Cary 3500 uv vis spectrometer

UV-visible spectroscopic analyses were performed in a 1 cm path length cuvette using an in-situ Agilent Cary 3500 UV-Vis Spectrometer. The extinction coefficient was determined by using five solutions of different 4-NP concentrations (1.5 × 10⁻⁴ M, 1.0 × 10⁻⁴ M, 5.0 × 10⁻⁵ M, 2.5 × 10⁻⁵ M, 1.0 × 10⁻⁵ M), and 4.5∙10⁻³ M in NaBH₄. The calibration curve was plotted and gave an extinction coefficient of 18,900 M⁻¹ cm⁻¹, which is close to the literature value of 18,000 M⁻¹cm⁻¹ [47 (link),48 (link)]. The calibration was repeated three times and the average ε was obtained.

All spectra were recorded in a SIRT assay buffer consisting of 20 mM of Tris-HCl, pH 7.8, 150 mM of NaCl, and 5 mg/mL of MgCl₂. The absorbance spectra were recorded at room temperature in a cuvette with a pathlength of 10 mm using an Agilent CARY 3500 UV-Vis spectrometer. A compound concentration of 30 µM for Mcm-containing compounds or 10 µM for fluorescein-containing compounds was used. The fluorescence spectra were recorded in a fluorescence cuvette with a pathlength of 10 mm × 5 mm with a Horiba Fluoromax 4 (Kyōto, Japan). The compound concentration was 3 µM for Mcm-containing peptides and 1 µM for fluorescein-containing peptides. Excitation wavelengths were chosen according to the absorbance maximum of the individual compounds.