Fluorolog 3 221 spectrofluorometer

The binding constants (KA) of inhibitors to the binding site of WT-LLO, Y427A-LLO, V428A-LLO, S466A-LLO and K433A-LLO were measured using the fluorescence-quenching method. Fluorescence spectrofluorimetry measurements were carried out using a Horiba Jobin-Yvon Fluorolog 3-221 spectrofluorometer (Horiba Jobin-Yvon, Edison, NJ). The measurements were acquired using a 280-nm excitation wavelength with a 5-nm band-pass and a 345-nm emission wavelength with a 10-nm band-pass. The details of the measurements were described previously13 14 (link).

Absorption and fluorescence spectra of the compounds were recorded in air-saturated solutions at ambient temperature with a Cary 300 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) and FluoroLog-3-221 spectrofluorometer (Horiba Jobin Yvon, Edison, NJ, USA). All measured fluorescence spectra were corrected for the nonuniformity of detector spectral sensitivity. Coumarin 481 in acetonitrile (fluorescence quantum yield is 0.08) [33 (link)] was used as a reference for the fluorescence quantum yield measurements.

In our paper, fluorescence quenching can be described by the following Scatchard equation: r/D_f  = nK − rK, where r is the ligand amount of substance per mole of protein binding (r ≈ ΔF/F₀), D_f is the free concentration of CUR, K is the binding constant and n is the number of binding sites. In this system, due to the CUR concentration being far greater than the concentration of protein; the D_f is replaced by the total CUR concentration, Q. According to experimental results, the linear fitting plots of r/D_f vs. r between CUR and WT-Hla or its two mutants (K163A-Hla and Q89A-Hla) can be made in Fig. 6 and based on the plots the values of K and n can be obtained. In this work, Hla can act as the Fluorescence and CUR act as the quencher. Fluorescence spectrofluorimetry measurements were carried out using a Horiba Jobin-Yvon Fluorolog 3-221 spectrofluorometer (Horiba Jobin-Yvon, Edison, NJ). The measurements were acquired using a 280-nm excitation wavelength with a 5-nm band-pass and a 345-nm emission wavelength with a 10-nm band-pass.