J 1500 magnetic circular dichroism spectrometer

The Jasco J-1500 magnetic circular dichroism spectrometer (JASCO International CO., Tokyo, Japan) was used to measure CD spectra. Circular dichroism spectra were collected for protein solutions and after adding small portions of analyzed phthalimide analogs. Phosphate buffer was used as a solvent here (pH 7.5), and due to this, physiological parameters were simulated here. The parameters of measurements of CD measurements were as follows: the range was 205–250 nm for BSA and AAG, and 210–250 nm for GG, scan speed rate was equal to 50 nm/min, with a response time of 1 s, path length—10 mm. The concentrations of solution used were 1.0 μM, and 1.0 μM for the proteins and phthalimide analogs, respectively. The molar ratios of proteins and ligands were equal to 1:0, 1:0.5, 1:1, 1:5, and 1:10. The CD Multivariate Calibration Creation and CD Multivariate SSE programs (JASCO International CO., Tokyo, Japan) were used for the analysis of the secondary structure elements. Mean residue molar concentrations of proteins have been included in this analysis.

Circular dichroism (CD) spectra were measured on the Jasco J-1500 magnetic circular dichroism spectrometer (JASCO International CO., Tokyo, Japan) at room temperature with a 10 mm path length. All spectra for the HSA and AAG solutions in the absence and presence of 5a, 5b, and 5c were measured under simulated physiological conditions in pH 7.4, in phosphate buffer as a solvent, and were baseline corrected (the spectrum phosphate buffer was used as a baseline). The spectra were measured in the range of 205–250 nm at a scan rate speed of 50 nm/min, with a response time of 1 s. The concentration of HSA and AAG was 1·10⁻⁶ M, while for 5a, 5b, and 5c, it was 1·10⁻³ M. Experiments were performed to obtain the appropriate protein to compound molar ratios equal to 1:0, 1:0.5, 1:1, 1:1, 1:3, and 1:5. They were respectively added into 3 mL of protein solution successive portions of the solutions of the analyzed compounds. The analysis of the obtained results was performed using the CD Multivariate Calibration Creation and CD Multivariate SSE programs (JASCO International CO., Tokyo, Japan). Protein concentrations were converted here for mean residue molar concentrations.

Circular dichroism (CD) spectroscopy was performed using the Jasco J-1500 magnetic circular dichroism spectrometer (JASCO International CO., Tokyo, Japan). All measurements for the protein solutions in the absence and presence of the analyzed peptides L1, L2, and L3 were made at room temperature under simulated physiological conditions in pH 7.4, in phosphate buffer as a solvent. The phosphate buffer not only controls the stable pH but also imitates the biological environments. However, its use leads to some limitations for CD measurements due to containing NaCl and KCl. The spectra were baseline corrected (phosphate buffer was used as a baseline) and were measured in the range of 205–250 nm at a scan rate speed of 50 nm/min, with a response time of 1 s, and a 10 mm or 5 mm path length for BSA and GGF, respectively. The concentrations of proteins and analyzed peptides were 1 × 10⁻⁶ mol/dm³ and 1 × 10⁻³ mol/dm³, respectively. Experiments were performed with protein to ligand molar ratios: 1:0, 1:0.25, 1:0.5, 1:0.75, 1:1, and 1:1.5. In total, 3 cm³ of a solution of each protein was titrated by successive additions of analyzed peptides. The results were analyzed by CD Multivariate Calibration Creation and CD Multivariate SSE programs (JASCO International CO., Tokyo, Japan), with the conversion of protein concentrations for mean residue molar concentrations.