ADA Kinetic Assay Preparations
- A weight of 0.6702 g of sodium phosphate dibasic was dissolved in 50 mL of water to obtain a molarity of 0.05 M. The pH of the solution was adjusted to pH 7.4.
.050 L of water × = .0025 mol of Na2HPO4 × = 0.6702 g
- The pH was was adjusted to 7.4 by the addition of 2 drops of 12 M HCl.
- To obtain 1 mM inosine, 1.5 mg of the solid was dissolved in 1 mL buffer. This was further diluted by collecting 89.3 µL of the 1.5 mg/ml inosine into 5 mL of the sodium phosphate buffer.
.0015 g of inosine × = .000005596 mol ÷ .001 L = .005596 M = 5.596 mM
5.596 mM (V1)= 0.1 mM (5 mL)
V1 = 0.08934 mL = 89.3 μL in 5 mL of buffer
- 3 mM adenosine was prepared by dissolving 0.0082 g of the solid into 10 mL sodium phosphate buffer. The stock concentration of adenosine was 3.07 mM.
.0082 g of adenosine × = 3.06840 × 10-5 ÷ .010 L = .00307 M = 3.07 mM
UV-visible scans of Reagents
- Mody and Nagle condcuted the runs for the UV-visible scans of ADA, adenosine, and inosine to verify the absorbance peaks for each.
- From the ADA Activity Assay protocol, it was specified to monitor the absorbance of each reagent at wavelengths 235 and 265.
- 1 mL of inosine was transferred to a cuvette. The final concentration of inosine in the cuvette was 1 mM.
- 10 μL of adenosine was diluted to 990 μL of the sodium phosphate buffer in a cuvette. The final concentration of adenosine was .0307 mM.
M1V1 = M2V2
(3 mM)(10 μL) = M2 (1000 μL)
M2 = .0307 mM
- ADA was diluted as follows:
3 μL of ADA × 65 μM = M2 (1000 μL)
M2 = .195 μM of ADA
500 μL × .195 μM = M2 (1000 μL)
M2 = .0975 μM = 97.5 nM
- 2 additional 500 μL dilutions of 97.5 nM to 1000 μL were executed using the dilution equation.
Second dilution M = 48.8 nM
Final Concentration = 24.38 nM
- Wavelength 235 and 265 were monitored for the absorbance of the reagents adenosine and inosine Their absorbance are listed on the table below.
- By manipulating Beer's Law, the molar absorptivity was calculated from the absorbance and concentration of the substances.
- The calculation for the molar absorptivities of adenosine and inosine are shown below.
- At 235 = 18566.78
- At 265 = 13029.32
- At 235 = 1400
- At 265 = 6000
- Evaluating the molar absorptivities for each wavelength, the results indicate that there is a greater difference of molar absorptivities between adenosine and inosine at wavelength 235. The molar absorptivity for adenosine was observed to be higher than inosine.
- We can hypothesized that before running the UV-Vis scans for the kinetic assay of ADA with adenosine, adenosine will produce a more pronounced absorption and signal. Therefore, adenosine should be monitored at 235.
- Adenosine is the substrate of ADA. As time passes, ADA catalyzes the formation of inosine from adenosine. Thus, the concentration of adenosine should decrease per unit time. When concentration of adenosine decreases, the absorbance monitored at 235 should also decrease per unit time.
| Absorbance at 235
| Absorbance at 265