User:Moira M. Esson/Notebook/CHEM-571/2013/10/08: Difference between revisions

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Revision as of 11:30, 9 October 2013

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Objectives

  1. Monitor the turnover kinetics of adenosine to inosine catalyzed by adenosine deaminase (ADA)


Kinetics

General Protocol:

  1. Prepare a 40μM solution of adenosine in 50mM phosphate pH 7.4 buffer.
    • Note: This solution was prepared via dilution of a 0.0123M ADA stock solution
  2. Add 3mL of 40μM adenosine to a cuvette with a stir bar
    • Note: All analysis should be carrier out in a temperature controlled environment. The temperature was kept at 25°C
  3. Allow the solution to reach a constant 25°C
  4. Begin taking absorbance spectra (take a spectrum every 15seconds)
  5. After 1min, add 30μL 1.1units/mL ADA
  6. Allow the reaction to run for 10 minutes, taking a spectrum every 15seconds.


Sample Preparation:
Table 1. Preparation of 0.0123M stock solution

Mass of adenosine (g) 0.0165
Volume of buffer(mL) 5


Table 2. Preparation of 40μM adenosine solution

Volume of stock(mL) 0.0098
Volume of buffer(mL) 2.9902

UV-vis:
Figure 1. Corrected Absorbance spectra of the conversion of adenosine to inosine by ADA

  • Using the molar absorptivity of inosine and adenosine determined on 2013/09/03, the conversion of adenosine to inosine can be represented as concentrations.The molar absorptivity of adenosine and inosine was also determined for all wavelengths.


Table 3. Molar Absorptivity of Adenosine at 260nm and Inosine at 250nm

Wavelength Adenosine Inosine
250 13333 11937.5
260 16866.7 7312.5


Table 4. Concentration of Adenosine during the conversion to inosine

Time (s) Concentration(M)
15 0.000039
30
45
60
75
90
105
120
135
150
165
180
195
210
225
240
255
270
285
300
315
330
345
360
375
390
405
420
435
450
465
480
495
510
525
540
555
570
585
600


Table 5. Monitoring the concentration of inosine during the conversion of adenosine to inosine by ADA

Time (s) Concentration(M)
15 0.0000
30 0.0000
45 0.0000
60 0.0000
75
90
105
120
135
150
165
180
195
210
225
240
255
270
285
300
315
330
345
360
375
390
405
420
435
450
465
480
495
510
525
540
555
570
585
600


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