Model j 810

The circular dichroism (CD) spectra were measured by a Jasco Model J810 spectro-polarimeter flushed with nitrogen gas. A quartz cuvette with a path length of 0.1 cm was used to contain protein samples. The protein samples were dissolved in Tris-HCl buffer (20 mM, pH 7.4) and the same Tris-HCl buffer supplemented with 2 or 10 mM CaCl₂. The final concentration of the protein was ~10 μM. The spectra were recorded at a scan rate of 50 nm/min and were corrected for buffer contributions.

Circular dichroism spectra were collected using a Jasco Model J-810 spectropolarimeter. Spectra were collected in the range 5–85°C, at intervals of 10°C, in the 320–200 nm range, at a resolution of 0.5 nm, a scanning rate of 50 nm/min, a bandwidth of 1 nm, a response of 2 s, 6 accumulations per scan, at standard sensitivity (100 mdeg). The SOMAmer reagent concentration was 1 mg/ml (0.1 mM) in 130 mM NaCl, 20 mM Na-phosphate, pH 7. The cell pathlength was 1 cm.

Data were recorded on a Jasco circular dichroism spectropolarimeter (Model J810) as described previously^{11 (link), 14 (link)}.

To test the structural integrity of catalase treated with acetone, the primary and secondary structures of treated catalase or PAAM-n(catalase) were characterized. Native catalase, PAAM-n(catalase), catalase treated with acetone, and PAAM-n(catalase) treated with acetone were heated for 5 min at 95 °C and analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gel electrophoresis was conducted for 2 h on 10% acrylamide gels with a 5% stacking gel at 120 V. The gels were run until the tracking dye had just exited the gel and then were stained with Coomassie blue brilliant R-250. In addition, circular dichroism (CD) analysis was performed on a spectrophotometer (Model J-810, Jasco, Tokyo, Japan). Native catalase, catalase treated with acetone and PAAM-n(catalase) treated with acetone were prepared at 0.2 mg/mL. Measurements were collected at 37 °C over the wavelength range of 250–190 nm.
The morphologies of catalase treated with acetone and PAAM-n(catalase) treated with acetone were assessed using a high-resolution transmission electron microscope (TEM, Jem-2100F, JEOL Ltd., Tokyo, Japan), and samples were stained with 2% phosphotungstic acid before observations.