0636 absorbance per minute since change in absorbance

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initial rate, with a calculated increase of 0.0636 absorbance per minute. Since change in absorbance is indicative of change in concentration, we can be sure that this buffer has the highest initial rate of product formation for our enzyme. With our optimum pH identified, we were ready to determine the v max and K m of yeast ADH with varying concentrations of ethanol. After 1 Gober, J. W. (2018). Introduction to Protein Science Laboratory Manual. 15-18. 17th edition
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assaying the test tubes with varying concentrations of ethanol we were able to use Beer’s Law to calculate the rate of change of concentration over time for each of the tubes using our known change in absorbance as a reference point (Fig. 1, Table 1). The molar absorptivity constant for NADH is 6.22 mM -1 cm -1 and the path length for our cuvette was 1cm. 2 Example Calculation: With the change in concentration calculated for each of the tubes, we were then able to get our v max and K m by calculating our change in product over time from our change in concentration. This involved taking into account the volume used to create the concentrations Example Calculation: 2 Gober, J. W. (2018). Introduction to Protein Science Laboratory Manual. 15-18. 17th edition
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Figure 1: A plotted graph of the absorbance values assayed over time in our 9 test tubes. Using Beer’s Law, the slope of these lines is equal to the change in absorption which has a linear relationship with change in concentration. The changes in absorption range from 0.0264 to 0.0641 The samples were prepared using the solutions shown in Table 1 Table 1: Molecular composition of samples prepared for assay Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Buffer (mL) 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 2M EtOH (mL) 0 0.02 0.04 0.06 0.08 0.1 0.2 0.3 0.4 25mM NAD + (mL) 1 1 1 1 1 1 1 1 1 H 2 O (mL) 0.4 0.38 0.36 0.34 0.32 0.30 0.20 0.10 0 [EtOH] in 3mL 0 0.013 0.026 0.04 0.053 0.066 0.133 0.2 0.26
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Table 2: Absorbance assay of samples over time Time (min) Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 0 0.0339 0.031 0.0198 0.0078 0.0679 0.0404 0.0342 0.0146 0.1833 0.0368 0.0373 0.0259 0.0146 0.0805 0.0503 0.0455 0.0261 0.3666 0.042 0.0435 0.0323 0.0215 0.0857 0.0601 0.0553 0.0382 0.55 0.0462 0.0499 0.0395 0.0273 0.0953 0.0695 0.0658 0.0497 0.7333 0.0512 0.0566 0.0458 0.0337 0.1025 0.0789 0.0743 0.0646 0.9166 0.0566 0.0635 0.0557 0.0393 0.1112 0.0891 0.0849 0.0751 1.1 0.0612 0.0712 0.0589 0.0471 0.1209 0.0986 0.0951 0.0865 1.283 0.0666 0.0761 0.0654 0.0516 0.1289 0.1084 0.1074 0.0988 1.466 0.0716 0.0827 0.072 0.0574 0.1376 0.119 0.1161 0.1101 1.65 0.0763 0.0891 0.0787 0.0666 0.1461 0.128 0.1291 0.1222 1.833 0.0813 0.0948 0.0855 0.0708 0.1545 0.137 0.1354 0.1347 2.016 0.0861 0.1019 0.0914 0.0775 0.1641 0.1464 0.1457 0.1459 2.2 0.091 0.1101 0.0988 0.0843 0.1721 0.1574 0.156 0.1583 2.383 0.0955 0.1146 0.1048 0.0916 0.1816 0.1655 0.1672 0.1694 2.567 0.1006 0.1212 0.1116 0.0973 0.193 0.1751 0.1763 0.1816 2.75 0.1057 0.1282 0.1181 0.1042 0.2024 0.1846 0.1874 0.193 2.933 0.1097 0.1345 0.1249 0.1112 0.2088 0.1941 0.1975 0.2046 3.116 0.1146 0.1408 0.1311 0.1177 0.2158 0.2032 0.208 0.215 3.3 0.119 0.1471 0.1383 0.1247 0.2246 0.2126 0.2177 0.2266 3.483 0.1244 0.1536 0.1452 0.1309 0.2322 0.2218 0.2285 0.2387 3.666 0.1291 0.1598 0.152 0.1374 0.2404 0.2313 0.2379 0.2492 3.85 0.134 0.1668 0.1591 0.1443 0.2492 0.2404 0.2477 0.2611 dA/dt (mM/min) 0.0264 0.0352 0.0359 0.0353 0.0471 0.052 0.0554 0.0641
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dc/dt (mM/min) 0.00424437 299 0.005659 163987 0.005771 70418 0.005675 241158 0.007572 347267 0.008360 128617 0.008906 752412 0.010305 46624 dP/dt (3mL, umol/min) 0.01273311 897 0.016977 49196 0.017315 11254 0.017025 72347 0.022717 0418 0.025080 38585 0.026720 25723 0.030916 39871 The lower part of the table shows the calculated values for change in absorption over time, change in concentration over time, and change in product concentration over time. These values were derived from
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  • Spring '08
  • Kim
  • pH, buffer solution, Alcohol dehydrogenase, Lineweaver–Burk plot, Protein Science Laboratory Manual.

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