Suprasil quartz cuvette

The CD spectra of native Hb (0.04 mg mL⁻¹), bare PLGA-NPs (1.00 mg mL⁻¹), and HbNPs (0.96 mg mL⁻¹) that were dispersed in TRIS 1 were recorded by using a JASCO J-815 Spectropolarimeter (JASCO, Essex, UK). A 0.5 mm path length suprasil quartz cuvette from Hellma was used for the measurements. The spectra were obtained as an accumulative average of 10 scans within the wavelength range of 190 to 260 nm, with a 2 nm bandwidth. The spectrum of the control sample (i.e., TRIS 1) was first subtracted and the resulting spectra were smoothed. The intensity of the ellipticity was normalized to the lowest band of the Hb spectrum. Two independent samples were analyzed.

Circular dichroism (CD) measurements were conducted with a J750 Spectropolarimeter (Jasco Inc, Maryés Court, Easton, USA) equipped with a Pelletier device. MtxA_Δ1−24 protein sample was prediluted to 0.2 mg/mL in buffer containing 20 mM Tris-HCl pH 8.5, 200 mM NaCl and measured with a 0.1 cm optical path Suprasil quartz cuvette (Hellma GmbH & Co., Müllheim, Germany). Spectra profiles of the samples were measured at a wavelength range of 190–240 nm at ambient temperature with bandwidth set to 1 nm, scan speed set to 10 nm/min and a time constant of 4 s. The thermal denaturation experiments of MtxA_Δ1−24 were conducted by monitoring the dichroic absorption at a wavelength of 222 nm as a function of increased temperature varying from 25°C to 75°C at a heating rate of 1.0°C × min⁻¹. The thermodynamic parameters associated with the temperature-induced denaturation were obtained by nonlinear, least-squares fitting analysis of the temperature dependence of CD, and a two-state denaturation process was assumed for the curve fit.

Measurements of DLS were performed using a Malvern Zetasizer Nano ZS ZEN3600 (Malvern, Herrenberg, Germany), equipped with a 633 nm laser. Each sample (70 μL) was measured in a Suprasil® quartz cuvette (Hellma GmbH, Muellheim, Germany) with a path length of 3 mm and 200–2500 nm spectral range. Monomeric and stressed samples at 1 mg/mL were measured at 25°C to determine the volume-based average protein particle diameter in solution.