Leo Forster
2213-018
IB Biology HL Internal Assessment
Effects of Temperature on Invertase Activity
Leo Forster, 2213-018 Taejeon Christian International School
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Leo Forster
2213-018
Research Question: How will changing the incubation temperature of sucrose affect its hydrolysis into glucose and fructose as a result of invertase reaction resulting from color changes due to reaction with DNS, measured with a spectrophotometer? Introduction & Hypothesis: DNS or 3,5-Dinitrosalicylic acid reacts with simple sugars (glucose and fructose) to produce 3amino-5-nitrosalicylic acid (ANS)1. This product absorbs light at 540nm, and hence, the quantity of nitrosalicylic acid can be determined by measuring the color change of the sample2. The enzyme Invertase catalyzes the hydrolysis of sucrose into fructose and glucose3. These two factors can be combined and used to measure invertase activity by measuring the changes in glucose concentration as a product of the color-change of the solution when mixed with DNS. In this experiment, a sucrose solution will be mixed with invertase to allow its hydrolysis into glucose and fructose. The enzyme will be subject to incubation at different temperatures before and during digestion. It is expected that the varying temperatures inhibit enzyme activity and allow for less glucose to be synthesized. This can be quantified using DNS to color the solutions, and measured using a spectrophotometer. Then, after measuring the absorbance of the pigments at their respective 位max, invertase activity can be found was follows:
Consequently, the expected graph from this experiment is as follows:
Graph 1: Expected changes in invertase activity at varying temperatures 1
Class handout; Author unknown. Ibid 3 http://en.wikipedia.org/wiki/Invertase 2
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Leo Forster
2213-018
Variables:
Variable
Description
Method of Measuring / Controlling
Independent
Incubation Temperature
Activated enzyme solutions will be incubated at 4°C, 25°C, 37°C, 80°C and 100°C
Dependant
Enzyme activity4
Measure the amount of ANS with the spectrometer at λmax, 410.5nm
Controlled
Cuvette used
Same cuvette will be used for all trials and will be washed after every trial
Volume of sucrose/buffer/enzyme/DNS used
Consistent amounts of solution will be used in each trial
Concentration of sucrose solution used
1M sucrose solution will be used for every trial
Duration of incubation
Enzyme and enzyme + substrate will be incubated at consistent temperatures throughout each trial
Temperature
Experiment will be conducted in one sitting, so that room temperature does not change and stays constant at 25°C
λmax
Absorbance will be measured at same λmax every time
Table 1: Variables and their methods of controlling/measuring
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Measured using spectrometer to estimate glucose concentration after adding DNS 2
Leo Forster
2213-018
Materials: -
Invertase & Buffer
-
Micropipettes
-
Visible Spectrometer
-
Distilled water
-
1M sucrose solution
-
100 ml Beaker
-
Logger Pro 3.7
-
DNS
-
Test tubes
-
Cuvettes
Procedures: 1. Prepare enzyme solutions. See Table 2 for instructions on preparation of solutions. Respective Volumes Volume 1M sucrose solution added / cm3
Volume of Invertase added / cm3
Volume of buffer added /cm3
Volume of Distilled Water added / cm3
Total Volume of Solution / cm3
1
0.5
0.1
0.4
0.0
1.0
2
0.5
0.1
0.4
0.0
1.0
3
0.5
0.1
0.4
0.0
1.0
4
0.5
0.1
0.4
0.0
1.0
5
0.5
0.1
0.4
0.0
1.0
Control
0.0
0.1
0.4
0.5
1.0
Trial #
Table 2: Preparation of invertase solutions 2. Noting the time, incubate the invertase solution at 25°C for 30 minutes. 3. Add sucrose to the solution, and incubate at 25°C for a further 20 minutes. 4. Add 0.1ml DNS to the solution and place in boiling water for 10 minutes, allowing color to change. 5. Repeat steps 2-4 with incubation temperatures of 37°C, 80°C, 100°C, and replacing sucrose with distilled water for the control.
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Leo Forster
2213-018
6. Place the contents of the test tube in a cuvette, and place the cuvette in the visible spectrometer, having previously calibrated said machine with a cuvette full of distilled water. 7. At 位max, measure the absorbance of the pigments, and plot it against the incubation temperature. 8. Repeat steps 6-9 with the remaining cuvettes, collecting triplicate samples of each one in order to aid in the calculation of the mean and standard deviation.
At this point, it should be noted that an additional standard plot was made of glucose concentration against its absorbance in a DNS solution. Later, this plot will be used to relate the calculated absorbance of the spectrophotometer to an actual concentration of glucose within the solution. It will be used to provide final empirical data concerning the ability for invertase to digest sucrose into simple sugars.
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Leo Forster
2213-018
Data Collection: Qualitative Data: The experiment was three times, and yet two out of the three times, the results were inconclusive. In the first and last times this experiment was repeated, the DNS solutions all turned into a dark orange color (which was not intended). Nonetheless, during the second repeat of the experiment, there was a visible graduation of the colors of the solutions, though it was nonsensical and did not agree with what had been predicted.
Quantitative Data: Raw Data: Absorbance at λmax, 410.5nm Incubation Temperature / °C
Trial 1
Trial 2
Trial 3
Average ± St. Dev.a
Control
1.671
1.546
1.566
1.594 ± 0.067
4
1.804
1.807
1.799
1.803 ± 0.004
25
1.804b
1.627
1.657
1.642 ± 0.021
37
1.791
1.787
1.772
1.783 ± 0.010
80
1.683
1.708
1.723
1.705 ± 0.020
100
1.652
1.589
1.664
1.635 ± 0.040
Table 3: Absorbance of DNS solutions at λmax, 410.5 nm
a
Standard Deviation and average have been computed using the available triplicate samples and Microsoft Excel
b
Data point was omitted in calculation of standard deviation and average, as it was considered an outlier
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Leo Forster
2213-018
Data Processing: The enzyme activity was measured by the amount of glucose and fructose present after digestion, and was made measurable by the DNS. The absorbance that was taken is shown in Table 3, and the calculations of the enzyme activity can then be made with the following formula:
Absorbance has no unit. The calculations can be seen in Table 4. Calculation of Enzyme Activity
Initial Absorbance, A
Average Absorbance after DNS treatment
Enzyme Activity / arbitrary units
Control
1.594
-
0
4
1.594
1.803
0.209
25
1.594
1.642
0.048
37
1.594
1.783
0.189
80
1.594
1.705
0.111
100
1.594
1.635
0.041
Incubation Temperature / 째C
Table 4: Finding the enzyme activity. Sample Calculation: Note that Absorbance has no unit, while Time is in hours.
This calculation was repeated for the remaining data values, and the results noted in Table 4.
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Leo Forster
2213-018
Error Analysis / Uncertainties: Table 5 shows the calculations for the uncertainties involved in the calculation of enzyme activity. For Amount of Solution used: Incubation Temperature / °C
Average Absorbance ± St. Dev.
Percent Uncertainty of Absorbancef / %
Enzyme Activity ± Percent Uncertainty
Absolute Uncertaintyh
Enzyme Activity ± Absolute Uncertainty
Control
1.594 ± 0.067
4.203
-
-
0
4
1.803 ± 0.004
0.222
0.209 ± 0.222
0.000
0.222 ± 0.000
25
1.642 ± 0.021
1.279
0.048 ± 1.279
0.001
1.279 ± 0.001
37
1.783 ± 0.010
0.561
0.189 ± 0.561
0.001
0.561 ± 0.001
80
1.705 ± 0.020
1.173
0.111 ± 1.173
0.001
1.173 ± 0.001
100
1.635 ± 0.040
2.446
0.041 ± 2.446
0.001
2.446 ± 0.001
Table 5: Calculating uncertainty in Amount of Solution Used
f
Sample calculation for Percent uncertainty in amount of 0.100M SDS used:
h
Sample calculation for Absolute Uncertainty:
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Leo Forster
2213-018
Data Presentation:
Graph 2: Standard Plot for Glucose Concentration 8
Leo Forster
2213-018
Data Processing, Part II Using the best fit line on the graph above, Y = 0.14445x, the values for the absorbance calculated above can be used to calculate the actual glucose concentration within the sample. It should be noted that the glucose concentration is recorded in moles, as the absorbances across the two sets of data were taken at different wavelengths and with different volumes of solute. Hence the results in terms of strict grams of glucose could be erroneous. Incubation Temperature / 째C
Enzyme Activity
Glucose Concentrationi / M
Control
0
0.000
4
0.209
1.447
25
0.048
0.332
37
0.189
1.308
80
0.111
0.768
100
0.041
0.284
Table 6: Calculating glucose concentration
i
Sample calculation for Glucose Concentration:
(Recall that
)
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Leo Forster
2213-018
Data Presentation, Part II
Graph 3: Glucose Concentration against varying Incubation Temperatures
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Leo Forster
2213-018
Graph 4: Data set with omission and best fit line 11
Leo Forster
2213-018
Conclusion: Given the data calculated and plotted above, it seems as though there is no clear relationship between the two variables. After omission of the 25°C point, it is possible to draw a line between the remaining points with an acceptable R2 value of 0.926. It is also reasonable to conclude that this line is indeed linear, as it has a constant slope of -0.118 and has a positive R2 value. Though the best fit line does not pass through all the error bars on all of the points, there is still a reasonably strong correlation between the data points and the line itself. There was one outlier, the 25°C trial, which originally hindered the creation of a best fit line; but upon being omitted, the line drawn was of acceptable quality. As it was found that the optimum temperature for invertase is 60°C, and yet no 60°C trial was carried out, it is possible that the data modeled above could look very much different if this point were considered. Nonetheless, it seems as though the data produced does give a generally relevant and accurate prediction of how the enzyme would function, as its activity is only really hindered when subject to high temperatures. This makes sense because it is at those high temperatures that the hydrogen bonds between atoms are broken, which causes a conformational change within the enzyme, rendering it denatured and unable to continue its digestion of the substrate5. This trait is evident in the data above, as enzyme activity begins to drop off after the 80°C trial. Hence, upon further investigation, the extent of enzyme denaturation could be determined by carrying out the experiment at a range of higher temperatures. Thus, for this given range of temperatures, as the temperature is increased, enzyme activity and sucrose hydrolysis (and subsequently glucose production) will decrease. With this, the research question is answered accordingly.
It should be noted that it was generally felt that this experiment was rather flawed. Throughout experimentation, there were many errors of unexplained and random origin. Samples of distilled water and DNS would routinely turn dark orange during boiling, something which should not have been happening despite the circumstances. Additionally, enzymes incubated at freezing temperatures also turned bright orange during boiling – another result which did not make sense. It is possible that these were due to a human error in carrying out the procedure, but it is more probable that it was due to some unforeseen error within the methodology of the experiment itself.
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http://www.elmhurst.edu/~chm/vchembook/161Ahydrogenbond.html
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Leo Forster
2213-018
Evaluation: Procedural Weakness
Hypothesized Improvement
Inconsistent or contaminated cuvette face
Thoroughly clean cuvette face with a wet paper towel after every use; and make sure that the cuvette is consistently placed into the colorimeter with the same face toward the spectrometer’s sensor.
Measurement of absorbance at incorrect λmax
Correct calibration and choosing of sample with which to find λmax is important in this case. If a sample that is only vaguely colored is used to find λmax, there is a rather large chance of it not exactly correlating to the actual λmax of the contents of the solution. It is advised that the λmax always be found with the darkest, deepest colored sample.
Inconsistencies or mistakes in spectrometer calibration
Always make sure that spectrometer is calibrated with the solution in which the samples will be dissolved – in this case DNSsolution – so as to remove any possibility of different absorbance based on the makeup of the solution.
Inconsistencies in DNS application
As DNS appeared to be extremely volatile and very sensitive, much care should be taken that the conditions under which it is used be controlled properly. During the first time this experiment was carried out, test tubes were reused from a previous experiment (with washing). It was found that these tubes were reacting with the DNS and causing some of the color-change. For the second and third times the experiment was carried out, brand new pyrex tubes were used and there was less discoloration. It is unknown what other sources of error may exist, so much caution must be made in future experimentation. Table 7: Procedural errors and their negation.
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