User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/10/08

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

Entry title

  • Insert content here...

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

  1. Make a 40μM solution of adenosine in buffer (50mM phosphate buffer, pH 7.4)
  2. Go to Dr. Hartings lab for enzyme kinetics measurements.
    1. Add 3mL of adenosine solution to the cuvette
    2. Start your kinetics measurement
      1. 1ms integration (on front panel)
      2. 10 scan average (on front panel)
      3. Set "Save the first available scan every" to 15 seconds (after clicking File>Save)
      4. Set "Stop after this amount of time" to 10 minutes (after clicking File>Save)
      5. Set "File Type" to Tab Delimited
      6. Give the files a directory and a name
      7. Click accept
      8. Just before 1 minute add 30ul of 0.01u/mL ADA

Data

  1. Buffer
    1. 0.7318 g of NaH2PO4·H2O and 5.2520g Na2HPO4·7H2O in 500mL water --> 50mM Phosphate buffer pH 74
  2. Adenosine deaminase (ADA) stock
    1. 1.1mg (24.0units in 1 mg) in 25mL of buffer --> 1.1 units/mL ADA
  3. ADA for experiments
    1. 100uL of stock ADA and 900uL buffer --> 0.11 units/mL ADA

After we collected the data, we worked up the data and we found the concentration of inosine and adenosine, based on beer's law, and the peaks 260 and 250, from which we know epsilon, b and the absorbance. After putting the files and working it up, we obtained the graph of Adenosine to Inosine.

"Figure 1. Adenosine to Inosine"

"Table 1. System of equation to calculate concentration of Adenosine"


Adenosine Absorbance at time 0 molar absorptivity Initial concentration
at 2600.511140253.64349*10^-5
at 2500.43311884.19773.64349*10^-5


"Table 2. System of equation to calculate concentration of Inosine"


Inosine Absorbance at time 0 molar absorptivity Initial concentration
at 26005254.513890
at 2500110070


Then after this, we set up a system of equations to find out the concentration at different points during the time the reaction took, and sketched a graph.

"Figure 2. Change in concentrations from Adenosine to Inosine"



Personal tools