Spectramanager 2 analysis software

Kinetic studies of cluster transfer experiments were performed based on the experimental design of Johnson and coworkers.^{32 (link),33 (link)} Reactions were performed on a JASCO J-815 CD spectrophotometer in a quartz 1 cm anaerobic cuvette from 600 – 300 nm at a scan rate of 200 nm/min at 25°C with a 2 min interval between each accumulation. Reactions that reached completion within the first 10 min were analyzed over a 10 nm wavelength scale based on the peak of interest, with 10 sec intervals between accumulations. Spectra were processed using JASCO Spectramanager II Analysis software and were represented in Origin 7.0.
Reactions in 50 mM HEPES, 100 mM NaCl, pH 7.5 were prepared by degassing a mixture of 40 μM apo protein, and 5 mM DTT, and were then transferred to an anaerobic cuvette via a gas tight syringe. Degassed holo protein at 20 μM was added to the cuvette to initiate the reaction. The concentration of [2Fe-2S] in the reaction for each holo protein was determined via standard iron quantitation methods.^{34 (link)}The kinetic rate constants for cluster transfer were obtained by monitoring and converting the change in CD signal to the percentage of cluster transferred over time. The plot of time versus percent cluster transfer was fit to an exponential to obtain an observed first-order rate constant k_obs that was used to obtain the second-order rate constant.

Iron-sulfur cluster transfer experiments were performed on a JASCO J-815 CD spectrometer in a quartz 1 cm anaerobic cuvette by collecting data from 600 to 300 nm at a scan rate of 200 nm/min at 25°C with a 2 min interval between each acquisition. Reactions that reached completion within the first 10 min, were analyzed over a 10 nm wavelength range based on the peak of interest with 10 sec intervals between acquisitions. Spectra were processed by use of JASCO Spectramanager II Analysis software and were represented in Origin 7.0.
Reactions involving Grx3 were prepared in 50 mM HEPES, 100 mM NaCl, pH 7.5, by degassing a mixture of 40 µM apo protein, 5 mM DTT, and 3 mM GSH, and were subsequently transferred to an anaerobic cuvette via a gas tight syringe. GSH was not included in reactions without Grx3. Degassed holo protein at 40 µM was added to the cuvette to initiate the reaction. The concentration of [2Fe-2S] in the reaction for each holo protein was determined via standard iron quantitation methods. Cluster transfer reactions were analyzed by converting the change in CD signal to the percentage of cluster transferred and fit using DynaFit (41 (link)) to determine the second-order rate constants for the various reactions by best-fit simulation to second-order kinetics.

CD scans of apo and holo proteins were recorded on a JASCO J-815 CD spectrometer in a quartz 1 cm anaerobic cuvette. CD scans from 300 nm to 600 nm were collected to analyze signature cluster-bound protein peaks at a scan rate of 200 nm/min at 25 °C. Data were processed using JASCO Spectramanager II Analysis software and were represented in Origin 7.0.