N-Phenylnaphthalen-1-amine

N-Phenyl-1-naphthylamine is a dye that fluoresces strongly when bound to the inner phospholipid bilayer of Gram-negative bacteria. N-Phenyl-1-naphthylamine can be used to measure outer membrane permeability. N-Phenylnaphthalen-1-amine is a fluorescence probe for odorant-binding proteins (OBP) with a dissociation constant of 1.67 μM. N-Phenylnaphthalen-1-amine exhibits an excitation wavelength of 337 nM and an emission wavelength of 407 nM^[1][2]. In Vitro:Odorant Binding Protein (OBP) Binding Affinity Assay^[1]
1. Materials Preparation
Fluorescent probe: N-phenyl-1-naphthylamine, dissolved in methanol (MeOH) to prepare a 1 mM stock solution
Protein solution: 2 μM odorant binding protein (OBP), dissolved in 50 mM Tris-HCl buffer (pH 7.4)
Experimental temperature: 25 °C
Fluorescence spectrophotometer: equipped with a 1 cm quartz cuvette, excitation and emission slit widths set to 5 nm
Detection wavelengths:
Excitation wavelength (λ_ex): 337 nm
Emission wavelength (λ_em): 407 nm
2. Fluorescence Binding Assay Procedure
Place 2 μM odorant binding protein solution into the cuvette.
Gradually add the N-phenyl-1-naphthylamine stock solution (1 mM in methanol), mixing gently after each addition.
Set the final concentration range to 1-20 μM.
After each addition, immediately record the fluorescence emission spectrum (range 380-460 nm).
Measure and record the emission intensity at 407 nm for plotting the binding curve.
3. Data Analysis
Plot fluorescence intensity against the concentration of free N-phenyl-1-naphthylamine.
Use a Scatchard plot for linearization and calculation of the binding constant.
Assume a 1:1 binding ratio and 100% protein activity.
The dissociation constant (K_d) of N-phenyl-1-naphthylamine with loc-OBP1 is 1.67 μM.
4. Competitive Binding Assay
Use a fixed concentration of 2 μM odorant binding protein and 1 μM N-phenyl-1-naphthylamine.
Add test ligands (dissolved in methanol) at final concentrations typically ranging from 1-20 μM.
Monitor the decrease in fluorescence intensity and plot the competitive binding curve.
Calculate the IC₅₀, and then convert it to the dissociation constant (K_d) of the test ligand.