User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/11/14

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ADA Kinetic Assay Runs During the previous lab entry, the molar absorptivities of adenosine and inosine were calculated. Adenosine was found to have a higher molar absorptivity at 235. It was concluded from the previous period that the absorption of adenosine should decrease over time. A kinetic assay scan of the reaction containing 2.7 mL of 0.05 M sodium phosphate buffer, 300 μL of 3.07 mM adenosine, and 15 μL of 42 μM ADA was taken. The final concentration for each reagent are 0.0450 M, 0.307 mM, and 0.21 μM, respectively. The program was set to Kinetics with the wavelength at 235 for a collection span of 600s. In the scan, it can be observed that the absorption of adenosine is not decreasing over time. Instead, the absorption is increasing. Also, the signal for the absorption of adenosine is over 1. It was decided that the next measurements, the concentration of adenosine should be decreased by decreasing the volume in th cuvette. This is to ensure that the absorbance is below 1. As seen above, decreasing the concentration of adenosine maintained an absorbance below 1. However, the absorbance is still increasing. The addition of 50 μL ADA intensified the increase of absorbance in comparison to the addition of 15 μL ADA. Volume of ADA (μL) Concentration of ADA (μM) Volume of phosphate buffer (mL) Concentration of phosphate buffer (M) Volume of adenosine (μL) Concentration of adenosine (mM) 15 0.21 2.95 0.0492 50 0.0512 50 0.70 2.95 0.0492 50 0.0512 UV-vis of Adenosine and Inosine Due to the inconsistent behavior observed from the data above, it was decided to remeasure the absorbance of adenosine and inosine in the UV-visible spectra. The program was returned to the Spectrum setting. The measurement was adjusted to 400-200 nm. The absorbance values are listed below in the table. The calculation for the molar absorptivities of adenosine and inosine are shown below. For adenosine At 235 [math]\displaystyle{ \frac{\epsilon = .099}{.0000307 M} }[/math] = 3224.75 At 265 [math]\displaystyle{ \frac{\epsilon = .24}{.0000307 M} }[/math] = 7817.59 For inosine At 235 [math]\displaystyle{ \frac{\epsilon = .754}{.0001 M} }[/math] = 7540 At 265 [math]\displaystyle{ \frac{\epsilon = .553}{.0001 M} }[/math] = 5530 Running the UV-vis scan for each reagent showed that the increasing trend observed on the ADA kinetic assays was inosine not adenosine. This was verified by recalculating the molar absorptivities. The molar absortivity of inosine at 235 is greater than adenosine. This explains the increase over time since as ADA catalyzes the formation of inosine from adenosine; the catalysis increases the concentration of inosine so as its absorbance. The kinetic assay with its corrected information will be continued a week after break. ' adenosine inosine A235 0.099 0.754 Molar Absorptivity 235 3224.75 7540 A265 0.24 0.553 Molar Absorptivity 265 7817.59 5530 Dialysis of ADA Fractions 2 and 8 of ADA were transferred to a dialysis tube and submerged in water for one week.