User:Moira M. Esson/Notebook/CHEM-571/2013/10/08: Difference between revisions
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== | ==Objectives== | ||
* | #Monitor the turnover kinetics of adenosine to inosine catalyzed by adenosine deaminase (ADA) | ||
<br> | |||
==Kinetics== | |||
'''General Protocol''': | |||
#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 | |||
#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 | |||
#Allow the solution to reach a constant 25°C | |||
#Begin taking absorbance spectra (take a spectrum every 15seconds) | |||
#After 1min, add 30μL 1.1units/mL ADA | |||
#Allow the reaction to run for 10 minutes, taking a spectrum every 15seconds. | |||
<br> | |||
'''Sample Preparation''': | |||
Table 1. Preparation of 0.0123M stock solution | |||
<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|Mass of adenosine (g) | |||
| align="center" style="background:#f0f0f0;"|0.0165 | |||
|- | |||
| Volume of buffer(mL)||5 | |||
|} | |||
<br> | |||
Table 2. Preparation of 40μM adenosine solution | |||
<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|Volume of stock(mL) | |||
| align="center" style="background:#f0f0f0;"|0.0098 | |||
|- | |||
| Volume of buffer(mL)||2.9902 | |||
|} | |||
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Objectives
KineticsGeneral Protocol:
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