UV-VIS of Apoprotein
- UV-VIS spectroscopy was run on the protein to determine if the extraction of the heme groups was successful. If so, there should be a peak at 280 nm. If there is heme left, there would be a peak at 400 nm.
- The spectrum revealed a large peak at 280 nm (roughly 0.7) and a very slight peak at 400 (0.06-0.07).
Reconstitution of Apoprotein
- In order to reconstitute the apoprotein with the Mn PPIX, the ratio used in other syntheses was 2 mg PPIX per 10 mg apoprotein product (assuming full yield); this is still an excess of PPIX. For this reaction, 200 mg of myoglobin was used, so the stoichiometric amount of PPIX is 40 mg. Of which, we have 500 mg.
- Next, 2 mL of 100mM Phosphate buffer at pH 7.0, and 0.1 N (0.1 M) NaOH were made.
- 0.0037 mol of Sodium Phosphate Monobasic and 0.0062 mol of Sodium Phosphate Dibasic need to be dissolved in 100 mL of H2O
- 0.0037 mol monobasic = 511 mg
- 0.0062 mol dibasic = 880 mg
- 40 mg of PPIX was dissolved in 100 mL of the buffer and 10 drops of NaOH were added, when it should have been dissolved in 2 mL of the buffer. 2 mL of the solution was removed and added to a second beaker to compensate, and 10 drops of NaOH were added. The resulting solution was left to cool in the cold room.
- Once the PPIX dissolved completely into solution, the apoprotein solution was added dropwise and let react overnight.