Difference between revisions of "User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/11/14"

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(UV-vis of Adenosine and Inosine)
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
 
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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[[Image:AdenoIno2.png]]
 
[[Image:AdenoIno2.png]]
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* The absorbance values are listed below in the table. The calculation for the molar absorptivities of adenosine and inosine are shown below.
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For adenosine
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* At 235 <math>\frac{\epsilon = .099}{.0000307 M}</math> = 3224.75
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* At 265 <math>\frac{\epsilon = .24}{.0000307 M}</math> = 7817.59
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For inosine
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* At 235 <math>\frac{\epsilon = .754}{.0001 M}</math> = 7540
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* At 265 <math>\frac{\epsilon = .553}{.0001 M}</math> = 5530
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* 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.
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* The kinetic assay with its corrected information will be continued a week after break.
  
 
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| A235||0.099||0.754
 
| A235||0.099||0.754
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| Molar Absorptivity 235||3224.75||7540
 
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| A265||0.24||0.553
 
| A265||0.24||0.553
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|Molar Absorptivity 265||7817.59||5530
 
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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
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__NOTOC__
  
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==Dialysis of ADA==
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* Fractions 2 and 8 of ADA were transferred to a dialysis tube and submerged in water for one week.
  
  

Latest revision as of 21:15, 26 September 2017

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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.


Adeno300time.png

  • 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.


Adaruns1114.png

  • 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.


AdenoIno2.png

  • 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 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{\epsilon = .099}{.0000307 M}} = 3224.75


  • At 265 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{\epsilon = .24}{.0000307 M}} = 7817.59

For inosine

  • At 235 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{\epsilon = .754}{.0001 M}} = 7540


  • At 265 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \frac{\epsilon = .553}{.0001 M}} = 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.