Spectroscopic Study on the Interaction between Naphthalimide-Polyamine Conjugates and Bovine Serum Albumin (BSA)

Abstract

The effect of a naphthalimide pharmacophore coupled with diverse substituents on the interaction between naphthalimide-polyamine conjugates 1-4 and bovine serum albumin (BSA) was studied by UV absorption, fluorescence and circular dichroism (CD) spectroscopy under physiological conditions (pH = 7.4). The observed spectral quenching of BSA by the compounds indicated that they could bind to BSA. Furthermore, caloric fluorescent tests revealed that the quenching mechanisms of compounds 1-3 were basically static type, but that of compound 4 was closer to a classical type. The Ksv values at room temperature for compound-BSA complexes-1-BSA, 2-BSA, 3-BSA and 4-BSA were 1.438 × 10⁴, 3.190 × 10⁴, 5.700 × 10⁴ and 4.745 × 10⁵, respectively, compared with the value of MINS, 2.863 × 10⁴ at Ex = 280 nm. The obtained quenching constant, binding constant and thermodynamic parameter suggested that the binding between compounds 1-4 with BSA protein, significantly affected by the substituted groups on the naphthalene backbone, was formed by hydrogen bonds, and other principle forces mainly consisting of charged and hydrophobic interactions. Based on results from the analysis of synchronous three-dimensional fluorescence and CD spectra, we can conclude that the interaction between compounds 1-4 and BSA protein has little impact on the BSA conformation. Calculated results obtained from in silico molecular simulation showed that compound 1 did not prefer either enzymatic drug sites I or II over the other. However, DSII in BSA was more beneficial than DSI for the binding between compounds 2-4 and BSA protein. The binding between compounds 1-3 and BSA was hydrophobic in nature, compared with the electrostatic interaction between compound 4 and BSA.

Keywords: bovine serum albumin (BSA); conjugates; molecular docking; naphthalimide-polyamine; spectroscopic methods.

Figure 1

Structures of naphthalimide-polyamine conjugates.

Figure 2

UV of compounds 1–4, BSA and the BSA-compound complexes. Conditions: c (compound) = 12 × 10⁻⁶ mol∙L⁻¹; c (BSA) = 1.04 × 10⁻⁶ mol∙L⁻¹.

Figure 2

UV of compounds 1–4, BSA and the BSA-compound complexes. Conditions: c (compound) = 12 × 10⁻⁶ mol∙L⁻¹; c (BSA) = 1.04 × 10⁻⁶ mol∙L⁻¹.

Figure 3

UV absorption spectra of compounds 1–4 with BSA. Numbers 1–13 indicated the concentrations of compounds 1–4: 0.0, 0.4 × 10⁻⁶, 0.8 × 10⁻⁶, 1.2 × 10⁻⁶, 2.4 × 10⁻⁶, 3.6 × 10⁻⁶, 4.8 × 10⁻⁶, 6.0 × 10⁻⁶, 7.2 × 10⁻⁶, 8.4 × 10⁻⁶, 9.6 × 10⁻⁶, 10.8 × 10⁻⁶ and 12 × 10⁻⁶ mol∙L⁻¹, respectively. BSA concentration applied was 1.04 × 10⁻⁶ mol∙L⁻¹.

Figure 3

UV absorption spectra of compounds 1–4 with BSA. Numbers 1–13 indicated the concentrations of compounds 1–4: 0.0, 0.4 × 10⁻⁶, 0.8 × 10⁻⁶, 1.2 × 10⁻⁶, 2.4 × 10⁻⁶, 3.6 × 10⁻⁶, 4.8 × 10⁻⁶, 6.0 × 10⁻⁶, 7.2 × 10⁻⁶, 8.4 × 10⁻⁶, 9.6 × 10⁻⁶, 10.8 × 10⁻⁶ and 12 × 10⁻⁶ mol∙L⁻¹, respectively. BSA concentration applied was 1.04 × 10⁻⁶ mol∙L⁻¹.

Figure 4

Fluorescence spectroscopy of compounds 1–4 and BSA. Numbers 1–13 indicated concentrations of compounds 1–4: 0.0, 0.4 ×10⁻⁶, 0.8 × 10⁻⁶, 1.2 × 10⁻⁶, 2.4 × 10⁻⁶, 3.6 × 10⁻⁶, 4.8 × 10⁻⁶, 6.0 × 10⁻⁶, 7.2 × 10⁻⁶, 8.4 × 10⁻⁶, 9.6 × 10⁻⁶, 10.8 × 10⁻⁶ and 12 × 10⁻⁶ mol∙L⁻¹, respectively. BSA concentration applied was 1.04 × 10⁻⁶ mol∙L⁻¹. Scan condition: Ex = 280 nm, Em = 290–550 nm; slits of both Ex and Em of compounds 1–3 were 5 nm while those of compound 4 were 5 nm and 10 nm, respectively.

Figure 4

Fluorescence spectroscopy of compounds 1–4 and BSA. Numbers 1–13 indicated concentrations of compounds 1–4: 0.0, 0.4 ×10⁻⁶, 0.8 × 10⁻⁶, 1.2 × 10⁻⁶, 2.4 × 10⁻⁶, 3.6 × 10⁻⁶, 4.8 × 10⁻⁶, 6.0 × 10⁻⁶, 7.2 × 10⁻⁶, 8.4 × 10⁻⁶, 9.6 × 10⁻⁶, 10.8 × 10⁻⁶ and 12 × 10⁻⁶ mol∙L⁻¹, respectively. BSA concentration applied was 1.04 × 10⁻⁶ mol∙L⁻¹. Scan condition: Ex = 280 nm, Em = 290–550 nm; slits of both Ex and Em of compounds 1–3 were 5 nm while those of compound 4 were 5 nm and 10 nm, respectively.

Figure 5

Fuorescence of compounds 1–4, BSA and BSA + compounds 1–4. c (compound) = 12 × 10⁻⁶ mol∙L⁻¹; c (BSA) = 1.04 × 10⁻⁶ mol∙L⁻¹.

Figure 5

Fuorescence of compounds 1–4, BSA and BSA + compounds 1–4. c (compound) = 12 × 10⁻⁶ mol∙L⁻¹; c (BSA) = 1.04 × 10⁻⁶ mol∙L⁻¹.

Figure 6

Fluorescence quenching ratio (F/F₀) of BSA by compounds 1–4 and MINS at room temperatures.

Figure 7

Stern-Volmer linear plot of fluorescence quenching of BSA by compounds 1–4 at different temperatures.

Figure 7

Stern-Volmer linear plot of fluorescence quenching of BSA by compounds 1–4 at different temperatures.

Figure 8

Linear plot of log [1/c_comp.] vs. log [F/(F₀ − F)] of the interaction between compounds 1–4 and BSA at different temperatures.

Figure 8

Linear plot of log [1/c_comp.] vs. log [F/(F₀ − F)] of the interaction between compounds 1–4 and BSA at different temperatures.

Figure 9

Synchronous fluorescence spectra of compounds-BSA system. (A: Δλ = 15 nm, B: Δλ = 60 nm). The concentrations of BSA and compounds were the same as those in Figure 4.

Figure 9

Synchronous fluorescence spectra of compounds-BSA system. (A: Δλ = 15 nm, B: Δλ = 60 nm). The concentrations of BSA and compounds were the same as those in Figure 4.

Figure 9

Synchronous fluorescence spectra of compounds-BSA system. (A: Δλ = 15 nm, B: Δλ = 60 nm). The concentrations of BSA and compounds were the same as those in Figure 4.

Figure 10

Three-dimensional fluorescence spectra of BSA in the absence (A) and presence (B) of compounds 1–4. Conditions: c (BSA) = 1.04 × 10⁻⁶ mol∙L⁻¹, c (compounds 1–4) = 12.0 × 10⁻⁶ mol∙L^–1.

Figure 10

Three-dimensional fluorescence spectra of BSA in the absence (A) and presence (B) of compounds 1–4. Conditions: c (BSA) = 1.04 × 10⁻⁶ mol∙L⁻¹, c (compounds 1–4) = 12.0 × 10⁻⁶ mol∙L^–1.

Figure 11

CD spectra of BSA in the presence of the compounds. Conditions: c (BSA): 40.0 × 10^–6 mol∙L^–1; c (conpounds): 0, 120.0 × 10^–6 mol∙L^–1.

Figure 11

CD spectra of BSA in the presence of the compounds. Conditions: c (BSA): 40.0 × 10^–6 mol∙L^–1; c (conpounds): 0, 120.0 × 10^–6 mol∙L^–1.

Figure 12

Thermal denaturation of 40 × 10⁻⁶ mol∙L^–1 BSA in the absence and presence of 120 × 10⁻⁶ mol∙L^–1 compounds at pH = 7.4.

Figure 12

Thermal denaturation of 40 × 10⁻⁶ mol∙L^–1 BSA in the absence and presence of 120 × 10⁻⁶ mol∙L^–1 compounds at pH = 7.4.

Figure 13

Docking experiment results showing the 2D diagrams of the binding modes of the complex DSI-compounds 1–4. Polar and non-polar residues were represented in red and green circles, respectively. H-bond acceptors or donors were shown in green-dotted arrows. Electrostatic interactions from ion pairs were presented in red-dotted lines. π-π and π-H interactions were represented in green-dotted lines.

Figure 14

Docking experiment results showing the 2D diagrams of the binding modes of the complex DSII-compounds 1–4. Polar and nonpolar residues were represented in the red and green circles, respectively. H-bond acceptors or donors were shown in green-dotted arrows. Electrostatic interactions from ion pairs were presented in red-dotted lines. π-π and π-H interactions were represented in green-dotted lines.