Critical Thinking

The 0.1460, and cuvette four at 0.1695. In

The Effects of Inhibitors on Lactase Activity. Alyssa Ramirez,2017, Functional Biology, Texas State University, San Marcos, TX 78666 Different sugars may act as competitive inhibitors and affect the enzyme catalyzed reaction and delay the reaction rate. We investigated the different sugars, glucose, sucrose and lactose to figure out if any of them could inhibit an enzyme catalyzed reaction. We did this by creating 0.1% dilutions of each of the sugars and adding the substrate ONPG. Four cuvettes were filled and ran through the spectrophotometer to see the change in absorption and when exactly the change in absorption happened for each solution. It was found that cuvette one had a change in  absorption rating of 0.1675, cuvette two had a change in absorption rating at 0.09, cuvette three had a change in absorption rating at 0.1460, and cuvette four at 0.1695. In conclusion, it is slightly proven that sugars act as competitive inhibitors in lactase activity.IntroductionThis experiment tested the effect of increasing concentrations of sugars and determined if they        acted as a competitive inhibitor for Lactase. Lactase is the primary ingredient in Lactaid and plays an vital part in the digestive system. (Bayless, Brown, Page 2017) Lactase is the enzyme found in the small intestine of mammals and catalyzes the breakdown of lactose, the sugar found in milk. Enzyme catalysts are responsible for decreasing the activation energy by increasing the rate of a chemical reaction at the activation site. (Agarwal 2006) Most enzymes are made perfect for their specific substrates. (Lashinski 2013) For example, the enzyme Lactase is broken down with the substrate Lactose and Sucrase by the substrate Sucrose which is the same thing as table sugar. Many things can delay the rate of a reaction such as pH, salt, temperature and competitive inhibitors. (Shu et la. 2016) Competitive inhibition occurs when the competitive inhibitor beats the substrate to the active site and binds with the enzyme. If the enzyme binds with a competitive inhibitor it can longer bind with a substrate. Though, the two have a similar structure (Singh et. al 2017) they do not do the same job; competitive inhibitors will decrease or sometimes stop catalysis (Worthington Biochemical Corporation 2017) while the substrate speeds up the rate of the reaction through enzyme catalysis. An example of a competitive inhibitor is Cyanide, it competes with the enzyme Cythochrome C Oxidase to prevent the transport chains of electrons in cellular respiration causing the cell to no longer produce ATP. (Li et al. 2014)  The purpose of our experiment was to figure out which sugars would or would not inhibit the enzyme catalysis reaction and to also see the effect that increasing the concentration of an enzyme will do to the reaction. The cofactors used for this experiment were the sugars Glucose, Sucrose and Lactose, a phosphate buffer and the substrate ONPG. It is noted that Lactose and Sucrose have a similar structure of 12-Carbon, 22-Hydrogen and 11-Oxoygen that differs from the rest of the substrates used in the experiment. (Oba, Mewis, Zhining 2015) Glucose is a carbohydrate that is important in the production of ATP which is a natural sugar that gives the body energy. (Li et al 2012) Sucrose also known as table sugar is made up of a combination of glucose and fructose which could influence this experimental reaction because of the different enzymes each sugar attaches with. (Baker 1975) In this experiment we used the substrate ONPG because, it is a colorimetric, which means it will allow for a yellow color to be observed when broken down and it is also a positive known substrate for Lactose. (Li et al. 2012)Methods and MaterialsIn this experiment, latex gloves were worn at all times to prevent any chemical getting on the skin, as well as goggles to protect the eyes incase of any splashes or spills. To start off the experiment, four flask were filled with a different solution to create the dilutions used. One held a glucose solution, one held a phosphate-buffer, one contained a sucrose solution and the last one contained a lactose solution. Then, four 10 mL pipettes were gathered and used for the solutions in each of the four flasks. Next, two 50 mL beakers were used to filter the lactase solution. After that, a ONPG substrate solution was gathered, as well as a micropipette for measuring the substrate. Next, four 4 mL cuvettes were collected and used to hold the dilutions for when it was time to go into the spectrophotometer. After that, we gathered a tablet of Lactaid. Lactase is the primary ingredient in Lactaid and is the enzyme that causes the catalyzed reaction being tested. A mortar and pestle was used for crushing the Lactaid tablet. We then used paper towels to lay over the 50 mL beaker to filter the solution with the enzyme. Next, we collected three test tubes and a test tube rack. These materials were used for mixing the liquids as we made the dilutions. Lastly, a spectrophotometer was used to measure the absorbance rate of each enzyme catalyzed reactions. The first step in this experiment, a Lactaid tablet was crushed with a mortar and pestle into a powder and added to a beaker with 10 mL of phosphate buffer. Then, a different beaker was covered with a paper towel to filter the soluble enzyme from the insoluble Lactaid powder. Next, three test tubes were filled with 9.0 mL of phosphate buffer for creating the following dilutions, using the micropipette 1.0 mL of the enzyme buffer solution was added to the first test tube, creating the dilution of 10%. Then, using a micropipette, 1.0 mL of the solution in test tube one was added to the second test tube creating the second dilution of 1%. The last dilution was created by taking 1.0 mL from test tube two with the micropipette and  placing it into test tube three creating the optimal enzyme dilution of 0.1%. Next, 1.0 mL of the optimal dilution was added into each of the four cuvettes. Once the optimal dilution was placed in each cuvette, they were then labeled one through four. Next using the correct pipettes, 1.0 mL of phosphate buffer was added to cuvette one, 1.0 mL of the glucose solution was added to cuvette two, 1.0 mL of the lactose solution was added to cuvette three and lastly, 1.0 mL of the sucrose solution was added to cuvette four. We then filled a fifth cuvette with 1.0 mL of phosphate buffer to use as a blank for the spectrophotometer. Next, 1.0 mL of ONPG was added to cuvette one and placed into the spectrophotometer quickly. Each cuvette was ran at a wavelength of 410.0 nm for five minutes, recording the enzyme catalyzed reaction every 15 seconds. The other three cuvettes also received 1.0 mL of the substrate ONPG and ran the same way as cuvette one, being blanked with the phosphate buffer between each of the different cuvettes to ensure accuracy. This process was done twice creating the four cuvettes over again.The data from both trials were averaged and used to find the change in absorbance in each solution. Lastly, graphs were created. The first graph showed the different absorption changes in each of the four cuvettes and the second graph displayed the exact point of when the change in absorbance occurred at 150 seconds.ResultsIn cuvette one which contained 1.0 mL of ONPG substrate, 1.0 mL of the enzyme dilution and 1.0 mL of phosphate buffer  had a change in absorption rating that ranged from 0.0 to 0.332 and at 150 seconds, cuvette one had a change in absorbance of 0.1675. It also had an average absorption rate that ranged from 0.041 to 0.373. Cuvette two contained the glucose solution and had an absorption average rating 0.0 to 0.1785 and average absorbance  that ranged 0.0295 to 0.1785. At 150 seconds, cuvette two had a change in absorbance rating of 0.09. Cuvette three contained the sucrose solution  and had a change in absorption rating from 0.0 to 0.2905 and an average absorbance that ranged 0.0275 to 0.3180. At 150 seconds, cuvette three had a change in absorption rating of 0.1460. Cuvette four contained the lactose solution and had an average change in absorption from 0.0 to 0.3215 and an average absorbance rating that ranged from 0.025 to 0.3465. At 150 seconds, cuvette four had a change in absorbance of 0.1695. The first graph displays the change in absorption over time and the second graph displays the exact point where absorption changed.LactoseGlucoseSucroseEnzyme ONPGBuffercuvette 1***cuvette 2***cuvette 3***cuvette 4***DiscussionIn this experiment the hypothesis was partially supported. The enzyme activity was only altered to a limited extent by the sugars. In this study four cuvettes were used and only 3 out were altered. The greatest change in enzyme activity took place in cuvette one, which contained PO4, the enzyme lactase, and the substrate ONPG. Cuvette one had a dilution of one to ten and had the highest change in absorption as well. The results also concluded cuvette four, which contained lactose was the only cuvette not altered in the experiment. Cuvettes two and three were altered in the experiment but did not have as big of a reaction as cuvette one. The hypothesis was also only supported in part because of possible limitations of this experiment. For example, more test could have been done to on each of the cuvettes to see if the results would be different, the spectrophotometers that were used during this experiment had been running all day and were not giving the most accurate readings. The time allotted for the experiment could have been longer, which could have allowed for more trials to be done. The measurements could have more precise for example, when making the solutions the chemicals going into the test tubes were not as precisely measured as they could have been. The dilutions could have also changed the reaction due to the idea of the theory that the reaction is proportional to the concentration of the enzyme-substrate compound. (Lineweaver and Burk 1934) Based on the findings in this experiment, in the future different things could be investigated like why certain sugars are inhibitors and why other sugars are not, the effect of adding a different substrate opposed to  ONPG, changing the concentration of the dilutions from one to ten to one to one hundred, and trying the experiment with a different enzyme. In conclusion, it is proven that yes, sugars are competitive inhibitors. IntroductionThis experiment tested the effect of increasing concentrations of sugars and determined if they        acted as a competitive inhibitor for Lactase. Lactase is the primary ingredient in Lactaid and plays an vital part in the digestive system. (Bayless, Brown, Page 2017) Lactase is the enzyme found in the small intestine of mammals and catalyzes the breakdown of lactose, the sugar found in milk. Enzyme catalysts are responsible for decreasing the activation energy by increasing the rate of a chemical reaction at the activation site. (Agarwal 2006) Most enzymes are made perfect for their specific substrates. (Lashinski 2013) For example, the enzyme Lactase is broken down with the substrate Lactose and Sucrase by the substrate Sucrose which is the same thing as table sugar. Many things can delay the rate of a reaction such as pH, salt, temperature and competitive inhibitors. (Shu et la. 2016) Competitive inhibition occurs when the competitive inhibitor beats the substrate to the active site and binds with the enzyme. If the enzyme binds with a competitive inhibitor it can longer bind with a substrate. Though, the two have a similar structure (Singh et. al 2017) they do not do the same job; competitive inhibitors will decrease or sometimes stop catalysis (Worthington Biochemical Corporation 2017) while the substrate speeds up the rate of the reaction through enzyme catalysis. An example of a competitive inhibitor is Cyanide, it competes with the enzyme Cythochrome C Oxidase to prevent the transport chains of electrons in cellular respiration causing the cell to no longer produce ATP. (Li et al. 2014)  The purpose of our experiment was to figure out which sugars would or would not inhibit the enzyme catalysis reaction and to also see the effect that increasing the concentration of an enzyme will do to the reaction. The cofactors used for this experiment were the sugars Glucose, Sucrose and Lactose, a phosphate buffer and the substrate ONPG. It is noted that Lactose and Sucrose have a similar structure of 12-Carbon, 22-Hydrogen and 11-Oxoygen that differs from the rest of the substrates used in the experiment. (Oba, Mewis, Zhining 2015) Glucose is a carbohydrate that is important in the production of ATP which is a natural sugar that gives the body energy. (Li et al 2012) Sucrose also known as table sugar is made up of a combination of glucose and fructose which could influence this experimental reaction because of the different enzymes each sugar attaches with. (Baker 1975) In this experiment we used the substrate ONPG because, it is a colorimetric, which means it will allow for a yellow color to be observed when broken down and it is also a positive known substrate for Lactose. (Li et al. 2012)References:Bayless, T. M., Brown, E., & Paige, D. M. (2017). Lactase Non-persistence and Lactose Intolerance. Current Gastroenterology Reports, 19(5), 23.doi:10.1007/s11894-017-0558-9Lashinski, E. M. (2013). Enzymes and enzyme activity: structure, biology and clinical significance. New York: Nova Science Publishers, ©2013.Li, W., Zhao, X., Zou, S., Ma, Y., Zhang, K., & Zhang, M. (2012). Scanning assay of ?-galactosidase activity. Applied Biochemistry &Microbiology, 48(6), 603-607. doi:10.1134/S0003683812060075Oba, M. m., Mewis, J. L., & Zhining, Z. (2015). Effects of ruminal doses of sucrose, lactose, and corn starch on ruminal fermentation and expression of genes in ruminal epithelial cells. Journal Of Dairy Science, 98(1), 586-594.Shu, G., Bowen, Z., Hong, L., Hongchang, W., & Qian, Z. (2016). Effect of Temperature, pH, Enzyme to Substrate Ratio, Substrate Concentration and Time on the Antioxidative Activity of Hydrolysates from Goat Milk Casein by Alcalase. Acta Universitatis Cibiniensis. Series E: FoodTechnology, 20(2), 29-38.Singh, A., Ekka, M., Kaushik, A., Pandya, V., Singh, R., Banerjee, S., & Kumaran, S. (2017). Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism. Biochemistry,56(37), 5011-5025. doi: 10.1021/acs.biochem.7b00500 Agarwal; licensee BioMed Central Ltd. (2006). Enzymes: An integrated veiw of structures, dynamics and function Microbial Cell Factories,doi: 10.1186/1475-2859-5-2Attar, A., Cubillana-Aquilera, L., Naranjo-Rodriguez, I., de Cisneros, JL, Palacios-Santander, JM., Amine, A. (2014). Amperometric inhibition biosensors based on horseradish peroxidase and gold sononanoparticles immobilized onto different electrodes for cyanide measurements. Bioelectrochemistry doi: 10.1016A.I Tarasou, F. Semplici, M.A Ravier, E.A Bellomo, T.J Pullen, P. gilon, I. Sekler, R. Rizzuto, G.A Rutter (2012). The Mitochondrial Ca2+ Uniporter MCU Is Essential for Glucose-Induced ATP Increases in Pancreatic ?-Cells doi: 10.1371/journal.pone.0039722H.G Baker (1975). Sugar Concentrations in Nectars from Hummingbird Flowers Biotropica Vol. 7, No. 1 (Apr., 1975), pp. 37-41

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