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Protein Re-engineering
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Mb catalysis DMSO
Following the description from Tuesday.
Stock solutions used
- 28.6mg of o-phenylenediamine in 26.5mL of DMSO
- 1.134 ml 30% H2O2 +8.866 ml DMSO
- 11.1mg of (5.7mg myoglobin +0.4861g KCl lyophilized from 20mL of 10mM citrate pH 3) in 1 mL of DMSO.
Samples were made by
- 2.025 mL of the OPD solution
- 0.225 mL of the Mb solution
- Start the scan
- 1,000 us integration time
- Average over 500 scans
- Run for 10 minutes (600,000,000 us)
- Add 0.75mL of the H2O2 solution
- Added after scan reached 1%
- Kinetics scans were run at 25, 35, and 45C (DMSO freezes at around 20C). Several before and after spectra were also taken, as were the dark and reference signal spectra.
- Absorbance measurements were calculated using the following equation: A = -log[(SampleSignal-DarkSignal)/(ReferenceSignal-DarkSignal)]
Mb Catalysis Isopropanol
Following the description from Tuesday.
Stock solutions used
- 29.2mg of o-phenylenediamine in 27mL of Isopropanol
- 1.134 ml 30% H2O2 +8.866 ml Isopropanol
- 15mg of (5.7mg myoglobin +0.4861g KCl lyophilized from 20mL of 10mM citrate pH 3) in 1.5 mL of isopropanol.
Samples were made by
- 2.025 mL of the OPD solution
- 0.225 mL of the Mb solution
- Start the scan
- 1,000 us integration time
- Average over 500 scans
- Run for 10 minutes (600,000,000 us)
- Add 0.75mL of the H2O2 solution
- Added after scan reached 1%
- Kinetics scans were run at 5, 15, 25, 35, and 45C. Several before and after spectra were also taken, as were the dark and reference signal spectra.
- Absorbance measurements were calculated using the following equation: A = -log[(SampleSignal-DarkSignal)/(ReferenceSignal-DarkSignal)]
All of the kinetics data taken today are no good because I didn't save the dark and reference spectra as Signal files. They were saved as processed absorption spectra. DMSO showed reactivity while there was no reactivity in isopropanol.
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