0.01 m phosphate buffer tablets

The synthesis of pyridazinone derivatives 1, 2, 3, 4, and 5 was described in our previous work [1 (link)]. All tested derivatives, based on their NMR and MS spectra and their physical properties, were determined to have purity of >95%. UV-Vis and fluorescence spectra of analyzed compounds are presented in Supplementary Materials (Figure S4). Studied double-stranded calf thymus DNA (ctDNA, powder), proteins: AAG and GG (powders), and 0.01 M phosphate buffer tablets were bought from Sigma-Aldrich Chemie GmbH, (St. Louis, MO, USA). Protein solutions of a given concentration were prepared by dissolving an appropriate amount of reagent in the form of powder in a phosphate buffer solution. For ctDNA, we prepared the stock solution with a concentration equal to 1 mg/mL, in phosphate buffer as a solvent. We have checked if ctDNA is free from protein. For this purpose, the absorbance in 260 and 280 nm was measured. The value of A₂₆₀/A₂₈₀ was higher than 1.8, which confirmed the purity of ctDNA [45 (link)]. The actual concentration of the solution was determined from Beer–Lambert law by measuring the absorbance at 260 nm and calculating it using the molar absorption coefficient equal to 6600 L/mol·cm [46 (link)]. The prepared solution was stored in a freezer, thawed, and diluted with buffer to appropriate concentrations before measurement.

The synthesis of compounds analyzed was performed in the Department of Medicinal Chemistry, Wroclaw Medical University, and described in work [3 (link)]. Studied proteins, BSA, AAG, GG, and 0.01 M phosphate buffer tablets were bought from Sigma-Aldrich Chemie GmbH, (St. Louis, MO, USA).