for the Effect of pH on Enzyme Activity Three 10 ml test tubes were selected and numbered 1, 2 and 3 and three 150 ml beakers were also numbered 1, 2 and 3. One potato cylinder was then placed in each test tube using a forceps. Each test tube was then pre-treated by adding hydrochloric acid into tube 1 just cover the potato cylinders and sodium hydroxide into tube 3 just to cover the potato cylinder. In tube 2, distilled water was added just to cover the potato cylinders in the tubes. The tubes were then left to pre-treat for 5 minutes before filling each tube with hydrogen peroxide to the brim. The three test tubes were then corked, turned upside down in its own number beaker, i.e. test tube 1, 2 and 3 in beakers 1, 2 and 3 respectively and the time the tube is inverted was noted. During the experiment, the pH for HCL, NaOH and distilled water tested by placing a drop of each solution on a strip of pH indicator paper. Color chart on the pH paper tube was used to determine the pH of each solution. The results were then recorded in table 3. After exactly 5 minutes, the lengths of gas formed on each tube were measured and results recorded in table 3. After that, the used hydrogen peroxide and solution were poured into the sink and potato cylinders thrown into trash and glassware washed. Average values from other groups within the class were collected, calculated and recorded in table 4. Procedure for the Relationship between Co2 Level and pH Of a Solution 75 ml of Bromthymal blue (BTB) solution was placed in a flask. Using a soda straw, exhaled breath was then bubbled into the green, neutral water and any color change observed and recorded. While swirling, a drop of NaOH was then added into the solution and continued until no further color change was observed. The results were then noted. While swirling, a drop of HCL was added into the flask and the results observed. The addition was then continued and for each drop of HCL that was added swirling was done until there was no color change of BTB. The pH of the flask was then adjusted to neutral (pH 7.0) using the acid and base as needed and was indicated by the green color. BTB samples were then pooled together with those from other members of the class and analyzed. RESULTS Effect of Temperature on Catalase Enzyme Table 1: Gas Production at Different Temperatures- Individual Team Result Tube Number Time Tube Inverted Bubble Length (mm) at 10 minutes Tube Temperature 1 6:17 5 00C 2 6:17 9 220C 3 6:17 13 370C 4 6:17 0 1000C Table 2: Gas Production at Different Temperatures- Averaged Class Team Result Tube Number Bubble Length (mm) Tube Temperature 1 7.5 00C 2 11.6 220C 3 13.8 370C 4 0 1000C Figure 1: Gas Production at Different temperatures- Average Class team Data Effect of pH on Catalase Enzyme Activity Table 3: Gas Production at Different pH treatments- Individual Team Result Tube Number Bubble Length (in mm) pH 1 3 1 2 9 7 3 3 11 Table 4: Gas Production at Different pH treatments- Averaged Class Team data Tube Number Bubble Length (in mm) pH 1 2.9 1 2 10 7 3 3 11 Figure 2: Optimal pH for Catalase Enzyme Relationship between carbon dioxide levels and pH of a solution Table 5: Relationship between carbon dioxide levels and pH of a solution Acidic solution Neutral solution Basic solution Amount of CO2 High Low pH 6.0 or Lower 7.0 7.6 or higher Color of BTB Yellow Green Blue DISCUSSIONS Effect of Temperature on Catalase Enzyme The results from this experiment show that extreme temperature has adverse effects on the activity of catalase enzyme as evidenced by the various amounts of gas produced. While at low temperatures there is some enzymatic activity, higher temperatures above 370C results into reduction in the activity of catalase enzyme and at 1000C, there is no activity at all. Catalase enzymes works better at 370C as shown in figure 1. The body temperature of potato just like many living plants and animals is 370C. The function of enzyme catalase in potatoes is to enhance biochemical reactions that lead to availability of food for the plant. As evident in table 2 and figure 1 above, the upper safe temperature for enzymes within this category is between 400C and 450C. This is because lower temperatures result into deactivation of the enzymes while higher temperatures denature the enzymes. Birds and mammals both evolved in similar body temperatures because evolution resulted in both species having the same body temperatures because they share a living environment and eating habitats. These are two important key aspects for the successful survival of animals living in this habitat. According to Onteh et al (2005), temperature play a vital role in selection of plants and animals thus certain plants can survive in other habitats while others cannot. Plants provide foods for animals and animals require the right temperature for its enzymes not only for digestion but also for metabolism and other biochemical reactions. As stated by (), extreme temperatures interfere with the structure of enzymes thus active sites are denatured. This inhibits enzyme activity since enzymes are not in their ideal state thus affecting their active sites, substrate-enzyme contact or both. This also explains why we experience fever at higher temperatures since; enzymes that are protein in nature are often denatured at extreme temperatures. Effect of pH on Enzyme Activity pH of an environment refers to the measure of hydrogen ions present in that particular environment. As stated by Das et al (2011), Each enzyme has an optimal pH at which it works the best because the enzyme’s active sites arrangements is partially fixed by the ionic bonds between -COOH and -NH2 groups of the polysaccharide forming the enzyme. In this respect, a change in pH affects these bonding thus affects the active sites of the enzyme. The pH of a solution has an impact on how well the catalase enzyme works as is evident by different gas productions in figure 2 and tables 3 and 4. Extreme pH of 1 and 11 recorded the lowest production of gas; 2.9mm and 3.1mm respectively while neutral pH recorded the highest gas production (10mm) as shown in table 4 and figure 2. This is an indication that catalase enzyme functions better at neither acidity nor alkalinity environment. This also explains the environment upon which this enzyme was extracted. Unlike changes in temperatures, small changes in pH have a greater impact than a change in temperature discussed in the previous section. This shows that catalase enzyme is pH specific and since it is found in potatoes, the internal pH of potato tissues and cells are neutral. This is because this enzyme can only function in neutral environments. During the digestion of food in the stomach, and particularly the digestion of proteins, pepsin and trypsin are often secreted followed by the secretion of sodium bicarbonate. This is because trypsin and pepsin function well under acidic conditions. It is therefore important for the body to adjust the pH to reach optimal for normal digestion of protein. Relationship between carbon dioxide levels and pH of a solution Pure water with a pH of 7.0 is considered as neither acidic nor basic. Lower pH indicates high acidity while high pH indicates strong base. An acid is a molecule that breaks down in water and release H+ while a base releases hydroxide (OH-) in water. As stated by Das et al (2011), when carbon dioxide dissolves in water, some molecules combine with water to form carbonic acid, hence the more carbon dioxide dissolved in water, the more carbonic acid is produced. However this reaction can be reversed by the removal of carbon dioxide from the solution. This explains why the color of the solution changes to yellow when exhaled air is vigorously bubbled through the neutral water. When NaOH was added the solution changed to blue which shows that the addition of NaOH lowered the lowered the acidity. When hydrochloric acid was added, the solution changed again to yellow showing that the solution is acidic or the level of acidity has been raised. The sensitivity of Bromthymol blue (BTB) is evident since it gives distinctive changes in color rapidly with each drop of either HCl or NaOH. Exhaled air contains carbon dioxide from our tissues and lungs thus when exhaled in water, it dissolves to produce acidity as evidenced by the change in color of the indicator. CONCLUSIONS This experiment shows that Catalase enzyme is specific to pH 7 and its performance is highly deteriorated at any other pH above or below. Increase in temperature of solution of enzyme however increases the rate of reaction with availability of substrate up to optimal upon which it begins to fall since they are denatured.. However, optimal reaction can be attained with the availability of enzyme in excess of substrate. The optimal state is achieved when the rate of action is steady despite increase in substrate concentration. The range of the data collected was not able to derive Vmax and Km and could be due to the differences in absorbance or errors in reading. References . Das, B., Chakraborty, A., Ghosh, S., & Chakrabarti, K. (2011). Studies on the effect of pH and carbon sources on enzyme activities of some pectinolytic bacteria isolated from jute retting water. Turkish Journal Of Biology, 25(6), 671-678. Onteh, F. A., Grandison, A. S., & Lewis, M. J. (2005). Factors affecting lactoperoxidase activity. International Journal Of Dairy Technology, 58(4), 233-236. Onzález, L. J., Moreno, D. M., Bonomo, R. A., & Vila, A. J. (2014). Host-Specific Enzyme-Substrate Interactions in SPM-1 Metallo-β-Lactamase Are Modulated by Second Sphere Residues. Plos Pathogens, 10(1), 1-12. Read More