Rate Of Reaction Lab Report
Abstract
This study was undertaken to determine the effects of different environments affecting the rate of reaction, PNPP (p-nitrophenyl phosphate) + H20 ? PNP (p-nitrophenol) + H3P04. This reaction is catalyzed by the enzyme phosphatase. Different environments produced different reaction rates as environmental factors affect the efficiency of phosphatase. This is because environmental factors can change the tertiary structure of phosphatase, which alters its active site, and thus changes its efficiency to catalyze the reaction. We measured the rate of reaction, by using a chromogenic substrate, PNPP, and a spectrometer to determine the amount of product produced in 15 min and also using our normal curve created of known PNP concentrations …show more content…
and absorbencies. The optimal conditions for phosphatase which produced the highest rate of reactions were pH of 5 and a temperature of 50ºC. We also discovered that increasing the concentration of phosphatase will increase the rate of reaction regardless at what our initial concentration of phosphatase is. However, by increasing PNPP, the substrate, there are significant increases in the rate of reaction when the initial concentrations of PNPP are low, but when concentrations of PNPP are already high, increases in PNPP concentration resulted in little or no increase in the rate of production of PNP.
Introduction
An enzyme is a protein that catalyzes a chemical reaction. An enzyme works by increasing the rate of reaction by bringing reactants together in the proper orientation and thus decreasing the activation energy. Activation energies drop because enzymes destabilize bonds in the reactant, stabilize the transition state, make acid-base reactions more favorable, and change the reaction mechanism through a covalent bonding interaction (Freeman 2002). Enzymes also catalyze specific reactions because its active site is able to accept one or a very limited number of substrate molecules that will be able to bind to its active site. This is referred to as the lock-and-key model of enzyme function (Freeman 2002).
The environment that the enzyme is in can affect its catalytic efficiency. Environmental conditions such as pH and temperature affect the rate at which the enzyme can catalyze a reaction. This is because at high kinetic energies or at unfavorable proton levels, the tertiary structure of the enzyme will be affected. Although the peptide bond is quite stable and does not even break under extreme temperatures, the forces that stabilize a protein 's tertiary structure are weakened under elevated temperature (Feldberg 2003). When the tertiary structure of an enzyme is altered, the active site also can be altered and can no longer effectively bind the substrate and catalyze the reaction.
In our pH and temperature variant experiments, we expect to find an optimal condition, a pH level and temperature at which rate of reaction is highest, phosphatase would operate. Likewise, there will be pH and temperature conditions which will denature the enzyme, changing phosphatase tertiary structure enough so that PNPP is unable to bind to the active site and the rate of reaction is only the result of spontaneous reactions. When we create graphical representations, of rate of production of PNP vs. pH level and rate of production of PNP vs. temperature, we expect a bell curve shape. At the apex of the bell curve is the optimal rate of reaction as it operates at the optimal pH or temperature level.
Enzymes can only operate at a maximum rate. Thus, in our experiment, when we begin to add more and more PNPP, substrate, the rate of production of PNP should level off as the concentration PNPP increases. Although more substrate is added to the reaction, the reaction is already saturated with substrate and the enzyme is working as fast as it can. The enzyme will not catalyze the extra substrate as it already has substrate in its active site. In the graph of rate of production of PNP vs. concentration of PNPP, there should be an quick increase, followed by a leveling off.
However, increasing phosphatase concentration, in our experiment, will increase the rate of reaction. Addition of more phosphatase should increase the rate of production of PNP because the more phosphatase in the reaction, the more active sites will be available for PNPP, and the reaction to PNP will occur faster. For this reason, I expect there should be a positive linear relationship of the rate of production of PNP and the concentration of phosphatase.
In the following experiment, we will observe the efficiency and determine the optimal environmental conditions for the enzyme, phosphatase, as it catalyzes the reaction PNPP (p-nitrophenyl phosphate) + H20 ? PNP (p-nitrophenol) + H3P04 under different conditions of enzyme concentration, PNPP concentration, temperature and pH.
Materials and Methods
To begin to explore the rate of the reaction, we needed a quantitative measurable way to determine what the rate of the reaction was. To do this, we used the chromogenic substrate, p-nitrophenyl phosphate, PNPP. A chromogenic substrate is a compound that is colorless but yields a colored product upon chemical reaction (Pechenik 2003). As the reactants were produced p-nitrophenol, PNP, gave off a yellow color in the presence of high pH, thus the need to stop the reaction with 3 ml of 0.1 M NaOH. The amount of product was measured by its color, as the more absorbance of light from the test tube, the more amount of product was present. A Spectronic 20 color meter was used measuring absorbance at 410 nm (A410).
However, absorbance is an indirect measurement of the amount of PNP formed. We developed a standard curve relationship of PNP produced and absorbance. We took 1 ml each of concentrations of 0, 30, 60, 120, 180, 240, and 300 µM p-nitrophenol and diluted it with 1 ml each of 5.0 pH citrate buffer and 0.1% BSA along with 3ml of .1M NaOH, we obtained known concentrations of 0, 5, 10, 20, 30, 40, and 50 µM PNP in a 6 ml total solution. The 0 concentration of PNP was obtained by replacing a concentration of PNP with water. Absorbances of these concentrations were recorded and a standard curve equation was obtained relating concentration of PNP in µM and their absorbances. This standard curve would then be used to take absorbance numbers, calculate concentration PNP and thus rate of reaction. The linear relationship equation is (absorbance) = .0172[p-nitrophenol] - .00250 or otherwise written as [p-nitrophenol] = 58.1395 x (absorbance) + .1453. For calculations of obtaining te standard curve, refer to Appendix 1.
We then tested the effect of increasing the amount of enzyme on the rate of production of PNP. We began our experiment with 6 tubes containing 1 ml of pH 5.0 citrate buffer and 1 ml of 1.33 mM PNPP and allowed the solutions to incubate at 37º C for five min. The incubation time was to ensure that all reactions occurred at the same temperature. After five minutes, 1 ml of phosphatase concentration of 0.005, 0.01, 0.02, and 0.04 mg/ml was added to tubes 1 through 5, respectively. In tube 6, no enzyme was added, however 1 ml of 0.1% BSA was added. The solutions were given 15 min in 37º C to react. At 15 min, 3 ml of 0.1 M NaOH were added to each of the tubes, which raised the pH high enough to stop the reaction and allowed PNP to turn yellow. Absorbances were then measured in each of the tubes. Tube 6 containing no varying level of enzyme was used as our control as it measured the amount of absorbance, PNP, produced in the spontaneous reaction--without enzyme. The absorbency of tube 6, the spontaneous reaction, was subtracted from the absorbances of tubes 1 through 5 as to only measure the amount of absorbency produced as a result of varying levels of enzyme and not of the spontaneous reaction. We controlled variables of pH buffer level, substrate concentration, preincubation and incubation time, and amount of 0.1 M NaOH so the effect of the product formed was only a change in enzyme concentration.
Next we tested the effects of varying substrate concentration on the rate of production of PNP. In this experiment, we used 1 ml of pH 5.0 citrate buffer and 1 ml of varying substrate concentrations of 0.2 mM, 0.66mM, 1.33mM, 2.66mM, and 5.33mM. We allowed these solutions to incubate for five min at 37ºC and after 1 ml of 0.03 mg/ml phosphatase was added. After 15 min of incubation at 37ºC, 3 ml of 0.1 M NaOH were added. Absorbances were then recorded. However, adding increasing amounts of substrate would increase the amount of PNP produced in the spontaneous reaction. In order to determine the effect of increasing the substrate with phosphatase present, we also conducted the same experiment again with no enzyme present, rather 1 ml of 0.1% BSA. This produced our control, the spontaneous effects on the rate of reaction by increasing the substrate. The absorbency was measured in this control and then subtracted from its corresponding substrate concentration tube to determine just the effects of increasing the substrate in the presence of phosphatase.
We also tested the effect on the rate of production of PNP by varying incubation temperature. The procedure was like that of varying substrate concentrations, however, controlled variables were 1 ml substrate concentration of 1.33 mM of PNPP, 1 ml of pH 5.0 of citrate buffer, preincubation of five minutes at 37º C, 1 ml of 0.03 mg/ml phosphatase, 15 min incubation time, and 3 ml of 0.1 M NaOH. The variable we changed was at what temperature the reaction was allowed to incubate at. The varying temperatures we used were 0º C, room temperature (22º C), 37º C, 50º C, 70º C, and 90ºC. We also needed to repeat this experiment replacing the enzyme with 1 ml of 0.1% BSA as our control. As with the increasing concentration experiment, we wanted to isolate the effects of temperature on the enzyme and its effect on catalyzing the rate of reaction without the effects of the spontaneous reactions of increasing the temperature in the reaction. Thus, absorbance from control tubes were subtracted from their corresponding experimental absorbance numbers.
We tested the rate of reaction affected by the pH level in which the reaction took place. We held all other variables constant: substrate at 1.33 mM PNPP, preincubation time at 5 min, temperature at 37º C, enzyme concentration at 0.03 mg/ml, preincubation and incubation temperatures at 37ºC, and 3 ml of 0.1 M NaOH. What we varied were the 1 ml of pH buffer level. We used pH 3.0 citrate, 4.0 citrate, 5.0 citrate, 6.0 citrate, and 6.9 phosphate. We repeated the set up and experiment using 0.1% BSA instead of enzyme as a control to the effects of varying pH on the spontaneous production of PNP so we can determine just the effects of varying pH on the effectiveness of the enzyme. Results
The rate of reaction, the rate of production of PNP increased when we increased the concentration of the enzyme, phosphatase. Refer to figure 1.
Figure 1.
Figure 1. Rate of Reaction vs. [phosphatase]. The rate of the reaction in (µmoles/min) vs. the concentration of enzyme (phosphatase) in the tube (mg/ml) showed a positive linear relationship. As we increased the concentration of phosphatase, the rate of production of PNP also increased. The best fit equation is (rate of reaction) = 1.18[phosphatase] + .00562. The graph is shown above.
The reaction rate increased by large amounts initially when we increased the amount of PNPP, substrate, however with further increases in the PNPP concentrations, the increases in the rate of reaction lowered. Eventually the increases in the rate of reaction were minimal and showed a leveling off of the reaction rate at large concentrations of PNPP. Refer to figure 2.
Figure 2.
Figure 2. Rate of Reaction vs. [PNPP]. The rate of the reaction in (µmoles/min) was plotted against the concentration of substrate, [PNPP], in the tube (µM). With small PNPP concentrations, increases in PNPP, caused large increases in rate of reaction. However, with initially large concentrations of PNPP, an increase in PNPP caused only small increases in the rate of reaction.
As we initially increased the pH level, the rate of reaction increased. However, after pH 5, increases in pH causes a decrease in the reaction rate. At pH 6.9, the rate of reaction was 0. Refer to figure 3.
Figure 3
Figure 3. Rate of Reaction vs. pH. The rate of the reaction in (µmoles/min) vs. the pH level is shown above. There is a bell curvature in the graph and shows a peak rate of production of PNP at around pH of 5.
Initial increases in the temperature caused increases in the rate of reaction. However, temperature increases after 50ºC, dramatically reduced the rate of reaction. Refer to figure 4.
Figure 4
Figure 4. Rate of Reaction vs. temperature. The rate of the reaction in (µmoles/min) vs. the temperature at which the reaction took place showed an increasing relationship until temperature of 50ºC. Then the rate of production of PNP decreased dramatically to near zero.
Discussion
The data that was collected in the experiments do support our hypothesis in each of the variables. All of the graphical relationships concur with our expected relationships of the rate of reaction, rate of production of p-nitrophenyl, and the variables.
In the first experiment, we tested the effect of varying amount of phosphatase on the rate of reaction. There seemed to be a positive relationship because as we increased the concentration of phosphatase, the rate of reaction also increased. This is an expected result because as we increase the concentration of phosphatase, there becomes more enzyme available, thus more active sites to interact with p-nitrophenyl phosphate, PNPP, and therefore a faster rate of reaction as collectively, phosphatase can breakdown more PNPP. Refer to figure 1. Our results for the rate of reaction vs. [phosphatase] weren 't exactly perfect in a linear relationship. This may be attributed to an incorrect incubation time. Experimental error could have played a small role; especially in larger concentrations of enzyme because the rate at which the enzyme catalyzes is very quick in high concentrations and thus a few seconds off could result in a significant difference of product formed.
In the next experiment, we tested the effect of increasing the concentration of substrate, PNPP, and its effect on the rate of reaction. As we increased amount of substrate, the rate of reaction did increase, however, we saw much larger increases in the rate of reaction when initial amounts of PNPP were relatively small. But when increases in the concentration of PNPP when concentration was already fairly large, only a small increase in the rate of reaction resulted. This is in agreement with our hypothesis. As we held the amount of phosphatase, enzyme, in the reaction constant, and by increasing the amount of PNPP when substrate concentrations were low, PNPP will find available active sites to enter. However, when PNPP amounts becomes overwhelming and phosphatase is catalyzing as fast as possible, adding more substrate won 't increase the rate of the reaction significantly. Refer to figure 2. This change in rate of reaction is because PNPP had a hard time finding an open active site because the active sites are already all full. Adding more PNPP won 't increase the rate at which the enzyme catalyses the reaction. In this case, at high concentrations of substrate, the enzyme becomes the limiting factor because adding more and more substrate won 't increase the rate of reaction. An interesting experiment to further investigate would be to try to find the maximum rate the reaction, PNPP + H20 ? PNP + H3P04, occurs with the available 1 ml of 0.03 mg/ml phosphatase. This could be done by increasing the amount of PNPP by a very large amount to where the rate of the reaction does not increase at all. That rate would be the maximum rate of reaction. Looking at this graph of rate of reaction vs. [PNPP], we could also determine the km of the reaction, the amount of PNPP needed to bring the rate of reaction to half of its max rate.
The phosphatase enzyme was also tested under different pH levels. The effect of different pH levels affect the phosphatase enzyme as a varying amount of protons can affect the tertiary structure of the protein and thus the efficiency of being a catalyst. Under the different pH levels that we tested the rate of reaction, the rate of reaction seemed to be the highest around pH 5. Refer to figure 3. At any pH lower or higher than this phosphatase didn 't catalyze the reaction as fast. Our results support our hypothesis that there exists an optimal pH at which the enzyme catalyzes the reaction the fastest. Enzymes usually operate at their optimal pH in nature and therefore we would expect the environmental pH to be around 5 for phosphatase found in nature. Phosphatase also didn 't catalyze any reaction in pH 6.9. This is to be expected that there are pH levels in which tertiary structure is disrupted to a point where the active site is altered and the enzyme can no longer catalyze the reaction. The enzyme is said to be denatured. With phosphatase, denaturization occurs at around pH of 6.9. An area of exploration in phosphatase which would be interesting is to find phosphatase in nature and test at what pH it actually does operate to see if it coincides with our optimal pH of around 5.
Lastly, we put phosphatase under different temperature conditions.
The increase in temperature increased the rate of reaction up until 50ºC. After 50ºC, the rate of reaction quickly reduced to near 0. Refer to figure 4. The data supports our hypothesis. At temperatures from 0ºC to 50ºC, as temperature increases, so does the kinetic energy of the molecules. With a higher kinetic energy the molecules move faster and more PNPP will collide into phosphatase 's active site more often and the reaction would be catalyzed faster. This explains our positive relationship of increasing rate of reaction as we increased temperature. However, temperatures after 50ºC, the rate of reaction due to phosphatase immediately drops to near 0. This can be explained that at high temperatures and high levels of kinetic energy, the tertiary structure of phosphatase is altered along with the active site so PNPP can no longer fit and be catalyzed. Phosphatase is no longer able to catalyze the reaction at these high temperatures and has become denatured. The highest rate of reaction occurred at a temperature at 50ºC. Around 50ºC would be the optimal temperature for phosphatase efficiency, and if found in nature, phosphatase is expected to be in an environment of 50ºC. A follow up experiment could involve finding phosphatase in nature and seeing its environmental temperature to see if it coincides with our optimal temperature. Also, to establish a better curve, we could test the rate of reaction of phosphatase at more temperatures around the suspected optimal, maybe 45ºC and 55ºC to more accurately determine the optimal
temperature.
Another interesting aspect we didn 't explore were inhibitors of phosphatase. If we were to add a competitive inhibitor to the reaction which competed with PNPP for the active site we would expect to see a drop in the rate of reaction as concentration of the inhibitor increased. It would be interesting to see how much concentration of an inhibitor it takes to significantly reduce the enzyme 's efficacy of catalyzing the reaction.
Literature Cited
Feldberg, R., E. Siegel and K. McLaughlin. 2003. Biology 13 Cells and Organisms:
Fall.
Freeman, S. 2002. Biological Science. New Jersey: Prentice Hall.
Pechenik, J. and M. Gaudette. 2001. Cells and Organisms Bio 13L: Custom Edition for
Tufts University. Boston: Pearson.
Appendix
Appendix - Figure 1. We increased the concentration of p-nitrophenol and recorded the absorbency at 410 nm by the solution. Refer to Appendix 1 - Figure 1.
Appendix - Figure 1
Appendix - Figure 1. Absorbance at 410 nm at Concentrations of p-nitrophenol. The absorbances at 410 nm were measured and plotted against their different concentrations of p-nitrophenol. The graph is shown in figure 1. With known values of absorbance at 410 nm vs. p-nitrophenol concentrations a linear normal curve was created to quantitatively explain the positive relationship. The normal curve equation is (absorbance) = .0172[p-nitrophenol] - .00250 or otherwise written as [p-nitrophenol] = 58.1395 x (absorbance) + .1453
Appendix - Table 1
tubephosphatase (enzyme) concentrationAbsorbance at 410 nm[p-nitrophenol] mass of product (in µmoles)rate of reaction(in µmoles/min)
11 ml of .005mg/ml0.1378.11041150.0486624690.003244165
21 ml of .01mg/ml0.5934.4476050.206685630.013779042
31 ml of .02mg/ml0.68239.7964390.2387786340.015918576
41 ml of .03mg/ml0.64837.8196960.2269181760.015127878
51 ml of .04mg/ml0.60835.4941160.2129646960.014197646
60 (1ml of 1% BSA)00.14530.00087180.00005812
Appendix - Table 1. After obtaining the absorbance at 410nm and using the normal curve equation obtained in Figure 1, [p-nitrophenol] was calculated. The mass of p-nitrophenol was calculated as we found the concentration and volume of [p-nitrophenol] was .006 L. Rate of reaction is determined by dividing the mass of the product (µmoles) by the time we allowed the reaction to take place (15 minutes).
Appendix - Table 2
tubeenzyme concentration (mg/ml)concentration enzyme in tube
10.0050.001666667
20.010.003333333
30.020.006666667
40.030.01
50.040.013333333
600
Appendix - Table 2. Using the equation (C1)(V1)=(C2)(V2) and of volumes of 1ml of different concentrations of phosphatase resulting in 3 ml of total solution, final concentration of enzyme was obtained.
Appendix - Table 3
Amount of substrateA410 (experimental tube) - A410 (of control) = Net A410[p-nitrophenol] mass of product (in µmoles)rate of reaction(in µmoles/min)
1 ml of .2 mM PNPP.219 - .003 = .21612.7034320.0762205920.005081373
1 ml of .66 mM PNPP.344 - .011 = .33319.50575350.1170345210.007802301
1 ml of 1.33 mM PNPP.422 - .009 = .41324.15691350.1449414810.009662765
1 ml of 2.66 mM PNPP.460 - .020 = .44025.726680.154360080.010290672
1 ml of 5.33 mM PNPP.474 - .024 = .45026.3080750.157848450.01052323
Appendix - Table 3. After obtaining the absorbance at 410nm and using the normal curve equation obtained in Figure 1, [p-nitrophenol] was calculated. The mass of p-nitrophenol was calculated as we found the concentration and volume of [p-nitrophenol] was .006 L. Rate of reaction is determined by dividing the mass of the product (µmoles) by the time we allowed the reaction to take place (15 minutes).
Appendix - Table 4
substratesubstrate concentration in µmolessubstrate concentration in tube
1 ml of .2 mM PNPP0.00000026E-10
1 ml of .66 mM PNPP0.000000661.98E-09
1 ml of 1.33 mM PNPP0.000001333.99E-09
1 ml of 2.66 mM PNPP0.000002667.98E-09
1 ml of 5.33 mM PNPP0.000005331.599E-08
Appendix - Table 4. Using the equation (C1)(V1)=(C2)(V2) and of volumes of 1ml of different concentrations of PNPP, substrate, resulting in 3 ml of total solution, final concentration of substrate was calculated.
Appendix - Table 5
tubepHA410 (experimental tube) - A410 (of control) = Net A410[p-nitrophenol] mass of product (in µmoles)rate of reaction(in µmoles/min)
13.147 - .005 = .1428.4011090.0504066540.003360444
24.319 - .009 = .31018.1685450.109011270.007267418
35.492 - .008 = .48428.2848180.1697089080.011313927
46.384 - .006 = .37822.1220310.1327321860.008848812
56.9.002 - .005 = -.003-0.0291185-0.000174711-1.16474E-05
Appendix - Table 5. After obtaining the absorbance at 410nm as an effect of varying pH and using the normal curve equation obtained in Figure 1, [p-nitrophenol] was calculated. The mass of p-nitrophenol was calculated as we found the concentration and volume of [p-nitrophenol] was .006 L. Rate of reaction is determined by dividing the mass of the product (µmoles) by the time we allowed the reaction to take place (15 minutes).
Appendix - Table 6
tubetemperature (ºC)A410 (experimental tube) - A410 (of control) = Net A410[p-nitrophenol] mass of product (in µmoles)rate of reaction(in µmoles/min)
10.055 - 0 = .0553.34297250.0200578350.001337189
222.221 - 0 = .22112.99412950.0779647770.005197652
337.502 - .007 = .49528.92435250.1735461150.011569741
450.728 - .008 = .72042.005740.252034440.016802296
570.228 - .195 = .0332.06390350.0123834210.000825561
690.504 - .476 = .0281.7732060.0106392360.000709282
Appendix - Table 6. After obtaining the absorbance at 410nm as an effect of different temperatures and using the normal curve equation obtained in Figure 1, [p-nitrophenol] was calculated. The mass of p-nitrophenol was calculated as we found the concentration and volume of [p-nitrophenol] was .006 L. Rate of reaction is determined by dividing the mass of the product (µmoles) by the time we allowed the reaction to take place (15 minutes).
This study was undertaken to determine the effects of different environments affecting the rate of reaction, PNPP (p-nitrophenyl phosphate) + H20 ? PNP (p-nitrophenol) + H3P04. This reaction is catalyzed by the enzyme phosphatase. Different environments produced different reaction rates as environmental factors affect the efficiency of phosphatase. This is because environmental factors can change the tertiary structure of phosphatase, which alters its active site, and thus changes its efficiency to catalyze the reaction. We measured the rate of reaction, by using a chromogenic substrate, PNPP, and a spectrometer to determine the amount of product produced in 15 min and also using our normal curve created of known PNP concentrations …show more content…
and absorbencies. The optimal conditions for phosphatase which produced the highest rate of reactions were pH of 5 and a temperature of 50ºC. We also discovered that increasing the concentration of phosphatase will increase the rate of reaction regardless at what our initial concentration of phosphatase is. However, by increasing PNPP, the substrate, there are significant increases in the rate of reaction when the initial concentrations of PNPP are low, but when concentrations of PNPP are already high, increases in PNPP concentration resulted in little or no increase in the rate of production of PNP.
Introduction
An enzyme is a protein that catalyzes a chemical reaction. An enzyme works by increasing the rate of reaction by bringing reactants together in the proper orientation and thus decreasing the activation energy. Activation energies drop because enzymes destabilize bonds in the reactant, stabilize the transition state, make acid-base reactions more favorable, and change the reaction mechanism through a covalent bonding interaction (Freeman 2002). Enzymes also catalyze specific reactions because its active site is able to accept one or a very limited number of substrate molecules that will be able to bind to its active site. This is referred to as the lock-and-key model of enzyme function (Freeman 2002).
The environment that the enzyme is in can affect its catalytic efficiency. Environmental conditions such as pH and temperature affect the rate at which the enzyme can catalyze a reaction. This is because at high kinetic energies or at unfavorable proton levels, the tertiary structure of the enzyme will be affected. Although the peptide bond is quite stable and does not even break under extreme temperatures, the forces that stabilize a protein 's tertiary structure are weakened under elevated temperature (Feldberg 2003). When the tertiary structure of an enzyme is altered, the active site also can be altered and can no longer effectively bind the substrate and catalyze the reaction.
In our pH and temperature variant experiments, we expect to find an optimal condition, a pH level and temperature at which rate of reaction is highest, phosphatase would operate. Likewise, there will be pH and temperature conditions which will denature the enzyme, changing phosphatase tertiary structure enough so that PNPP is unable to bind to the active site and the rate of reaction is only the result of spontaneous reactions. When we create graphical representations, of rate of production of PNP vs. pH level and rate of production of PNP vs. temperature, we expect a bell curve shape. At the apex of the bell curve is the optimal rate of reaction as it operates at the optimal pH or temperature level.
Enzymes can only operate at a maximum rate. Thus, in our experiment, when we begin to add more and more PNPP, substrate, the rate of production of PNP should level off as the concentration PNPP increases. Although more substrate is added to the reaction, the reaction is already saturated with substrate and the enzyme is working as fast as it can. The enzyme will not catalyze the extra substrate as it already has substrate in its active site. In the graph of rate of production of PNP vs. concentration of PNPP, there should be an quick increase, followed by a leveling off.
However, increasing phosphatase concentration, in our experiment, will increase the rate of reaction. Addition of more phosphatase should increase the rate of production of PNP because the more phosphatase in the reaction, the more active sites will be available for PNPP, and the reaction to PNP will occur faster. For this reason, I expect there should be a positive linear relationship of the rate of production of PNP and the concentration of phosphatase.
In the following experiment, we will observe the efficiency and determine the optimal environmental conditions for the enzyme, phosphatase, as it catalyzes the reaction PNPP (p-nitrophenyl phosphate) + H20 ? PNP (p-nitrophenol) + H3P04 under different conditions of enzyme concentration, PNPP concentration, temperature and pH.
Materials and Methods
To begin to explore the rate of the reaction, we needed a quantitative measurable way to determine what the rate of the reaction was. To do this, we used the chromogenic substrate, p-nitrophenyl phosphate, PNPP. A chromogenic substrate is a compound that is colorless but yields a colored product upon chemical reaction (Pechenik 2003). As the reactants were produced p-nitrophenol, PNP, gave off a yellow color in the presence of high pH, thus the need to stop the reaction with 3 ml of 0.1 M NaOH. The amount of product was measured by its color, as the more absorbance of light from the test tube, the more amount of product was present. A Spectronic 20 color meter was used measuring absorbance at 410 nm (A410).
However, absorbance is an indirect measurement of the amount of PNP formed. We developed a standard curve relationship of PNP produced and absorbance. We took 1 ml each of concentrations of 0, 30, 60, 120, 180, 240, and 300 µM p-nitrophenol and diluted it with 1 ml each of 5.0 pH citrate buffer and 0.1% BSA along with 3ml of .1M NaOH, we obtained known concentrations of 0, 5, 10, 20, 30, 40, and 50 µM PNP in a 6 ml total solution. The 0 concentration of PNP was obtained by replacing a concentration of PNP with water. Absorbances of these concentrations were recorded and a standard curve equation was obtained relating concentration of PNP in µM and their absorbances. This standard curve would then be used to take absorbance numbers, calculate concentration PNP and thus rate of reaction. The linear relationship equation is (absorbance) = .0172[p-nitrophenol] - .00250 or otherwise written as [p-nitrophenol] = 58.1395 x (absorbance) + .1453. For calculations of obtaining te standard curve, refer to Appendix 1.
We then tested the effect of increasing the amount of enzyme on the rate of production of PNP. We began our experiment with 6 tubes containing 1 ml of pH 5.0 citrate buffer and 1 ml of 1.33 mM PNPP and allowed the solutions to incubate at 37º C for five min. The incubation time was to ensure that all reactions occurred at the same temperature. After five minutes, 1 ml of phosphatase concentration of 0.005, 0.01, 0.02, and 0.04 mg/ml was added to tubes 1 through 5, respectively. In tube 6, no enzyme was added, however 1 ml of 0.1% BSA was added. The solutions were given 15 min in 37º C to react. At 15 min, 3 ml of 0.1 M NaOH were added to each of the tubes, which raised the pH high enough to stop the reaction and allowed PNP to turn yellow. Absorbances were then measured in each of the tubes. Tube 6 containing no varying level of enzyme was used as our control as it measured the amount of absorbance, PNP, produced in the spontaneous reaction--without enzyme. The absorbency of tube 6, the spontaneous reaction, was subtracted from the absorbances of tubes 1 through 5 as to only measure the amount of absorbency produced as a result of varying levels of enzyme and not of the spontaneous reaction. We controlled variables of pH buffer level, substrate concentration, preincubation and incubation time, and amount of 0.1 M NaOH so the effect of the product formed was only a change in enzyme concentration.
Next we tested the effects of varying substrate concentration on the rate of production of PNP. In this experiment, we used 1 ml of pH 5.0 citrate buffer and 1 ml of varying substrate concentrations of 0.2 mM, 0.66mM, 1.33mM, 2.66mM, and 5.33mM. We allowed these solutions to incubate for five min at 37ºC and after 1 ml of 0.03 mg/ml phosphatase was added. After 15 min of incubation at 37ºC, 3 ml of 0.1 M NaOH were added. Absorbances were then recorded. However, adding increasing amounts of substrate would increase the amount of PNP produced in the spontaneous reaction. In order to determine the effect of increasing the substrate with phosphatase present, we also conducted the same experiment again with no enzyme present, rather 1 ml of 0.1% BSA. This produced our control, the spontaneous effects on the rate of reaction by increasing the substrate. The absorbency was measured in this control and then subtracted from its corresponding substrate concentration tube to determine just the effects of increasing the substrate in the presence of phosphatase.
We also tested the effect on the rate of production of PNP by varying incubation temperature. The procedure was like that of varying substrate concentrations, however, controlled variables were 1 ml substrate concentration of 1.33 mM of PNPP, 1 ml of pH 5.0 of citrate buffer, preincubation of five minutes at 37º C, 1 ml of 0.03 mg/ml phosphatase, 15 min incubation time, and 3 ml of 0.1 M NaOH. The variable we changed was at what temperature the reaction was allowed to incubate at. The varying temperatures we used were 0º C, room temperature (22º C), 37º C, 50º C, 70º C, and 90ºC. We also needed to repeat this experiment replacing the enzyme with 1 ml of 0.1% BSA as our control. As with the increasing concentration experiment, we wanted to isolate the effects of temperature on the enzyme and its effect on catalyzing the rate of reaction without the effects of the spontaneous reactions of increasing the temperature in the reaction. Thus, absorbance from control tubes were subtracted from their corresponding experimental absorbance numbers.
We tested the rate of reaction affected by the pH level in which the reaction took place. We held all other variables constant: substrate at 1.33 mM PNPP, preincubation time at 5 min, temperature at 37º C, enzyme concentration at 0.03 mg/ml, preincubation and incubation temperatures at 37ºC, and 3 ml of 0.1 M NaOH. What we varied were the 1 ml of pH buffer level. We used pH 3.0 citrate, 4.0 citrate, 5.0 citrate, 6.0 citrate, and 6.9 phosphate. We repeated the set up and experiment using 0.1% BSA instead of enzyme as a control to the effects of varying pH on the spontaneous production of PNP so we can determine just the effects of varying pH on the effectiveness of the enzyme. Results
The rate of reaction, the rate of production of PNP increased when we increased the concentration of the enzyme, phosphatase. Refer to figure 1.
Figure 1.
Figure 1. Rate of Reaction vs. [phosphatase]. The rate of the reaction in (µmoles/min) vs. the concentration of enzyme (phosphatase) in the tube (mg/ml) showed a positive linear relationship. As we increased the concentration of phosphatase, the rate of production of PNP also increased. The best fit equation is (rate of reaction) = 1.18[phosphatase] + .00562. The graph is shown above.
The reaction rate increased by large amounts initially when we increased the amount of PNPP, substrate, however with further increases in the PNPP concentrations, the increases in the rate of reaction lowered. Eventually the increases in the rate of reaction were minimal and showed a leveling off of the reaction rate at large concentrations of PNPP. Refer to figure 2.
Figure 2.
Figure 2. Rate of Reaction vs. [PNPP]. The rate of the reaction in (µmoles/min) was plotted against the concentration of substrate, [PNPP], in the tube (µM). With small PNPP concentrations, increases in PNPP, caused large increases in rate of reaction. However, with initially large concentrations of PNPP, an increase in PNPP caused only small increases in the rate of reaction.
As we initially increased the pH level, the rate of reaction increased. However, after pH 5, increases in pH causes a decrease in the reaction rate. At pH 6.9, the rate of reaction was 0. Refer to figure 3.
Figure 3
Figure 3. Rate of Reaction vs. pH. The rate of the reaction in (µmoles/min) vs. the pH level is shown above. There is a bell curvature in the graph and shows a peak rate of production of PNP at around pH of 5.
Initial increases in the temperature caused increases in the rate of reaction. However, temperature increases after 50ºC, dramatically reduced the rate of reaction. Refer to figure 4.
Figure 4
Figure 4. Rate of Reaction vs. temperature. The rate of the reaction in (µmoles/min) vs. the temperature at which the reaction took place showed an increasing relationship until temperature of 50ºC. Then the rate of production of PNP decreased dramatically to near zero.
Discussion
The data that was collected in the experiments do support our hypothesis in each of the variables. All of the graphical relationships concur with our expected relationships of the rate of reaction, rate of production of p-nitrophenyl, and the variables.
In the first experiment, we tested the effect of varying amount of phosphatase on the rate of reaction. There seemed to be a positive relationship because as we increased the concentration of phosphatase, the rate of reaction also increased. This is an expected result because as we increase the concentration of phosphatase, there becomes more enzyme available, thus more active sites to interact with p-nitrophenyl phosphate, PNPP, and therefore a faster rate of reaction as collectively, phosphatase can breakdown more PNPP. Refer to figure 1. Our results for the rate of reaction vs. [phosphatase] weren 't exactly perfect in a linear relationship. This may be attributed to an incorrect incubation time. Experimental error could have played a small role; especially in larger concentrations of enzyme because the rate at which the enzyme catalyzes is very quick in high concentrations and thus a few seconds off could result in a significant difference of product formed.
In the next experiment, we tested the effect of increasing the concentration of substrate, PNPP, and its effect on the rate of reaction. As we increased amount of substrate, the rate of reaction did increase, however, we saw much larger increases in the rate of reaction when initial amounts of PNPP were relatively small. But when increases in the concentration of PNPP when concentration was already fairly large, only a small increase in the rate of reaction resulted. This is in agreement with our hypothesis. As we held the amount of phosphatase, enzyme, in the reaction constant, and by increasing the amount of PNPP when substrate concentrations were low, PNPP will find available active sites to enter. However, when PNPP amounts becomes overwhelming and phosphatase is catalyzing as fast as possible, adding more substrate won 't increase the rate of the reaction significantly. Refer to figure 2. This change in rate of reaction is because PNPP had a hard time finding an open active site because the active sites are already all full. Adding more PNPP won 't increase the rate at which the enzyme catalyses the reaction. In this case, at high concentrations of substrate, the enzyme becomes the limiting factor because adding more and more substrate won 't increase the rate of reaction. An interesting experiment to further investigate would be to try to find the maximum rate the reaction, PNPP + H20 ? PNP + H3P04, occurs with the available 1 ml of 0.03 mg/ml phosphatase. This could be done by increasing the amount of PNPP by a very large amount to where the rate of the reaction does not increase at all. That rate would be the maximum rate of reaction. Looking at this graph of rate of reaction vs. [PNPP], we could also determine the km of the reaction, the amount of PNPP needed to bring the rate of reaction to half of its max rate.
The phosphatase enzyme was also tested under different pH levels. The effect of different pH levels affect the phosphatase enzyme as a varying amount of protons can affect the tertiary structure of the protein and thus the efficiency of being a catalyst. Under the different pH levels that we tested the rate of reaction, the rate of reaction seemed to be the highest around pH 5. Refer to figure 3. At any pH lower or higher than this phosphatase didn 't catalyze the reaction as fast. Our results support our hypothesis that there exists an optimal pH at which the enzyme catalyzes the reaction the fastest. Enzymes usually operate at their optimal pH in nature and therefore we would expect the environmental pH to be around 5 for phosphatase found in nature. Phosphatase also didn 't catalyze any reaction in pH 6.9. This is to be expected that there are pH levels in which tertiary structure is disrupted to a point where the active site is altered and the enzyme can no longer catalyze the reaction. The enzyme is said to be denatured. With phosphatase, denaturization occurs at around pH of 6.9. An area of exploration in phosphatase which would be interesting is to find phosphatase in nature and test at what pH it actually does operate to see if it coincides with our optimal pH of around 5.
Lastly, we put phosphatase under different temperature conditions.
The increase in temperature increased the rate of reaction up until 50ºC. After 50ºC, the rate of reaction quickly reduced to near 0. Refer to figure 4. The data supports our hypothesis. At temperatures from 0ºC to 50ºC, as temperature increases, so does the kinetic energy of the molecules. With a higher kinetic energy the molecules move faster and more PNPP will collide into phosphatase 's active site more often and the reaction would be catalyzed faster. This explains our positive relationship of increasing rate of reaction as we increased temperature. However, temperatures after 50ºC, the rate of reaction due to phosphatase immediately drops to near 0. This can be explained that at high temperatures and high levels of kinetic energy, the tertiary structure of phosphatase is altered along with the active site so PNPP can no longer fit and be catalyzed. Phosphatase is no longer able to catalyze the reaction at these high temperatures and has become denatured. The highest rate of reaction occurred at a temperature at 50ºC. Around 50ºC would be the optimal temperature for phosphatase efficiency, and if found in nature, phosphatase is expected to be in an environment of 50ºC. A follow up experiment could involve finding phosphatase in nature and seeing its environmental temperature to see if it coincides with our optimal temperature. Also, to establish a better curve, we could test the rate of reaction of phosphatase at more temperatures around the suspected optimal, maybe 45ºC and 55ºC to more accurately determine the optimal
temperature.
Another interesting aspect we didn 't explore were inhibitors of phosphatase. If we were to add a competitive inhibitor to the reaction which competed with PNPP for the active site we would expect to see a drop in the rate of reaction as concentration of the inhibitor increased. It would be interesting to see how much concentration of an inhibitor it takes to significantly reduce the enzyme 's efficacy of catalyzing the reaction.
Literature Cited
Feldberg, R., E. Siegel and K. McLaughlin. 2003. Biology 13 Cells and Organisms:
Fall.
Freeman, S. 2002. Biological Science. New Jersey: Prentice Hall.
Pechenik, J. and M. Gaudette. 2001. Cells and Organisms Bio 13L: Custom Edition for
Tufts University. Boston: Pearson.
Appendix
Appendix - Figure 1. We increased the concentration of p-nitrophenol and recorded the absorbency at 410 nm by the solution. Refer to Appendix 1 - Figure 1.
Appendix - Figure 1
Appendix - Figure 1. Absorbance at 410 nm at Concentrations of p-nitrophenol. The absorbances at 410 nm were measured and plotted against their different concentrations of p-nitrophenol. The graph is shown in figure 1. With known values of absorbance at 410 nm vs. p-nitrophenol concentrations a linear normal curve was created to quantitatively explain the positive relationship. The normal curve equation is (absorbance) = .0172[p-nitrophenol] - .00250 or otherwise written as [p-nitrophenol] = 58.1395 x (absorbance) + .1453
Appendix - Table 1
tubephosphatase (enzyme) concentrationAbsorbance at 410 nm[p-nitrophenol] mass of product (in µmoles)rate of reaction(in µmoles/min)
11 ml of .005mg/ml0.1378.11041150.0486624690.003244165
21 ml of .01mg/ml0.5934.4476050.206685630.013779042
31 ml of .02mg/ml0.68239.7964390.2387786340.015918576
41 ml of .03mg/ml0.64837.8196960.2269181760.015127878
51 ml of .04mg/ml0.60835.4941160.2129646960.014197646
60 (1ml of 1% BSA)00.14530.00087180.00005812
Appendix - Table 1. After obtaining the absorbance at 410nm and using the normal curve equation obtained in Figure 1, [p-nitrophenol] was calculated. The mass of p-nitrophenol was calculated as we found the concentration and volume of [p-nitrophenol] was .006 L. Rate of reaction is determined by dividing the mass of the product (µmoles) by the time we allowed the reaction to take place (15 minutes).
Appendix - Table 2
tubeenzyme concentration (mg/ml)concentration enzyme in tube
10.0050.001666667
20.010.003333333
30.020.006666667
40.030.01
50.040.013333333
600
Appendix - Table 2. Using the equation (C1)(V1)=(C2)(V2) and of volumes of 1ml of different concentrations of phosphatase resulting in 3 ml of total solution, final concentration of enzyme was obtained.
Appendix - Table 3
Amount of substrateA410 (experimental tube) - A410 (of control) = Net A410[p-nitrophenol] mass of product (in µmoles)rate of reaction(in µmoles/min)
1 ml of .2 mM PNPP.219 - .003 = .21612.7034320.0762205920.005081373
1 ml of .66 mM PNPP.344 - .011 = .33319.50575350.1170345210.007802301
1 ml of 1.33 mM PNPP.422 - .009 = .41324.15691350.1449414810.009662765
1 ml of 2.66 mM PNPP.460 - .020 = .44025.726680.154360080.010290672
1 ml of 5.33 mM PNPP.474 - .024 = .45026.3080750.157848450.01052323
Appendix - Table 3. After obtaining the absorbance at 410nm and using the normal curve equation obtained in Figure 1, [p-nitrophenol] was calculated. The mass of p-nitrophenol was calculated as we found the concentration and volume of [p-nitrophenol] was .006 L. Rate of reaction is determined by dividing the mass of the product (µmoles) by the time we allowed the reaction to take place (15 minutes).
Appendix - Table 4
substratesubstrate concentration in µmolessubstrate concentration in tube
1 ml of .2 mM PNPP0.00000026E-10
1 ml of .66 mM PNPP0.000000661.98E-09
1 ml of 1.33 mM PNPP0.000001333.99E-09
1 ml of 2.66 mM PNPP0.000002667.98E-09
1 ml of 5.33 mM PNPP0.000005331.599E-08
Appendix - Table 4. Using the equation (C1)(V1)=(C2)(V2) and of volumes of 1ml of different concentrations of PNPP, substrate, resulting in 3 ml of total solution, final concentration of substrate was calculated.
Appendix - Table 5
tubepHA410 (experimental tube) - A410 (of control) = Net A410[p-nitrophenol] mass of product (in µmoles)rate of reaction(in µmoles/min)
13.147 - .005 = .1428.4011090.0504066540.003360444
24.319 - .009 = .31018.1685450.109011270.007267418
35.492 - .008 = .48428.2848180.1697089080.011313927
46.384 - .006 = .37822.1220310.1327321860.008848812
56.9.002 - .005 = -.003-0.0291185-0.000174711-1.16474E-05
Appendix - Table 5. After obtaining the absorbance at 410nm as an effect of varying pH and using the normal curve equation obtained in Figure 1, [p-nitrophenol] was calculated. The mass of p-nitrophenol was calculated as we found the concentration and volume of [p-nitrophenol] was .006 L. Rate of reaction is determined by dividing the mass of the product (µmoles) by the time we allowed the reaction to take place (15 minutes).
Appendix - Table 6
tubetemperature (ºC)A410 (experimental tube) - A410 (of control) = Net A410[p-nitrophenol] mass of product (in µmoles)rate of reaction(in µmoles/min)
10.055 - 0 = .0553.34297250.0200578350.001337189
222.221 - 0 = .22112.99412950.0779647770.005197652
337.502 - .007 = .49528.92435250.1735461150.011569741
450.728 - .008 = .72042.005740.252034440.016802296
570.228 - .195 = .0332.06390350.0123834210.000825561
690.504 - .476 = .0281.7732060.0106392360.000709282
Appendix - Table 6. After obtaining the absorbance at 410nm as an effect of different temperatures and using the normal curve equation obtained in Figure 1, [p-nitrophenol] was calculated. The mass of p-nitrophenol was calculated as we found the concentration and volume of [p-nitrophenol] was .006 L. Rate of reaction is determined by dividing the mass of the product (µmoles) by the time we allowed the reaction to take place (15 minutes).