User:Michael F. Nagle/Notebook/Chem 571/2012/11/14

Objectives

• Test the ability of gold to form nanoparticles with Adenosine Deaminase
• Start dialysis with ADA solutions to remove salts
• Run ADA kinetic assay
  • This is so next we can compare it to an activity assay with varying concentrations of AuNPs to determine whether they interact with the active site, deactivating it.

Procedure

1. Nucleation of Au with ADA
   1. 8.42mM HAuCl₄ solution and 42μM ADA were used to prepare solutions with 1μM ADA and 25μM, 50μM, 75μM, 100μM, 125μM, 150μM, 175μM, and 200μM HAuCl₄. Water was used as a solvent and enough was added for each solution to be 2mL.
   2. The solutions were capped with tin foil and heated for 4 hours at 80^oC
2. Dialysis of ADA
   1. To remove the salt in ADA solution, the solution was placed in dialysis tubing. The tubing was clipped at each end.
   2. A foam piece was attached to one clip so the tubing would float. The tube was left in 2L water, which will be changed later this week.
3. ADA Kinetics
   1. Puja Moody calculated molar absortivity of adenosine, inosine and ADA at λ265 and determined that wavelength should be monitered because shows the most significant increase relative to adenosine concentration.
   2. Adenosine and inosine were analyzed just as they were yesterday and molar absortivity was recalculated, this time resulting in values contrary to those before. It was determined from this that the increase in absorbance actually signified an increase in inosine, and a decrease in adenosine.
   3. The samples analyzed contained 2.7mL sodium phosphate buffer at pH 7.4 and .3mL 1mM adenosine with 15µL, 25µL, 40µL, and 50µL 65µM ADA solution.