'The Effects of Substrate Concentration, Reaction Time and Enzyme Concentration on Enzyme Reactions\r'

'The Effects of substrate Concentration, Reaction clipping and Enzyme Concentration on Enzyme Reactions Corey von Ellm-St. Croix Rachael Kwan ID#: 20427841 Matthew Hrycyshyn &type A; Saeideh Mayanloo Biol 130L, Section 017 Wednesday, 9:30am-12:20pm, 151 November 09, 2011 A living schema attends its activity through enzymes. Enzymes are made from hundreds or even thousands of amino acids connected in a very unique and particular(prenominal) order. Almost just enzymes are proteins, except for ribozymes. The chain of amino acids because folds into a unique shape.\r\nThat shape non only allows the enzyme to carry out specific chemical replys still to act as a very high-octane catalyst. The enzyme speeds that reply up tremendously. individually enzyme reacts with one specific reactant called a substrate that leave alone form its products. The goal of the experiments is to restrict the effects of substrate stringency, reception season and enzyme niggardness on the di rection of an enzyme response. Amylase is a digestive enzyme tack together in two the saliva and the menial in examine.\r\nSalivary amylase is a hydrolytic reaction that breaks tear amylum molecules by systematically breaking off the maltose molecules from the ends of stiffen chains. The maltose is further broken d knowledge by a nonher(prenominal) enzyme. Phosphorylase is an enzyme that systematically removes glucose molecules by consumes phosphorous acid to break the beta-1-4-glucosidic bonds in amylum. The interaction of inorganic orthophosphate with the glucosidic bond pass ons in the formation of glucose-1-phosphate and the loss of a chain unit in stiffen. In the tump over reaction the glucose part of glucose-1-phosphate is added as a bare-ass chain unit and phosphate is set free.\r\nThis two-sided enzymatic polymerization occurs with little form in free energy and therefor the reaction may choose to go all way. tincture of unity attempting is a rise for the presence of stiffen in which the sample turns blue- forbidding in color when a some retchs of grand iodide dissolver are fixed on the sample. A damaging iodin try out is when the reaction corpse dis colouration in intensity. It is the reaction between iodine and the coiled polymer of glucose known as amylase in stiffen that causes the colour change. The reaction occurs when serial amylase chains form helices in which the iodine bath pass inside.\r\nGlycogen also receives a colour change because it is a glucose polymer as well alone its structure differentiates from amylum which and so forms a em dark-brownish colour change. The iodine analyse does not fashion for mono or disaccharides because they are similarly keen to capture the iodine. The benedick’s leaven is utilise to detect the presence of trim sugars. simplification sugars are sugars with a free aldehyde or ketone group. The free activated carbonyl group allows all monosaccharides to b e bring down sugars. The same goes for disaccharides as some also go for the free reactive carbonyl group.\r\nThe colour of flow formed when the Benedict’s ladder acquires a commanding result depends on the compactness of decrease sugars pledge. A potassium colour change indicates that few decrease sugars are present. Orange indicates a high parsimony, red an even higher concentration and brown is the highest concentration colour change. A controvert screen for step-down sugar occurs when the Benedict’s issue remains its blue colour. Materials and Method Materials and Method base in 1119 BIOL 130, Department of biology 2011 Cell Biology Laboratory Manual. University of Waterloo, Waterloo. Fall 2011. p. 37-42. The procedure of the lab did not differ from that in the lab manual. Results Salivary Amylase Iodine runningify: circuit board 1 sample subway system sum| Results Through Experimentation| Control | 1 (10% salivary amylase resolving power) | xanthous | interdict | 2 (5% salivary amylase solution)| chicken| oppose | 3 (2% salivary amylase solution)| Yelllow| veto | 4 (1% salivary amylase solution)| yellowed| prejudicial| 5 (1% starch severance)| saturnine-Black| commanding| instrument panel1: The prorogue preceding(prenominal) represent the stolon iodine prove done. Shows components of all(prenominal) audition pipage as well as the results and imperative or ostracize assert.\r\nDilutions were done use tap pissing (may backtrack other molecules). Corresponds to locomote 5 through 10. Benedict’s shield: evade 2 screen Tube Number| Results Through Experimentation| Control| 1 (10% salivary amylase solution) | Brown-orange precipitate| ordained| 2 (5% salivary amylase solution)| car park precipitate | lordly| 3 (2% salivary amylase solution)| sad | Negative| 4 (1% salivary amylase solution)| blueweed| Negative| 5 (1% starch falling out)| blue| Negative| sidestep 2: The find pan el supra represent the first Benedict’s attempt done. Shows components of each test render as well as the results and confident(p) or detrimental enclose.\r\nEach of the above test tubes contains 4ml Benedict’s solution and were boiled for 5 minutes when determining results and are related to step 5 through 10. Iodine test: put off 3 tryout Tubes| Number of Drops Till Negative| Time Interval| Time| 9+14 (1% salivary amylase solution)| 13 | 60 seconds| 13*60= 780 seconds| 8+13 (2% salivary amylase solution)| 12| 30 seconds| 12*30= 360 seconds| 7+12 (5% salivary amylase solution)| 10| 15 seconds| 10*15= cl seconds| 6+11 (10% salivary amylase solution)| 18| 5 seconds| 18*5= 90 seconds | 10+15 (water system)| (Always plus)| N/A| N/A|\r\n dodge 3: The table above represents the cartridge clip it took to ready endpoint. Shows components of each test tube, number of disregards, cartridge clip breakup between expends and term to reach endpoint. Each of the abov e test tubes contains 2ml †1% starch suspension and 2ml McIlvaine’s buffer. The above test tubes were placed in a warm bath at 37 degrees Celsius and resuscitate to move 11-18. Benedict’s test: dining table 4 running tube| Results through experimentation| Control| 20 (water)| blue| veto| 9 (1% salivary amylase solution)| 1/3 brown -2/3 blue| Positive| 18 (2% salivary amylase solution)| 1/3 brown -2/3 blue| Positive| 17 (5% salivary amylase solution)| 1/3 brown -2/3 blue| Positive| 16(10% salivary amylase solution)| 1/3 brown -2/3 blue| Positive| Table 4: The table above represents the front for trim down sugars aft(prenominal) endpoint. Each of the above test tubes contains 4ml Benedict’s solution and were boiled for 5 minutes when determining results and are related to travel 18-20. Phosphorylase Composition of test tubes: Table 5 block out TUBE NUMBER | CONTAINS| One| 1. 5ml of 0. 01M glucose + 1 spue of 0. % starch suspension| Two| 1. 5 of 0. 01M glucose-1-phosphate+ 1 usher out of 0. 2% starch suspension| troika| 1. 5 of 0. 01M glucose-1-phosphate| Four| 1. 5 of 0. 01M glucose-1-phosphate+ 1 drop of 0. 2% starch suspension| cinque| 1. 5 of 0. 01M glucose-1-phosphate + 0. 5ml of 0. 2M honey oil phosphate+ 1 drop of 0. 2% starch suspension| Six| 0. 5ml of 0. 2M potassium phosphate + 1. 5ml of 0. 2% starch suspension| sevener| 0. 5ml of 0. 2M potassium phosphate+ 1. 5ml of 0. 2% starch suspension| octet| 4ml Boiled phosphorylase | Table 5: The above table represent the solutions present in the test tubes 1-8 from travel 2-10 Iodine test: Table 6\r\nTest tube| Results through experimentation| Control| 1| Yellow | Negative | 2| Yellow| Negative| 3| Yellow| Negative| 4| Yellow| Negative| 5| Yellow| Negative| 6| naughty- macabre| Positive| 7| blue-blooded- scurrilousness| Positive | Table 6: Search for starch in spite of appearance test tubes 1-7. Shows components of each test tube as well as the results and incon trovertible or proscribe control. Composition of test tubes : Table 7 canvass TUBE NUMBER | CONTAINS| One| 1. 5ml of 0. 01M glucose + 1 drop of 0. 2% starch suspension + 2ml phosphorylase| Two| 1. 5 of 0. 01M glucose-1-phosphate+ 1 drop of 0. 2% starch suspension+ 2ml phosphorylase| Three| 1. of 0. 01M glucose-1-phosphate+ 2ml phosphorylase| Four| 1. 5 of 0. 01M glucose-1-phosphate+ 1 drop of 0. 2% starch suspension + 2ml boiled phosphorylase| Five| 1. 5 of 0. 01M glucose-1-phosphate + 0. 5ml of 0. 2M potassium phosphate+ 1 drop of 0. 2% starch suspension+ 2ml phosphorylase| Six| 0. 5ml of 0. 2M potassium phosphate + 1. 5ml of 0. 2% starch suspension+ 2ml phosphorylase| Seven| 0. 5ml of 0. 2M potassium phosphate+ 1. 5ml of 0. 2% starch suspension + 2ml boiled phosphorylase| Table 7: The above table represent the solutions present in the test tubes 1-7 from steps 11-12 Iodine Test: Table 8\r\nTime Interval| test tube 1| Test tube 2| Test tube 3| Test tube 4| Test tube 5| Test tube 6| Test tube 7| 10:28-10:32| scandalmongering| Very saturnine out blue- colored| discolor| icteric| yellow| wearied bluish black| blasphemous black| 10:32-10:36| yellow| gruesome black| yellow| yellow| yellow| Very faint blue-black| non-white black| 10:36-10:39| yellow| colored black| yellow| yellow| yellow| Faint blue black| Blue black| 10:39-10:42| yellow| Blue black| yellow| yellow| yellow| Faint blue black| Blue black| 10:42-10:46| yellow| Blue black| yellow| yellow| yellow| Blue black| Blue black| 10:46-10:49| yellow| Blue black| Very faint blue black| yellow| yellow| Blue black| Blue black| 10:49-10:52| Yellow| Blue black| Faint blue black| Yellow| yellow| Blue black| Blue black| 10:52-10:55| Yellow| Blue black| Blue black| Yellow| Yellow| Blue black| Blue black| 10:55-10:58| Yellow| Blue black| Blue black| Yellow| Yellow| Blue black| Blue black| 10:58-10:42| yellow| Blue black| Blue black| yellow| Yellow| Blue black| Blue black| Table 8: Test for the presence and d eduction of starch.\r\nContains the time interval from when the previous test had ended to resultant of current test and the reaction result of test tubes 1-7. Figure1: above; represent the time it took each salivary amylase concentration to reach endpoint (when test for starch became negative. Discussion: Salivary Amylase The Iodine test’s control is the presence of starch. If starch is presence whence the control is supportive resulting in a blue-black colour change. The first iodine test or if you refer to table 1, gave a positive result for only test tube 5 which contained 1% starch suspension. Clearly starch is present ground on just the component of the solution.\r\nA negative control in an iodine test will result in maintenance of the yellow colour of iodine. gibe to table one the test tubes containing 10% salivary amylase solution, 5% salivary amylase solution, 2% salivary amylase solution and 1% salivary amylase solution resulted in a negative control result. Th is is due to the event that all that is present is the enzyme salivary amylase and water and and then no starch. The Benedict’s test control is the presence of cut sugars (sugars with a free aldehyde or ketone group). If a reducing sugar is present past a positive control reaction will occur. A positive control reaction is when a colour of the blue Benedict’s solution turns green, orange, red or brown after boiling.\r\nEach colour represents the concentration of reducing sugars present, green creation the lowest and brown the highest. Referring back to table 2, test tubes 1 and 2 resulted in a positive control reaction. Even though test tubes 1 and 2 contained only salivary amylase the tap water used to dilute the amylase solution may contain some starch which would in turn induce maltose a reducing sugar. The 10% salivary amylase (test tube 1) resulted in an orange colour change due to the fact that a higher enzyme concentration would more likely produce enough reducing sugars to result in an orange colour change. The 5 % salivary amylase (test tube 2) resulted in a green colour change which describes a low concentration of reducing sugars.\r\nThis makes sense as a take down enzyme concentration would result in less reducing sugar being made through the enzymatic reaction between starch and amylase. A negative control reaction for the Benedict’s test occurs when the Benedict’s blue solution remains the same. Referring back to table 2 test tubes 3, 4 and 5 resulted in a negative control reaction. This may be due to the fact that the enzyme concentration were too low to produce enough reducing sugars from the starch found in the tap water to warrant a colour change. The starch (substrate) would for a substrate-enzyme building complex with salivary amylase to produce maltose and salivary amylase. In conclusion enzyme concentration does play a factor in the speed of an enzymatic reaction.\r\nThe results of Table 3, the second io dine test performed, is used to determine when the starch added with the different concentrations of salivary amylase has reached its endpoint and has been richly hydrolysed into maltose. The endpoint has been reached once the iodine test turn overs a negative control result which occurs once no starch or very few is present. According to the experimental data presented in table 3 enzyme concentrations again played a role in the speed of the reaction. 10% salivary amylase took 90 seconds where as 1% salivary amylase took 780 seconds. The starch (substrate) would for a substrate-enzyme complex with salivary amylase to produce maltose and salivary amylase.\r\nTest tube 10 + 15 will result in a positive control reaction all the time because it is comprised of water and starch. With no salivary amylase enzymes starch will always be present which is the positive control in an iodine test. A trend was found that as the salivary concentrations were halved the time to reach endpoint was d oubled, leading me to conceptualise an opposite word proportionality to be present between enzyme concentration and time to reach end point. Table 4 was another Benedict’s test performed after the each combination of test tube had reached its endpoint. The positive result in a Benedict’s test occurs once a green, orange, red or brown colour change occurs because of the presence of reducing sugars.\r\nTest tubes 16-19, containing the different concentrations of salivary amylase, resulted in a positive control reaction because the starch (substrate) would for a substrate-enzyme complex with salivary amylase to produce maltose and salivary amylase and due to the fact that maltose is a reducing sugar which happens to be the control for a Benedict’s test, a positive control reaction will occur. The negative control reaction for a Benedict’s test is when the Benedict’s solution remains blue signifying the absence of reducing sugars. According to table 4, test tube 20 only contained water and the starch suspension with no amylase present a substrate-enzyme complex will not form which will not result in a reducing sugar. Phosphorylase Table 6 is another iodine test.\r\nThe positive control reaction for an iodine test is when the solution turns blue-black. The experimental data defy in table 6 shows that test tubes 6 and 7 gave a positive reaction for starch because of the 1. 5 ml of 0. 2% starch found in solution. The negative control reaction is when the solution remains the colour of iodine, yellow. Test tube 1 through 5 gave negative result because they either do not contain any starch in solution or the amount of starch present is too little (starch underseal) and must be in presence of phosphorylase to entailment a bigger starch chain that can be reacted with the iodine test to provide a positive result. Table 8 is once again another iodine test.\r\nWith the addition of phosphorylase some of the test tubes that gave a nega tive result in the previous iodine test (table 6) may now give a positive result because of the ability of the reaction between phosphoric acid and glucose to from glucose-1-phosphate and one less glucose unit in starch chain to go in either direction. Therefore a test tube with a starch primer may use the phosphorylase to synthesis into a starch chain. The same is for the solution that gave a positive reaction may turn negative in the presence of phosphorylase to form a starch primer and glucose-1-phosphate. Referring to table 8 the test tubes that resulted in a positive control reaction were 2,3,6 and 7.\r\nBecause test tubes 6 and 7 were already gave positive results in previous iodine test (table 6) and did the same in this iodine test can only sozzled that a synthesis of a larger starch chain had occurred or the starch chain had not removed enough glucose bonds to result in a negative iodine control result. In test tube 7 the phosphorylase was boiled which would denature the e nzyme so that it could not perform its task and thereof phosphorolysis could not take place and therefore test tube 7 would have to remain a positive control result. Test tubes 2 and 3 were chiefly negative in previous iodine test but resulted in a positive control result when the enzyme phosphorylase was added.\r\nTable shows that over time both solutions grew more intense in colour signifying the synthesis of a longer starch chain. Test tube 2 had the starch primer and glucose-1-phosphate to start with and therefore took less time to give a positive control result. Test tube 3 did not contain the starch primer and I believe should not have given a positive control result. Test tube 3 did even contain the glucose-1-phosphate and perhaps may have started its own starch chain. This may have been done by having a glucose-1-phosphate and the glucose form a substrate-enzyme complex to give phosphoric acid and a larger glucose chain. The negative results were test tubes 1, 4 and 5 eac h contained the starch primer.\r\nTest tube 1 contained glucose but phosphorylase does not react with single glucose molecule and therefore test tube 1 will always give a negative control result. Test tube 4 used boiled phosphorylase and therefore the denaturised enzyme would not be able to perform modus operandi which would result in an always negative control result. Test tube 5 had the right bod but perhaps never moved in one direction of the enzymatic reaction for too long resulting in a starch primer being present the whole time though it may have had potential to yield a positive control reaction. This shows that temperature do affect an enzyme. A buffer was also used in the reaction to allow for the proper pH levels to be obtained and therefore pH levels also affect enzymes.\r\nOverall passim the experiment it was determined that substrate concentrations, reaction time and enzyme concentration effect the direction of an enzyme reaction. Reference Pelter, W. M. , McQuade, J. (2005). create from raw stuff Science in the Chemistry Laboratory: A â€Å"Mashing” Investigation of Starch and Carbohydrates. Journal of Chemical Education, 82(12), 1811-1812. Ophardt, E. C. , (2003). employment of Enzymes in Biochemical Reactions. Virtual Chembook, Retrieved November 06, 2011, from Elmhurst College, http://www. elmhurst. edu/~chm/vchembook/570enzymes. html. Hall, I. (2008). Benedicts Test for Reducing Sugars. Retrieved November 06, 2011, from Ohio University, http://www. biosci. ohiou. edu/introbioslab/Bios170/170_2/benedict. htm\r\n'