Molecular Interactions of 1-Hydroxypyrene with Catalase Revealed by Spectroscopic Methods Combined with Molecular Docking†

doi:10.7503/cjcu20150367

Abstract

Abstract:

The interactions between 1-hydroxypyrene(1-OHP) and catalase(CAT) were investigated by fluorescence, UV visible absorption(UV-Vis) spectra and molecular docking methods in aqueous solutions. The experimental results showed that the intrinsic fluorescence of CAT was quenched by the addition of 1-OHP through a static quenching mechanism. 1-OHP can bind with CAT to form 1:1 complex, with a binding constant of 2.96×10⁵ L/mol at 290 K. The result of thermodynamic analysis indicated that hydrogen bonds and van der Waals’ force were the dominant intermolecular forces in stabilizing the complex. Based on the Förster’s non-radiation energy transfer theory, the binding distance between 1-OHP and CAT was determined to be 3.48 nm. The synchronous fluorescence and UV-Vis absorption spectral results showed that the formation of 1-OHP-CAT complex can induce some conformation changes of CAT. And in the presence of high concentrations of 1-OHP, the CAT activity can be inhibited obviously. Molecular docking was further employed to seek the optimum binding site of 1-OHP in CAT, and get the detailed binding information, which is identical to the experiment results. This work contributes to understand the interaction mechanism between 1-OHP and CAT at the molecular level, and provides important insights to study the toxicity mechanism of PAHs and their metabolites on antioxidant enzyme system in organisms.

Key words: 1-Hydroxypyrene, Catalase, Spectroscopic method, Molecular docking, Conformation change

CLC Number:

O657
O625

TrendMD:

CHEN Linfeng, ZHANG Jing, ZHU Yaxian, ZHANG Yong. Molecular Interactions of 1-Hydroxypyrene with Catalase Revealed by Spectroscopic Methods Combined with Molecular Docking^†[J]. Chem. J. Chinese Universities, 2015, 36(12): 2394.

Figures/Tables 14

Fig.1 Fluorescence quenching spectra of CAT in the presence of various amounts of 1-OHP(pH=7.00) a—g. 106 c(1-OHP)/(mol·L-1): 0, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0; c(CAT)=2.0×10-6 mol/L; λex=280 nm.

Fig.2 Stern-Volmer curves for the quenching of CAT by 1-OHP at 290, 300 and 308 K

Table 1 Stern-Volmer quenching constants and the correlation coefficient of 1-OHP-CAT systems at 290, 300 and 308 K

T/K	10^-4K_sv/ (L·mol^-1)	10^-12K_q/ (L·mol^-1·s^-1)	R²
290	7.92	7.92	0.995
300	6.88	6.88	0.994
308	6.64	6.64	0.995

Table 2 Fluorescence lifetime constants of 1-OHP-CAT system*

10⁶c(1-OHP)/(mol·L^-1)	Lifetime/ns		Amplitude(%)		τ_AV	χ²
10⁶c(1-OHP)/(mol·L^-1)	τ₁	τ₂	a₁	a₂	τ_AV	χ²
0	1.94	5.29	35.47	64.53	4.10	0.980
2.0	1.91	5.19	34.52	65.48	4.06	1.030
4.0	2.05	5.25	36.71	63.29	4.08	1.059
6.0	1.70	5.05	30.22	69.79	4.04	0.913
8.0	1.91	5.25	35.62	64.38	4.06	1.080

Table 3 Binding constants(K) of 1-OHP-CAT interactions

T/K	10^-5K/(L·mol^-1)	n	R²
290	2.96	1.11	0.995
300	1.62	1.07	0.994
308	0.554	0.98	0.995

Fig.3 Correlation between the binding constant(lgK) for binding to CAT and the octanol/water partition coefficients lgKow Pearson correlation factor r is 0.316.

Fig.4 Van’t Hoff plot of the temperature dependence of the binding constant

Table 4 Thermodynamic parameters of 1-OHP-CAT interaction

T/K	ΔG/ (kJ·mol^-1)	ΔS/ (J·mol^-1·K^-1)	ΔH/ (kJ·mol^-1)
290	-30.60	-118.87	-65.07
300	-29.41	-118.87	-65.07
308	-28.34	-118.87	-65.07

Fig.5 Overlap of UV-Vis absorption spectrum of 1-OHP(a) and the fluorescence emission spectrum of CAT(b) c(CAT)=c(1-OHP)=2.0×10-6 mol/L.

Fig.6 Synchronous fluorescence spectra of 1-OHP-CAT system(pH=7.00) (A) Δλ=15 nm; (B) Δλ=60 nm. c(CAT)=2.0×10-6 mol/L; a—g. 106c(1-OHP)/(mol·L-1): 0, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0.

Fig.7 UV-Vis absorption spectra(A) and difference spectra(B) of CAT in the absence and presence of different concentrations of 1-OHP(pH=7.00) (A) a. c(1-OHP)=6×10-6 mol/L; b. c(1-OHP)=1.0×10-5 mol/L; c. c(CAT)=1.0×10-6 mol/L, c(1-OHP)=6×10-6 mol/L; d. c(CAT)=1.0×10-6 mol/L, c(1-OHP)=1.0×10-5 mol/L; (B) a. c(CAT)=1.0×10-6 mol/L; b. difference spectrum of c-a in (A); c. difference spectrum of d-b in (A).

Fig.8 Effects of different concentrations of 1-OHP on the activity of CAT(pH=7.00) c(CAT)=2.0×10-6 mol/L.

Table 5 Energy-ranked results of five 1-OHP-CAT binding conformations

Energy-ranked result	Conformation data
Energy-ranked result	1	2	3	4	5
Binding energy/(kJ·mol^-1)	-35.09	-34.71	-33.75	-33.75	-32.95
Ligand efficiency/(kJ·mol^-1/non-hydrogen atom)	-2.05	-2.05	-1.96	-1.96	-1.92
Inhibition constant/(nmol·L^-1)	706.77	830.92	1220	1220	1670
Intermolecular energy/(kJ·mol^-1)	-36.34	-35.92	-35.00	-35.00	-34.20

Fig.9 Docking results of the 1-OHP and CAT system (A) Binding site of 1-OHP to CAT; (B) detailed illustration of the binding between 1-OHP and CAT.

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