User:Michael F. Nagle/Notebook/Chem 571/2013/02/06

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==Objective==
 
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*Complete Activity Assay for ADA
 
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==Procedure==
 
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#UVProbe was opened.
 
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##Window > 1. Kinetics
 
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##Methods Icon
 
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###Wavelength: 265nm
 
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###Duration: 300 seconds (5minutes)
 
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###OK
 
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#Shimadzu CPS-Controller was set to 25°C.
 
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##wait for the temperature to raise to 25°C
 
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#place the sample in the cell and click start.
 
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*Phosphate buffer was used as a blank and was used to create the baseline.
 
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*Kinetics was run at 265nm using 2.38mL phosphate buffer, 600μL of 500μM stock adenosine, and 20μL ADA (as outlined in the table below).
 
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**The velocity was determined by clicking Operations > Activity Table > dAbs. The average of the dAbs values is the average slope (velocity).
 
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*5mM adenosine stock solution was too concentrated and exceeded the capacity of the UV-Vis therefore it was diluted:
 
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**150 uM adenosine stock solution was used; (stock) 150uL 5mM stock + 850 uL buffer =750 uM ->use .6mL new stock for ADA assay
 
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**200 uM adenosine stock solution was used: (stock) 200 uL 5mM stock+800 uL buffer = 1000uM ->use 0.6mL new stock for ADA assay
 
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**This table is the volumes of 5mM adenosine and phosphate buffer used to make the new adenosine stock solutions.
 
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***[[Image:Screen_Shot_2013-02-05_at_9.01.20_PM.png]]
 
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**This table is the updated table of the volumes and concentrations of solutions that will be added in the cuvette.
 
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***[[Image:Screen_Shot_2013-02-05_at_9.01.33_PM.png]]
 
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==Data==
 
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{| {{table}}
 
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| align="center" style="background:#f0f0f0;"|'''Concentration of Adenosine (uM)'''
 
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| align="center" style="background:#f0f0f0;"|'''Average Velocity over 30s (nm/sec)'''
 
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|-
 
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| 200||0.00021
 
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|-
 
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| 100||0.000183333
 
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|-
 
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| 40||0.00012
 
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|-
 
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| 16||0.0001
 
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|-
 
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| 6.4||0.000047
 
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|-
 
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| 2.56||0.000033
 
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|}
 
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{| {{table}}
 
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| align="center" style="background:#f0f0f0;"|'''1/[adenosine] (1/uM)'''
 
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| align="center" style="background:#f0f0f0;"|'''1/[velocity] (sec/nm)'''
 
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|-
 
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| 0.005||4761.904762
 
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|-
 
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| 0.01||5454.545455
 
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|-
 
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| 0.025||8333.333333
 
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|-
 
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| 0.0625||10000
 
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|-
 
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| 0.15625||21276.59574
 
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|-
 
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| 0.390625||30303.0303
 
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|}
 
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*[[Image:Graphsmn1.jpg]]
 
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{| {{table}}
 
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| -1/Vmax||6163.1
 
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|-
 
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| '''Vmax'''||'''0.000162256 nm/sec'''
 
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|-
 
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| -1/Km||-0.093
 
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|-
 
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| '''Km'''||'''10.75 uM'''
 
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|}
 

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