Spectroscopic Study on Ternary Complexes of Pyoverdine-terbium(Ⅲ)-ciprofloxacin†

doi:10.7503/cjcu20170667

Abstract

Abstract:

The binding properties of ciprofloxacin(CIP), terbium and pyoverdine(Pvd) were studied by UV-Vis spectroscopy, fluorescence spectroscopy, fluorescence lifetime measurement and isothermal titration calorimetry(ITC) in pH=8.0, 50 mmol/L Tris-HCl buffer. The results show that the characteristic sensitization peak of terbium is generated at 544 nm upon CIP, forming a 3∶1 complex with terbium, the binding constant is approach to 10¹³ L³/mol³. Pvd binds to terbium at 1∶1, representing no characteristic sensitization peak at 544 nm. More importantly, it is founded that Pvd-Tb³⁺-CIP ternary complex forms based on the different fluorescence sensitization properties of Pvd and CIP on terbium. The ITC data reveal that the binding constant for the binding of CIP to Pvd-Tb³⁺ is (3.10±0.39)×10³ L/mol. Consequently, fluorescence lifetime of CIP changes much as a result of the formation of ternary complexes. The characteristic fluorescence sensitization effect of CIP to Tb³⁺ provides a feasible method for the further study of the antibiotic resistance of pseudomonas aeruginosa.

Key words: Pyoverdine, Ciprofloxacin, Terbium, Fluorescence sensitization

CLC Number:

O614

TrendMD:

SONG Kaili, YANG Binsheng. Spectroscopic Study on Ternary Complexes of Pyoverdine-terbium(Ⅲ)-ciprofloxacin^†[J]. Chem. J. Chinese Universities, 2018, 39(3): 408.

Figures/Tables 11

Fig.1 Structures of pyoverdine(A) and ciprofloxacin(B)

Fig.2 Difference UV spectra(A), titration curve(B) of 1.2 mL ciprofloxacin(0.05 mmol/L) with Tb3+(0.60 mmol/L) at pH=8.0, 50 mmol/L Tris-HCl, and fitting of ΔA/[CIP]t at 284 nm as a function of [Tb3+]f(C)V(Tb3+)/μL: a. 0; b. 10; c. 20; d. 30; e. 35; f. 40; g. 50; h. 60; i. 80; j. 100.

Fig.3 Fluorescence spectra(A), titration curve(B) of 1.2 mL ciprofloxacin(0.2 mmol/L) with Tb3+(2.4 mmol/L) at pH=8.0, 50 mmol/L Tris-HCl, and fitting of (F0-Fi)/(F0-F∞) as a function of [Tb3+]f(C)V(Tb3+)/μL: a. 0; b. 10; c. 20; d. 30; e. 40; f. 50; g. 60; h. 70; i. 80; j. 100; k. 120; l. 140.The inset in (A) is the enlarged part of the fluorescence spectra of ciprofloxacin with Tb3+.

Fig.4 Isothermal titration calorimetry measurements produced by the titration of Tb3+(4 mmol/L) to 0.4 mmol/L CIP at pH=8.0 and 25 ℃The panel shows the integrated heat curve points normalized per mole of injectant as a function of molar ratio.

Table 1 Binding parameters of Tb3+ complexes from ITC at pH=8.0 and 25 ℃

Compound	Binding number, n	10^-4 K_i/(L·mol^-1 )	ΔH/(kJ·mol^-1)	TΔS/(kJ·mol^-1)	ΔG/(kJ·mol^-1)
CIP-Tb³⁺	1	15.39±2.60	-7.99±0.24	21.65	-29.64
	1	3.21±0.11	12.15±0.19	37.92	-25.77
	1	1.20±0.50	-50.19±0.11	-26.78	-23.41
Pvd-Tb³⁺	0.90±0.03	2.97±0.03	-9.50±0.20	16.15	-25.65
CIP-Tb³⁺-Pvd	1	0.31±0.04	1.77±0.58	21.78	-20.01
	1	0.27±0.04	-222.55±10.82	-202.79	-19.79

Fig.5 Difference UV-Vis spectrum of Pvd-Tb3+ at 1∶1(A) and fluorescence spectra generated by the titration approximately of 1 mL of Pvd(5 μmol/L) with Tb3+(100 μmol/L)(B) at pH=8.0 in 50 mmol/L Tris-HClV(Tb3+)/μL: a. 0; b. 10; c. 20; d. 30; e. 40; f. 50; g. 60; h. 70; i. 90. Inset in (B) is the fluorescence titration curve of Pvd with Tb3+.

Fig.6 Isothermal titration calorimetry measurements for the titration of Tb3+(4 mmol/L) to 0.4 mmol/L Pvd at 25 ℃

Fig.7 Fluorescence spectra for the addition of CIP-Tb3+(0.18 mmol/L) to the solution of 1.2 mL Pvd(0.01 mmol/L)(A), fluorescence spectra of CIP, CIP-Tb3+, Pvd-Tb3+-CIP, and 2Pvd-Tb3+-CIP(B), and fluorescence spectra for the addition of CIP(0.2 mmol/L) to Pvd-Tb3+(0.01 mmol/L) at pH=8.0 in 50 mmol/L Tris-HCl(C)(A) V(Tb3+)/μL: a. 0; b. 10; c. 20; d. 30; e. 40; f. 50; g. 60; h. 70; i. 80; j. 90; k. 100. Inset in (A) is the fluorescence titration curve of CIP-Tb3+ with Pvd. (B) a. CIP; b. CIP-Tb3+; c. Pvd-Tb3+-CIP; d. 2Pvd-Tb3+-CIP.

Fig.8 Isothermal titration calorimetry measurements for the titration of 1 mmol/L CIP to 0.08 mmol/L Pvd-Tb3+ in pH=8.0, 50 mmol/L Tris-HCl at 25 ℃

Fig.9 Fluorescence decay curves for CIP(a), CIP-Tb3+(1∶1)(b), CIP-Tb3+-Pvd(1∶1∶1)(c)(A) and Pvd(a), Pvd-Tb3+(1∶1)(b) and Pvd-Tb3+-CIP(1∶1∶1)(c)(B)(A) λex=405 nm, λem=418 nm; (B) λex=405 nm, λem=457 nm.

Table 2 Fluorescence decay parameters for CIP, Tb3+ and Pvd ternary system*

System	<τ>/ns	χ²	System	<τ>/ns	χ²
CIP	10.74±0.05	1.01	Pvd-Tb³⁺	4.27±0.02	1.05
Pvd	4.48±0.02	1.06	Pvd-Tb³⁺-CIP(418 nm)	4.64±0.03	1.09
CIP-Tb³⁺	5.17±0.04	1.10	Pvd-Tb³⁺-CIP(457 nm)	4.15±0.02	1.02

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