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

The Effects of Substrate Concentration, Reaction Time and Enzyme Concentration on Enzyme Reactions Corey von Ellm-St. Croix Rachael Kwan ID 20427841 Matthew Hrycyshyn & Saeideh Mayanloo Biol 130L, Section 017 Wednesday, 930am-1220pm, 151 November 09, 2011 A living system entertains its activity by means of and through with(predicate) enzymes. Enzymes be make from hundreds or even thousands of amino acids connected in a very unique and specific order. Almost all enzymes are proteins, except for ribozymes. The reach of amino acids then folds into a unique exploit.That shape non lone(prenominal) al firsts the enzyme to carry out specific chemical responses barely to act as a very efficient catalyst. The enzyme velocitys that reaction up tremendously. to each adept enzyme reacts with one specific reactant called a substrate that ordain configuration its products. The purpose of the experiments is to determine the effects of substrate assiduity, reaction time and enzyme a bsorption on the direction of an enzyme reaction. Amylase is a digestive enzyme found in twain the saliva and the small in sortine.Salivary amylase is a hydrolytic reaction that breaks down stiffen molecules by systematically breaking off the maltose molecules from the ends of amylum gyves. The maltose is further broken down by approximately other enzyme. Phosphorylase is an enzyme that systematically removes glucose molecules by consumes phosphoric acid to break the beta-1-4-glucosidic chemical bonds in stiffen. The interaction of orthophosphate with the glucosidic bond ensues in the formation of glucose-1-phosphate and the loss of a chain unit in stiffen. In the reverse reaction the glucose part of glucose-1-phosphate is added as a refreshed chain unit and phosphate is set free.This reversible enzymatic polymerization occurs with little change in free energy and therefor the reaction may choose to go both way. Iodine streamlet is a adjudicate for the heraldic be aring of stiffen in which the sample puzzle outs grimy- pitch-dark in color when a few deteriorates of kibibyte iodide dissolving agent are fit(p) on the sample. A ostracise unity evidence is when the reaction mud chickenhearted in colour. It is the reaction between tincture of atomic number 53 and the coiled polymer of glucose cognize as amylase in starch that causes the colour change. The reaction occurs when straight amylase chains form helices in which the atomic number 53 digest pass inside.Glycogen overly receives a colour change because it is a glucose polymer as well provided its structure differentiates from starch which therefore forms a chocolate-brown colour change. The one tally does not work for mono or disaccharides because they are as well small to capture the iodine. The Benedicts campaign is used to line up the presence of reduction abrasions. Reducing scrawls are boodles with a free aldehyde or ketone group. The free research laboratory ile carbonyl group allows all monosaccharides to be cut back sugars. The same goes for disaccharides as some also contain the free reactive carbonyl group.The colour of precipitate formed when the Benedicts mental turn outing acquires a convinced(p) declaration depends on the stringency of trim down sugars portray. A green colour change indicates that few reducing sugars are present. orangeness indicates a higher concentration, red an even higher concentration and brown is the highest concentration colour change. A prejudicious audition for reducing sugar occurs when the Benedicts ascendant corpse its grimy colour. Materials and method Materials and Method found in 1119 BIOL 130, Department of Biology 2011 Cell Biology research lab 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 tryout delay 1 discharge pipework Number Results by Experimentation Con trol 1 (10% salivary amylase solution) chickenhearted cast out 2 (5% salivary amylase solution) yellownessed Negative 3 (2% salivary amylase solution) Yelllow Negative 4 (1% salivary amylase solution) Yellow Negative 5 (1% starch suspension) inconsolcapable-Black Positive Table1 The shelve to a higher place check the first iodine see done. Shows components of each stress supply as well as the tops and decreed or negative nurse.Dilutions were done using tap wet (may contain other molecules). Corresponds to step 5 through 10. Benedicts running game Table 2 turn out Tube Number Results Through Experimentation Control 1 (10% salivary amylase solution) Brown-orange precipitate Positive 2 (5% salivary amylase solution) Green precipitate Positive 3 (2% salivary amylase solution) dour Negative 4 (1% salivary amylase solution) black Negative 5 (1% starch suspension) blasphemous Negative Table 2 The duck above represent the first Benedicts establish done. Show s components of each test resistance as well as the resolvings and positive or negative restrict.Each of the above test organ pipe-shaped structures contains 4ml Benedicts solution and were boiled for 5 minutes when find directs and are related to steps 5 through 10. Iodine test Table 3 Test Tubes Number of Drops till Negative Time Interval Time 9+14 (1% salivary amylase solution) 13 60 certifys 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= 150 seconds 6+11 (10% salivary amylase solution) 18 5 seconds 18*5= 90 seconds 10+15 ( urine system) (Always positive) N/A N/ATable 3 The table above represents the time it took to tense up endpoint. Shows components of each test thermionic vacuum provide, scrap of bemuses, time interval between drops and time to elapse endpoint. Each of the above test tubes contains 2ml 1% starch suspension and 2ml McIlvaines buffer. The above te st tubes were placed in a warm bath at 37 degrees Celsius and pertain to steps 11-18. Benedicts test Table 4 Test tube Results through experimentation Control 20 (water) blue negative 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 search for reducing sugars later endpoint. Each of the above test tubes contains 4ml Benedicts solution and were boiled for 5 minutes when determining results and are related to steps 18-20. Phosphorylase Composition of test tubes Table 5 TEST TUBE NUMBER CONTAINS One 1. 5ml of 0. 01M glucose + 1 drop of 0. % starch suspension Two 1. 5 of 0. 01M glucose-1-phosphate+ 1 drop of 0. 2% starch suspension Three 1. 5 of 0. 01M glucose-1-phosphate Four 1. 5 of 0. 01M glucose-1-phosphate+ 1 drop of 0. 2% starch suspension Fi ve 1. 5 of 0. 01M glucose-1-phosphate + 0. 5ml of 0. 2M kelvin phosphate+ 1 drop of 0. 2% starch suspension hexad 0. 5ml of 0. 2M thousand phosphate + 1. 5ml of 0. 2% starch suspension Seven 0. 5ml of 0. 2M potassium phosphate+ 1. 5ml of 0. 2% starch suspension Eight 4ml Boiled phosphorylase Table 5 The above table represent the solutions present in the test tubes 1-8 from steps 2-10 Iodine test Table 6Test tube Results through experimentation Control 1 Yellow Negative 2 Yellow Negative 3 Yellow Negative 4 Yellow Negative 5 Yellow Negative 6 blue(a)- colored Positive 7 Blue- fatal Positive Table 6 Search for starch deep down test tubes 1-7. Shows components of each test tube as well as the results and positive or negative authorization. Composition of test tubes Table 7 TEST 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 8Time Interval test tube 1 Test tube 2 Test tube 3 Test tube 4 Test tube 5 Test tube 6 Test tube 7 1028-1032 yellow genuinely indistinct blue- unrelentingamoor yellow yellow yellow kick the bucket blue-black Blue black 1032-1036 yellow Blue black yellow yellow yellow Very faint blue-black Blue black 1036-1039 yellow Blue black yellow yellow yellow Faint blue black Blue black 1039-1042 y ellow Blue black yellow yellow yellow Faint blue black Blue black 1042-1046 yellow Blue black yellow yellow yellow Blue black Blue black 1046-1049 yellow Blue black Very faint blue black yellow yellow Blue black Blue black 1049-1052 Yellow Blue black Faint blue black Yellow yellow Blue black Blue black 1052-1055 Yellow Blue black Blue black Yellow Yellow Blue black Blue black 1055-1058 Yellow Blue black Blue black Yellow Yellow Blue black Blue black 1058-1042 yellow Blue black Blue black yellow Yellow Blue black Blue black Table 8 Test for the presence and synthesis of starch.Contains the time interval from when the anterior test had ended to termination 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 tests halt is the presence of starch. If starch is presence then the concur is positive resulting i n 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. all the way starch is present based on just the component of the solution.A negative control in an iodine test will result in maintenance of the yellow colour of iodine. According 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. This is due to the fact that all that is present is the enzyme salivary amylase and water and therefore no starch. The Benedicts test control is the presence of reducing sugars (sugars with a free aldehyde or ketone group). If a reducing sugar is present then a positive control reaction will occur. A positive control reaction is when a colour of the blue Benedicts solution turns green, orange, red or brown after boiling.Each colour represents the co ncentration of reducing sugars present, green being the final 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 alloy the amylase solution may contain some starch which would in turn become 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 set off 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.This makes sense as a lower enzyme concentration would result in less(prenominal)(prenominal) reducing sugar being made through the enzymatic reaction between starch and amylase. A negative control reaction for the Benedicts test occurs when the Benedicts blue solution remains the same. Ref erring 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 complex with salivary amylase to produce maltose and salivary amylase. In conclusion enzyme concentration does profligacy a factor in the speed of an enzymatic reaction.The results of Table 3, the second iodine test commited, is used to determine when the starch added with the different concentrations of salivary amylase has reached its endpoint and has been fully hydrolysed into maltose. The endpoint has been reached once the iodine test gives 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 s econds 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.Test 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 forever 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 doubled, leading me to confide an opposite word proportionality to be present between enzyme concentration and time to reach end point. Table 4 was another Benedicts test performed after the each combination of test tube had reached its endpoint. The positive result in a Benedicts test occurs once a green, orange, red or brown colour change occurs because of the presence of reducing sugars.Test tubes 16-19, containing the different concentrations of salivary amylase, resulted in a pos itive 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 Benedicts test, a positive control reaction will occur. The negative control reaction for a Benedicts test is when the Benedicts 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.The positive control reaction for an iodine test is when the solution turns blue-black. The experimental data given 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 fare of starch present is too little (starch undercoat) and must be in presence of phosphorylase to synthesis a larger starch chain that can be reacted with the iodine test to provide a positive result. Table 8 is once again another iodine test.With the addition of phosphorylase some of the test tubes that gave a negative 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 t hat resulted in a positive control reaction were 2,3,6 and 7.Because 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 mean 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 enzyme so that it could not perform its task and therefore phosphorolysis could not take place and therefore test tube 7 would halt to remain a positive control result. Test tubes 2 and 3 were primarily negative in previous iodine test but resulted in a positive control result when the enzyme phosphorylase was added.Table shows that over time both solutions grew more incisive 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 however 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 each contained the starch primer.Test 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 denatured enzyme would not be able to perform function which would result in an always negative control result. Test tube 5 had the right condition 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 possible 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 veracious pH levels to be obtained and therefore pH levels also affect enzymes. general throughout 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). Brewing Science in the alchemy Laboratory A Mashing Investigation of Starch and Carbohydrates. Journal of Chemical Education, 82(12), 1811-1812. Ophardt, E. C. , (2003). usage 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