Effect of on the activity of amylase using visible spectrophtometer
Seung Soo (Jason) Lee002213-065Internal Assessment – Investigating the Relationship between Concentration ofSodium Chloride and the Rate of Reaction of Enzyme AmylaseResearch Question:How will changing the percentage of sodium chloride concentration affect the rate of reaction ofenzyme amylase, measured using the absorbance of starch and iodine with a spectrophotometer.Introduction:Amylase is an enzyme that is involved in the human digestive process. Found in both the humanpancreas and the human saliva, amylase breaks down starch into sugar so that large molecules can beeasily digested1. Like all enzymes, amylase must be kept in a certain condition in order to functionproperly. In this experiment, the effect of sodium chloride concentration on the rate of reaction ofamylase will be investigated with the use of starch and iodine.When starch is mixed with iodine, the coils of beta amylose molecules found in starch trap iodine,causing the mixture to turn into a shade of blue-black. 2 When starch is broken down into glucose,however, the monosaccharide does not react with iodine. Therefore, glucose does not change coloreven when it’s mixed with iodine. Correspondingly, when drops of amylase are inputted into a blue-black mixture of starch and iodine, the starch molecules will be broken down into glucose molecules,causing the mixture to turn colorless. Thus, the rate of reaction of amylase correlates to the absolutevalue of the rate of change in absorbance of the solution. A rapid decrease in the absorbance of theblue-black color equates to a high rate of reaction of amylase, whereas a slow decrease in absorbancesignifies a low rate of reaction. In this experiment, an external variable of sodium chloride will bemanipulated into the amylase enzyme to determine the effect the concentration of sodium chloride onthe rate of reaction of amylase. Rate of Reaction = │ │1 "Amylase." Wikipedia. N.p., n.d. Web. 12 Jan 2011.<http://en.wikipedia.org/wiki/Amylase>.2 Senese, Fred. "How Does Starch Indicate Iodine?." N.p., 15 Feb 2010. Web. 6 Jan 2011.<http://antoine.frostburg.edu/chem/senese/101/redox/faq/starch -as-redox-indicator.shtml>.
Seung Soo (Jason) Lee002213-065Hypothesis:As aforementioned, amylase, like all enzymes, must be kept under a certain set of conditions in order tofunction properly. Factors such as pH level, temperature, and salt concentration could all denature theenzyme and decrease its activity. . When a substrate can no longer bind to the active site of an enzymedue to its conformational change, the enzyme activity and the rate of reaction of the enzyme dropssignificantly. For instance, a high concentration of sodium chloride would alter the electrostaticinteractions between charged amino acids, causing conformational change in the enzyme anddestroying its active site.3 Furthermore, the presence of sodium chloride will only have little impact onthe enzyme structure unless the sodium chloride concentration is very high, when it could completelydenature the enzyme. Therefore, an enzyme should experience an exponential decrease in its rate ofreaction as the concentration of sodium chloride is increased Rate of Reaction of Amylase, Abss-1 Concentration of Sodium Chloride, %Figure 1: Prediction of the Effect of Sodium Chloride Concentration on Rate of Reaction of Amylase EnzymeThus, the hypothesis for this experiment is that if the sodium chloride concentration is increased, thenthe rate of reaction of amylase will decrease. A high concentration of sodium chloride will denature theenzyme amylase and, as a result, it will no longer be able to break down starch into glucose. The figureabove demonstrates that the average rate of change in absorbance will undergo an exponentialdecrease as the concentration of sodium chloride is increased.3 "Rule of Protein Structure." N.p., n.d. Web. 6 Jan 2011.<http://users.rcn.com/jkimball.ma.ultranet/BiologyP ages/D/DenaturingProtein.html>.
Seung Soo (Jason) Lee002213-065Variables: Variable Description Units / range Method of Measuring / Manipulating Independent Concentration of % The independent variable will be sodium chloride manipulated by a process of serial dilution, from 20% concentration of sodium chloride to 10%, 10% to 5%, 5% to 1%, and 1% to 0.1%. Dependent Rate of reaction This will be measured with a of amylase │ │ spectrophotometer and Logger Pro. Because amylase breaks down starch into (ΔAbss-1) glucose, and glucose does not react with iodine, the enzyme activity of amylase will lower the blue-black absorbance of starch+iodine. Therefore, the rate of decrease in absorbance over time correlates to the absolute value of the rate of reaction of amylase. The change in absorbance will be measured from 0-20 seconds, and the rate of reaction can be calculated by finding the slope of the absorbance vs. time graph. The uncertainty can be considered negligible. Controlled Concentration of % This will be kept constant by using the starch & iodine same mixture created through steps 1-3 of procedures for every trial. (0.05% starch + 300μl of iodine) Amount of μl For every trial, 2.5ml of starch & iodine solutions inside solution and 500μl of sodium chloride & the cuvette amylase solution is put inside the cuvette. Temperature °C Temperature is kept constant by conducting the experiment at room temperature (about 25 °C) for every trial.Table 1: List of Variables
Seung Soo (Jason) Lee002213-065Apparatus and Materials: Electronic balance (±0.001g) 1 medium sized beaker 100 cm3 & 10 cm3 volumetric flasks Sodium Chloride 10 cm3 pipette (±0.02 cm3) Iodine 1000 μl & 50 μl micropipettes Starch 3 cm3 cuvettes Hot plate 2 cm3 micro tubes Vernier Spectrophotometer Microcentrifuge Logger Pro Five small beakers for serial dilutionProcedures:Preparation of 0.05% starch mixed with iodine 1. 0.05g of starch and 100cm3 of distilled water are poured into a medium sized beaker. 2. The beaker is placed on a hot plate, and then stirred several times using a plastic stirrer until a homogenous solution is made. 3. 300μl of iodine is put into the starch solution. It is stirred several times using a plastic stirrer until a blue-black solution is made.Preparation of sodium chloride of various concentrations (serial dilution) 8 cm3 distilled 9 cm3 distilled 3 5 cm distilled water water water 5 cm3 5 cm3 2 cm3 1 cm3 20% 10% 5% 1% 0.1% Figure 2: Serial Dilution of Sodium Chloride Solution
Seung Soo (Jason) Lee002213-065 4. 2g of sodium chloride and 10cm3 of distilled water is poured into a small beaker. 5. The beaker is stirred several times using a plastic stirrer until a homogenous solution is made, creating a 20% sodium chloride solution. 6. 5 cm3 of the obtained solution is transferred into another small beaker using a 10 cm3 pipette, and another 5 cm3 of distilled water is added into the beaker. The beaker is then stirred using a stirrer until a homogenous solution is made, creating a 10% sodium chloride solution. 7. The serial dilution of sodium chloride is continued, according to the layout in figure 2, to obtain 5%, 1%, and 0.1% concentrations of sodium chloride solutions.Conducting the experiment 8. The Vernier spectrophotometer is calibrated using distilled water. Then, the wavelength at which to measure the absorbance is determined using the maximum wavelength of the blue- black mixture of starch and iodine. 9. 450μl of 20% sodium chloride solution is put inside a micro tube using a 1000μl micropipette. 50μl of amylase solution is added into the micro tube using a 50μl micropipette. The micro tube is then placed inside a microcentrifuge so that the solution will mix together. 10. Step 9 is repeated three times for all concentrations of sodium chloride, creating three mixtures of sodium chloride and amylase for each of the five variables. 11. 2.5cm3 of the starch & iodine mixture is put into a 3cm3 cuvette using a micropipette. 500μl of the sodium chloride & amylase mixture is added into the cuvette using a micropipette. 12. The solution is squeezed in and out three times using the micropipette to ensure that amylase spreads throughout the starch solution. After mixing three times, the “start” button on Logger Pro is clicked. Steps 11 and 12 are performed with the cuvette placed inside the spectrophotometer to minimize error. 13. The rate of change in absorbance of the mixture is measured using Logger Pro for 20 seconds. 14. Steps 11-13 are repeated for triplicate trials for all five concentrations of sodium chloride.
Seung Soo (Jason) Lee002213-065Data Collection:Qualitative Data: Even with the naked eye, one could observe the disappearance of color inside the cuvette, from a dark, blue-black coloration to a clear, colorless state.Quantitative Data:*** Refer to the Appendix for a complete table of raw data from Logger Pro.
Seung Soo (Jason) Lee002213-065Data Processing:Sodium Chloride Rate of Decrease in Absorbance / ΔAbss-1 Concentration Trial 1 Trial 2 Trial 3 /% 20.0 -0.001240 -0.001280 -0.001223 10.0 -0.001517 -0.001299 -0.001402 5.0 -0.001812 -0.001523 -0.0012304 1.0 -0.001836 -0.001703 -0.001714 0.1 -0.001845 -0.001985 -0.001931 Control (no sodium chloride): -0.002830Table 2: Rate of Decrease in Absorbance for All Trials5 Sodium Chloride Calculation Average Rate of Reaction Concentration / % (±Standard Deviation)6 / ΔAbss-1 20.0 0.001248 ± 0.000029 10.0 0.001406 ± 0.000109 5.0 0.001668 ± 0.000204 1.0 0.001751 ± 0.000074 0.1 0.001920 ± 0.000071Control (no sodium chloride): 0.002830 ΔAbss-1Table 3: Calculation of Average Rates of Reaction**Because the rate of reaction must be a positive value, the average rate of reactionwas taken as an absolute value.4 This value was neglected in data processing because it was considered as an outlier.5 The rate of decrease in absorbance was determined by finding the slope of absorbance vs. time graphusing linear regression on Logger Pro software.6 The processing of standard deviation is shown in table 4
Seung Soo (Jason) Lee002213-065Data Presentation: LEGEND Run 4: Control (no sodium chloride) Run 5: 20% sodium chloride Run 10: 10% sodium chloride Run 11: 5% sodium chloride Run 14: 1% sodium chloride Run 19: 0.1% sodium chlorideFigure 4: Graph of Raw Data from Logger Pro77 Slopes of lines that have values closest to the average slope value for each concentration of sodium chloride is shown in boxes.
Seung Soo (Jason) Lee002213-065 Effect of Sodium Chloride Concentration on the Rate of Reaction of Amylase Average Rate of Reaction / ΔAbss-1 0.00185 0.0016 y = -3E-05x + 0.0018 R² = 0.914 0.00135 0.0011 0 5 10 15 20 Sodium Chloride Concentration / %Figure 5: Graph of Average Rates of Reaction against Concentration of Sodium Chloride8 98 Vertical error bars represent standard deviation for triplicate trials.9 Though they are difficult to discern, horizontal error bars represent the absolute uncertainty of sodium chloride concentration.
Seung Soo (Jason) Lee002213-065Uncertainties:Standard Deviation:Sodium Chloride Rate of Reaction of Amylase / ΔAbss-1 Average / ΔAbss-1 Concentration (±Standard /% Deviation) Trial 1 Trial 2 Trial 3 20.0 -0.001240 -0.001280 -0.001223 0.001248 ± 0.000029 10.0 -0.001517 -0.001299 -0.001402 0.001406 ± 0.000109 5.0 -0.001812 -0.001523 -0.00123010 0.001668 ± 0.000204 1.0 -0.001836 -0.001703 -0.001714 0.001751 ± 0.000074 0.1 -0.001845 -0.001985 -0.001931 0.001920 ± 0.000071Table 4: Standard Deviation at Different Concentrations of Sodium ChlorideExample of Standard Deviation Calculation:[Sodium Chloride Concentration] = 20% ≒ 0.000029Same calculations were done for 10%, 5%, 1%, and 0.1% sodium chloride concentrations.10 This value was neglected in data processing because it was considered as an outlier.
Seung Soo (Jason) Lee002213-065Uncertainty due to dilution of glucose solution:*Uncertainty due to 10cm3 pipette = ±0.02 cm3Concentration of Uncertainties Glucose / % Volume of Volume of distilled Total percentage Absolute uncertainty 3 sodium chloride water added / cm error for for concentration of solution added / concentration of glucose / % cm3 glucose / % 20.000 – – – – 3 3 10.000 5.00 ± 0.02cm = 5.00 ± 0.02cm = ±0.80 0.008 5.00 ± 0.4% 5.00 ± 0.4% 5.000 5.00 ± 0.02cm3 = 5.00 ± 0.02cm3 = ±0.80 0.004 5.00 ± 0.4% 5.00 ± 0.4% 1.000 2.00 ± 0.02cm3 = 8.00 ± 0.02cm3 = ±1.25 0.013 2.00 ± 1% 8.00 ± 0.25% 0.100 1.00 ± 0.02cm3 = 9.00 ± 0.02cm3 = ±2.22 0.011 1.00 ± 2% 9.00 ± 0.22%Table 5: Uncertainty for Concentration of Glucose Solution Sodium Chloride Concentration Average Rate of Reaction (±Standard (±Uncertainty) / % Deviation) / ΔAbss-1 20.000 0.001248 ± 0.000029 10.000 ± 0.008 0.001406 ± 0.000109 5.000 ± 0.004 0.001668 ± 0.000204 1.000 ± 0.013 0.001751 ± 0.000074 0.100 ± 0.011 0.001920 ± 0.000071 Table 6: Combined Uncertainties for Independent & Dependent Variables
Seung Soo (Jason) Lee002213-065Conclusions:The hypothesis was supported by the results to the extent that an increase in sodium chlorideconcentration decreased the rate of reaction of enzyme amylase. However, the decrease in the rate ofreaction was not exponential; rather, the relationship between NaCl concentration and average rate ofreaction was pretty linear. As sodium chloride concentration increased, the average rate of reactiondecreased at a fairly constant rate. Furthermore, once extrapolated, the graph in figure 5 demonstratesthat the rate of reaction will be 0 when the sodium chloride concentration is at 60%. From this data, onecould conclude that the enzyme amylase will completely cease to catalyze reactions at NaClconcentration of 60%.In the hypothesis, it was stated that a slight presence of sodium chloride will not affect the rate ofreaction of amylase significantly, but as the concentration of sodium chloride increases, the enzyme willundergo a rapid decrease in its rate of reaction. This is due to the fact that, as more sodium chloride ionsare present in amylase, the ions associate with oppositely charged groups in the enzyme protein,increasing protein hydration and denaturing the enzyme.11 Contrary to the hypothesis, where the rate ofreaction was predicted to undergo a slight decrease up until a certain concentration of sodium chloride,then a rapid decrease as the concentration is at a level high enough to denature the enzyme, the graphbelow displays the fact that even 0.1% of sodium chloride was enough to largely decrease the rate ofreaction of amylase. Although the 0.1% sodium chloride did not completely denature amylase, it wasstill enough to cause the greatest decrease in the rate of reaction of amylase. Effect of Sodium Chloride Concentration on the Rate of Reaction of Amylase Average Rate of Reaction / ΔAbss-1 0.003 0.0025 0.002 0.0015 0.001 0 5 10 15 20 Sodium Chloride Concentration / %Figure 6: Graph Demonstrating the Relationship between Sodium Chloride Concentration and Rate ofReaction, Including the Control11 "Protein Denaturation." N.p., n.d. Web. 7 Jan 2011. <http://class.fst.ohio -state.edu/FST822/lectures/Denat.htm>.
Seung Soo (Jason) Lee002213-065Evaluation:Overall, the results of this experiment seem fairly accurate and reliable. There are no striking outliers –except for the one value shown in table 2 – and although the standard deviations are bit sizeable forsome values, they are not critical enough to negate the conclusions drawn. As the model in figure 5represents, the relationship between sodium chloride concentration and the rate of reaction of amylaseis clearly a negative correlation. On a separate note, while it is true that the best-fit line in figure 5 is alinear one, the best-fit line for the graph in figure 6 would more likely be an exponential one. Going backto one of the conclusions drawn, the relationship shown in figure 6 represents an exponential decreasebecause of the fact that the control is also included in the graph. The jump from 0% sodium chloride to0.1% sodium chloride is largely significant – more significant than any of the other increases in sodiumchloride concentration. Thus, such results encourage the next experiment to, perhaps, incorporate aneven smaller concentration of sodium chloride. The results of this experiment support the idea that aminiscule NaCl concentration such as 0.1% was still significant enough to disrupt the electrostatic bondswithin the enzyme. In order to observe the effect of NaCl concentration on the activity of enzyme moreefficiently, it would be apt to utilize even more miniscule concentrations of sodium chloride.The sizeable nature of the standard deviation could be caused by the discrepancy created by humanerror. Although a standard was set at the beginning of the experiment, to mix the amylase and starch inthe cuvette – in & out using the micropipette three times – then pressing “start” on Logger Pro, thisprocess posed the biggest error throughout the experiment. The time taken between the moment whenenzyme amylase was put into the cuvette – thus starting to interact and break down starch – and themoment when the “start” button was clicked varied, though only by little, for every trial. Furthermore,mixing the solutions in the cuvette three times – and taking up time in the process – may have been abad idea, for that time could have been sufficient for the enzyme to do all of its work. Moreover,another problem during the procedures could have occurred with the mixing of amylase with sodiumchloride. Because 15 separate micro tubes had to be filled one by one, and then mixed through themicrocentrifuge one by one, some of the amylase solutions in the micro tubes had longer time tointeract with sodium chloride. This could have meant longer time for the sodium chloride to denaturethe enzyme, thus lowering its rate of reaction. Though it’s not certain, this could have been anothersource of error in the experiment.Overall, however, this investigation was successful in terms of the accuracy of its results. The increasedpresence of sodium chloride did lower the enzyme activity of amylase, as predicted in the hypothesis,and as accepted as a scientific fact. Although improving on minor errors could strengthen theinvestigation, the experiment successfully produced consistent and reliable data, leading up to a solidconclusion.
Seung Soo (Jason) Lee002213-065Improving the Investigation: Error Impact ImprovementTime discrepancy It could have allowed more time for There are a few ways to improve thisbetween the moment amylase to break down starch in error. One way would be to get a helpamylase is inserted some trials than in others, causing of another person, allowing him tointo the cuvette and differences in rate of reaction from press the “start” button on Logger ProLogger Pro reading is trial to trail and increasing standard as soon as the amylase is mixed threestarted deviation times. Another method would be to simply take out the mixing process, and start the Logger Pro reading as soon as amylase is inserted into the cuvette.Time discrepancy in It allowed more time for the sodium All 15 micro tubes could be incubatedthe amount of time chloride in some micro tubes to for an allotted amount of time – aroundsodium chloride was denature amylase than in other 30-40 minutes – to equalize theallowed to interact micro tubes, allowing the possibility amount of time that sodium chloride iswith amylase for further decrease in the enzyme allowed to interact with amylase.between each trial activity for amylase used in some trials compared to other trials.450μl of sodium As mentioned in the conclusion, the The amount of sodium chloridechloride solution was results demonstrate a huge decrease solution and the amount of amylaseinputted into the from no sodium chloride to 0.1% solution could be balanced, to aboutmicro tube, while only sodium chloride. This suggests that 250μl each used for every trial. This50μl of amylase too much sodium chloride was change could perhaps produce resultssolution was inputted, incorporated throughout the that are closer to those that werecausing imbalance experiment, compared to the hypothesized. amount of amylase. The abundance of sodium chloride could have disrupted the enzyme activity of amylase too much.10 cm3 pipette used Decreased precision & increased Since only about 1.5ml of each sodiumduring serial dilution range of uncertainty chloride concentration was necessaryof sodium chloride for the experiment, a micropipette could have been used to perform the serial dilution, which would have lowered the range of uncertainty.Table 7: Ways to Improve the Investigation