Project name
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Objective
To observe and measure ADA turnover kinetics in the absence of an inhibitor. This work will be the basis of our comparison to ADA-AuNP turnover studies.
Description
- Make a 40μM solution of adenosine in buffer (50mM phosphate buffer, pH 7.4)
- Go to Dr. Hartings lab for enzyme kinetics measurements.
- Add 3mL of adenosine solution to the cuvette
- Start your kinetics measurement
- 1ms integration (on front panel)
- 10 scan average (on front panel)
- Set "Save the first available scan every" to 15 seconds (after clicking File>Save)
- Set "Stop after this amount of time" to 10 minutes (after clicking File>Save)
- Set "File Type" to Tab Delimited
- Give the files a directory and a name
- Click accept
- Just before 1 minute add 30ul of 0.01u/mL ADA
Data
- Buffer
- 0.7318 g of NaH2PO4·H2O and 5.2520g Na2HPO4·7H2O in 500mL water --> 50mM Phosphate buffer pH 74
- Adenosine deaminase (ADA) stock
- 1.1mg (24.0units in 1 mg) in 25mL of buffer --> 1.1 units/mL ADA
- ADA for experiments
- 100uL of stock ADA and 900uL buffer --> 0.11 units/mL ADA
Notes
Table 1. Preparation of 0.0123M stock solution
Mass of adenosine (g)
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0.0165
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Volume of buffer(mL) |
5
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Table 2. Preparation of 40μM adenosine solution
Volume of stock(mL)
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0.0098
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Volume of buffer(mL) |
2.9902
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Figure 1. Corrected Absorbance spectra of the conversion of adenosine to inosine by ADA
Table 3. Molar Absorptivity of Adenosine at 260nm and Inosine at 250nm
Wavelength
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Adenosine
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Inosine
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250 |
13333 |
11937.5
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260 |
16866.7 |
7312.5
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Figure 2. Monitoring concentration versus time of the conversion of adenosine to inosine using ADA as a catalyst.