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Effect Of Water Temperature On Amylase Using Colorimetric Starch Assay

1540 words - 7 pages

Timothy Pratt
Wednesday, 12:20
PBIO 1210L
Scientific Paper:
Effect of Water Temperature on Amylase Using Colorimetric Starch Assay

Introduction

For plants to function properly, chemical reactions must take place within the cells of each individual plant. Energy is necessary for each reaction to occur. The amount of energy that is necessary for a reaction is called the activation energy. Enzymes are used to reach the correct activation energy in plants. They lower the activation energy to the correct amount for the reaction to take place. Certain enzymes catalyze certain reactions, which means that many enzymes to operate the cell. On each cell, there is a spot where the substrate ...view middle of the document...

Methods

Iodine-Potassium-Iodide (IKI) was used to perform a colorimetric starch assay. The IKI interacts with the starch chains, forming a blue color. The concentration of the starch molecule is shown by how concentrated the blue color is.
In our control enzyme activity assay, we collected ten test tubes, labeled from zero to nine, and placed one drop of IKI into each tube. Next we took 2.5mL of our solution without any enzyme and added it to the test tube labeled zero. We then took a flask of our starch solution and added 1.0 mL, we immediately started the stopwatch and mixed the enzyme into the solution. We removed 2.5 mL of the reaction and placed it into one test tube with IKI in minute intervals. After nine minutes, all test tubes were filled with the enzymatic solution. The more starch in each test tube, the more concentrated the blue color is and vice versa. However this only gave us qualitative data. To obtain concrete, quantitative results, we took the test tubes over to the spectrophotometer. We wiped off each test tube off with a tissue then placed it into spectrophotometer two and recorded the readings. The results are shown in the table below.
After the control, we started the experiment. We collected twenty test tubes and labeled two different sets zero to nine and placed a drop of IKI into each tube. This time we put the flask that contained the solution in water that was forty degrees Celsius. Once the temperature of the solution was the same as that of the water, we collected 2.5mL from the starch solution and placed it into the test tube labeled zero. Then we added the 1.0 mL of amylase into the flask. At minute intervals, we placed 2.5mL of the reaction into a test tube. We again recorded the absorbance of the test tubes in spectrophotometer two. The same process was used for the second set of tubes. The tables below represent the data we collected.

Results

First Control Trial
Average Reaction Rate per Minute: 4.25 (μg/ml)
Time Absorbance Reading (OD at 580 nm) Starch Concentration (μg/ml) Reaction Rate (μg digested starch/min)
0 0.738 67.65 0
1 0.548 50.23 17.42
2 0.538 49.31 0.92
3 0.474 43.44 5.87
4 0.465 42.62 0.82
5 0.444 40.70 1.92
6 0.399 36.57 4.13
7 0.354 32.45 4.12
8 0.335 30.71 1.74
9 0.321 29.42 1.29

Second Control Trial
Average Reaction Rate per Minute: 5.61 (μg/ml)
Time Absorbance Reading (OD at 580 nm) Starch Concentration (μg/ml) Reaction Rate (μg digested starch/min)
0 0.735 67.37 0
1 0.470 43.08 24.29
2 0.459 42.07 1.01
3 0.364 33.36 8.71
4 0.308 28.23 5.13
5 0.296 27.13 1.10
6 0.246 22.55 4.58
7 0.228 20.90 1.65
8 0.213 19.52 1.38
9 0.184 16.86 2.66

First Experimental Trial (40 Degrees Celsius)
Average Rate per Minute: 5.00 (μg/ml)
Time Absorbance Reading (OD at 580 nm) Starch Concentration (μg/ml) Reaction Rate (μg digested starch/min)
0 0.587 53.80 0
1 0.454 41.61 12.19
2 0.321 30.06 11.55
3 0.239 21.90 8.10
4 0.191 17.50 4.40
5 0.135 12.37...

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