User:Alexander Cvitan/Notebook/Experimental Biological Chemistry Lab/2013/10/09

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Objective

  • To complete the procedure outline by Dr.Hartings HERE.
  • We hope to outline the kinetics of the conversion of a solution of Adenosine to Inosine using ADA and a EHNA inhibitor.

Procedure

  • Measured .1065 g Adenosine and added it to 10 ml buffer to create approx 40 mM adenosine solution. 3 micro-liters 40mM adenosine was added to 2996 micro-liters of buffer and 4 micro-liter of EHNA inhibitor to create a 40uM solution of adenosine.<br.>
  • In Dr.Hartings lab we added 30 ul of ADA after data was recorded 5 times. Data was recorded every 15 sec for 10 min.<br.>
  • Dilutions were made with 50mM Phosphate buffer pH 7.4 <br.>
  • ADA Stock Solution 0.11 units/mL ADA<br.>
  • EHNA Stock 3μM <br.>

Data

Spectra of ADA Conversion of Adenosine to Inosine

  • This graph represents the conversion of Adenosine to Inosine using ADA and inhibitor. Shown in the graph, there is a shift in the spectra which were recorded every 15 sec for 10 min. The shift; however is less prominent than it was yesterday without the inhibitor.
  • Overall it was observed that the adenosine peak shrunk more slowly overtime, and everything appeared to shift to the right.

Screen shot 2013-10-19 at 8.38.29 PM.png

  • At peak 250 nm; the molar absorptivity of adenosine and inosine are 10224 (1/M*cm) and 11007 (1/M*cm) respectively.
  • At Peak 260 nm; the molar absorptivity of adenosine and inosine are 14025 (1/M*cm) and 6630 (1/M*cm) respectively.

Concentration vs. Time for ADA Conversion of Adenosine to Inosine using an Inhibitor

  • The graph below represents the change in concentration from adenosine to inosine over time with the presence of an inhibitor of ADA. In order to calculate this from the spectra above, molar absorptivities from a previous day were utilized. As seen in the graph it isn't perfect because the concentration of inosine at time zero should ideally be zero.

Oct 9 Con vs. time ADA and Inhib.png