Preparation of ADA Kinetic Assay Reactants
- Adenosine deaminase was ordered from Worthington (19.8 u/mg). To obtain 0.5 M per mL, the calculation below was made amounting to 0.0253.
= 0.0253 mg of ADA in 1 mL phosphate buffer
- For convenience, it was decided to dissolve 2.5 mg into 100 mL of phosphate buffer. The final amount weighed 2.0 mg which will be dissolved into 80 mL of phosphate buffer. ADA was refrigerated between 2-8°C temporarily.
- In preparation of the sodium phosphate buffer, 3.1093 g of sodium phosphate monobasic (chempure, FW 268.07 g/mol) and 10.9019 g of sodium phosphate dibasic (fisher scientific, FW 137.99 g/mol) were dissolved in 1 L water to obtain a 0.1 M sodium phosphate buffer solution.
- The pH of the buffer was adjusted to 7.4 by the addition of 1 M sodium hydroxide.
- A concentration of 30 mM adenosine free base (amresco, MW 267.24 g/mol) was made from 0.0802 g of the white, needle-like solid dissolved into 10 mL of sodium phosphate buffer. However, this solution was saturated preventing the complete dissolution of adenosine.
- After preparing a 15 mM insoluble solution of adenosine, it was decided to prepare a 5 mM solution of adenosine. This was achieved by dissolving 0.0402 g of adenosine in 30 mL of the sodium phosphate buffer. This step was completed during the following lab period.
- After the use of reagents, the chemical and the newly-made stock solutions were refrigerated.