Study on the interaction behavior of lysozyme with lanthanide ions by flow injection chemiluminescence analysis

Abstract

The interaction behavior of lysozyme with trivalent lanthanide ions (Ln^III) was investigated by flow injection chemiluminescence (FI-CL) analysis. It was found that lysozyme with Ln^III could form a 1 : 1 association complex that remarkably quenched CL intensity from the luminol–lysozyme reaction, and the quantitative correlation equation of CL intensity decrements versus the logarithm of the concentration of Ln^III ΔI = A ln C_Ln + B was established. The results showed the sensitive factor A varied as atomic number Z increased monotonically, and A_LL of light lanthanides (LL) from La^III to Eu^III was less than A_HL of heavy lanthanides (HL) from Gd^III to Lu^III. The relationships of A vs. Z plot for LL and HL with a Gd^III break at f⁷ were clearly presented, and the correlations of A with physical parameters (γ_±, ΔH_hyd and E^o) of Ln^III were discussed. Using a homemade FI-CL model, the binding constants (K = 10⁵ to 10⁶ level) and binding sites (n = 1.0) were obtained, and the binding affinity abilities increased with increasing Z along the Ln^III series. The thermodynamic parameters of lysozyme with Ln^III were obtained, and the results showed that the reaction was a spontaneous process by electrostatic interaction. The possible mechanism and application of the luminol–lysozyme–Ln^III CL system were given.

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1. Introduction

The lanthanide family (Ln: La–Lu, 57–71) has a consequence of their peculiar and similar electronic configuration [Xe]4fⁿ (n = 0–14),¹ which is divided into light lanthanides (LL) from La to Eu and heavy lanthanides (HL) from Gd to Lu.² The chemical, optical, and magnetic properties as well as all aspects of lanthanide ions (Ln^III) were summarized in detail by J. C. G. Bünzli.³ Although they exist merely in nature, they play a significant role in many areas of contemporary techniques and scientific applications.^4,5 For example, as catalysts, lanthanum salts and cerium oxide are used in the production of petroleum and synthetic products;^6,7 Nd^III, Er^III and Yb^III are used in lasers and upconverting nanoparticles;^8–10 and La^III, Ce^III and Sm^III are applied to promote seed germination and seedling growth.^11–13 Eu^III, Tb^III and Dy^III have been utilized extensively in biological systems, and the applications range from luminescent labels of biological molecules and the detection of cellular functions in vivo to the elucidation of the structure and function of enzymes and proteins.^14–16

It is well known that study on the interaction of protein with metal ions has become a hot spot in the fields of chemistry, biochemistry and clinical research.¹⁷ For example, the interaction of lysozyme with Ce^III, Sm^III, Eu^III, Tb^III, Cu^II, Zn^II and Hg^II using nuclear magnetic resonance (NMR),^18,19 inductively coupled plasma mass spectrometry (ICP-MS),^20,21 fluorescence (FL),^22,23 inductively coupled plasma-atomic emission spectrometry (ICP-AES),²⁴ and ultraviolet and visible spectrometry (UV-Vis)²⁵ have been reported. Nevertheless, the interaction of lysozyme with lanthanide family members from La to Lu by flow injection chemiluminescence (FI-CL) analysis has not been reported up to now.

Lysozyme, a small monomeric, low molecular weight (14.4 kDa) globular protein with 129 amino acid residues and active sites of Glu35, Trp62 and Asp52,^26,27 could accelerate the electron transferring rate of excited 3-aminophthalate, and enhanced luminol CL intensity has been reported.²⁸ In this work, using luminol as a luminescence probe, the interaction of lysozyme with Ln^III was studied at nanomole levels, which provided an effective way for investigating luminescence behavior of the lanthanide family.

It was found that Ln^III could quench the CL intensity of the luminol–lysozyme system; the decrement of CL intensity was linearly proportional to the logarithm of Ln^III concentration in the range of 0.1–50.0 nmol L⁻¹ with a detection limit of 0.03 nmol L⁻¹ (3σ). At a flow rate of 2.0 mL min⁻¹, complete determination of Ln^III including sampling and washing could be accomplished in 0.5 min, giving a maximum sample throughput of 120 h⁻¹. The possible CL mechanism of luminol–lysozyme–Ln^III reaction was also studied, and the application of lysozyme with Ln^III was given.

2. Materials and methods

2.1. Apparatus

The FI-CL system used in this work is shown schematically in Fig. 1. A peristaltic pump of the IFFM-E Luminescence Analyzer (Xi'an Remax Analysis Instrument Co. Ltd., Xi'an, China) was applied to deliver all streams. PTFE tubing (1.0 mm i.d.) was used throughout the manifold for carrying the CL reagents. A six-way valve with loops of 100 μL was used for sampling. The flow cell was made by coiling 15 cm of a colorless glass tube (1.0 mm i.d.) into a spiral-shape disk (2.0 mm i.d.) and placing it close to the photomultiplier tube (PMT). The CL signal produced in the flow cell was detected without wavelength discrimination, and PMT output was recorded by a PC with an IFFE-E client system. Inductively coupled plasma atomic emission spectroscopy (ICP-AES, IRIS Advantage, Thermo Scientific, America) was used as a reference technique for the comparison of the results among the samples. A water bath (HW.SY11-K6B/C, Leici, Shanghai, China) was used for controlling the temperature.

Fig. 1 Schematic diagram of the FI-CL system for the determination of Ln^III. Luminol: 2.5 × 10⁻⁵ mol L⁻¹; lysozyme: 1.0 × 10⁻⁷ mol L⁻¹; NaOH: 0.025 mol L⁻¹; flow rate: 2.0 mL min⁻¹; high voltage: −700 V.

2.2. Reagents

All reagents used in this work were of analytical reagent grade unless specified, and doubly deionized water was purified in a Milli-Q system (Millipore, Bedford, MA, USA) and used for the preparation of solutions in the whole procedure. Luminol (Fluka, Biochemika, Switzerland) was purchased from Xi'an Medicine Purchasing and Supply Station, China. Lysozyme (Sigma-Aldrich, St. Louis, MO, USA) was used without any further purification. Ln₂O₃ (99.99%) was purchased from Beijing Founde Star Science and Technology Co., Ltd (Beijing, China). Samples with different La^III content were supplied by Dr Zhao in the College of Chemistry and Materials Science, Northwest University.

Luminol (2.5 × 10⁻² mol L⁻¹) was prepared by dissolving 0.44 g luminol in 100 mL of 0.1 mol L⁻¹ NaOH solution in a brown calibrated flask. Lysozyme solution (1.0 × 10⁻⁴ mol L⁻¹) was prepared by dissolving 35.00 mg lysozyme in 25 mL purified water. A series of LnCl₃ stock solutions (1.0 × 10⁻³ mol L⁻¹) were prepared by dissolving Ln₂O₃ in 12 mol L⁻¹ HCl with subsequent heating to deplete the excess HCl and dilute with purified water.²⁹ All working standard solutions were prepared daily from the above stock solutions by appropriate dilution as required, and the stock solutions were stored at 4 °C.

Sample preparation: five samples were weighed exactly and placed in five crucibles, and then every sample (50.0 mg) was heated at 700 °C for 4 h in muffle furnace. After that, each sample was dissolved in 12 mol L⁻¹ HCl. Thereafter, the sample solutions were heated at 80 °C to dry. After cooling to the room temperature, each sample was prepared with purified water in a 25 mL calibrated flask. Suitable aliquots were taken and diluted with appropriate multiples for the determination of La^III by the proposed CL and ICP-AES methods.

2.3. Molecular docking

The crystal structure of lysozyme was taken from the Protein Data Bank (PDB, http://www.rcsb.org/pdb/). The 3D conformer structure of luminol was retrieved from the National Center for Biotechnology Information and converted to PDB format by OpenBabel 2.3.2. These PDB structures of lysozyme and luminol were used for docking by AutoDock 4.2 software. Docking simulation was performed using the Lamarckian Genetic Algorithm (LGA) to search for the optimum binding energy of lysozyme with luminol. Docking parameters included AutoGrid using a box 40 Å × 40 Å × 40 Å. Luminol could therefore be completely contained in the box.

2.4. Procedures

A schematic profile of the equipment in the flow system is described in Fig. 1, and flow lines were inserted into luminol/NaOH, carrier (purified water), lysozyme and Ln^III solutions at a constant speed of 2.0 mL min⁻¹. The whole flow system was washed with purified water until a stable baseline was recorded. The luminol standard solution (100 μL) was injected into the carrier stream by an injection valve and merged with a mixture flow of lysozyme and Ln^III. The mixed solution was delivered into the CL cell, producing CL emission, and detected by the PMT at a high voltage of −700 V. The concentration of Ln^III could be quantified on the basis of the decrement of luminol–lysozyme CL intensity, ΔI = I₀ − I, where I and I₀ were luminol–lysozyme system CL signals in the presence and absence of Ln^III, respectively. The temperature of working solutions was controlled by a water bath, which was kept in a certain range (T ± 0.1 °C) throughout the experiment. Since the speed of detection was rapid, the time that the solution was flowing in the tube was short and the temperature of solutions was kept constant, so during detection, the error of temperature was negligible.

2.5. The optimization of experimental conditions

The effect of luminol concentration on CL intensity was investigated at concentrations ranging from 5.0 × 10⁻⁷ to 5.0 × 10⁻⁵ mol L⁻¹. It was found that the CL intensity increased steadily with increasing luminol concentration up to 2.5 × 10⁻⁵ mol L⁻¹ and then tended to be stable. Thus, 2.5 × 10⁻⁵ mol L⁻¹ was chosen as the optimum luminol concentration. Similarly, the influence of lysozyme concentration on CL intensity was tested from 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ mol L⁻¹. The CL intensity rose drastically with increasing lysozyme concentration up to 1.0 × 10⁻⁷ mol L⁻¹, and then increased slowly from a higher concentration. Therefore, 1.0 × 10⁻⁷ mol L⁻¹ was chosen as the optimum lysozyme concentration in the following experiments. Because luminol reaction is more favored under basic conditions, NaOH was introduced to improve the sensitivity of the system. A series of NaOH solutions with different concentrations were tested. The CL intensity arrived at a peak with the concentration of NaOH arriving at 0.025 mol L⁻¹, and thus, this concentration was employed in subsequent experiments.

The CL intensity was related to the flow rate and mixing tube length. The effect of flow rate on CL intensity was investigated with flow rates ranging from 0.5–5.0 mL min⁻¹. It was found that the CL intensity increased with the augmentation of flow rate, while the CL signal was unstable at a high flow rate. As a compromise between less reagent consumption and higher sensitivity, a flow rate of 2.0 mL min⁻¹ was recommended. The effect of mixing tube length on CL intensity was also tested with lengths from 5.0 to 20.0 cm, and it was observed that the CL intensity was strong and stable using a mixing tube of 10.0 cm. Accordingly, 10.0 cm was selected as the optimum length for the mixing tube.

The pH effect of Ln^III solutions on luminol–lysozyme CL intensity was tested at pH values ranging from 4.0 to 9.0, which were adjusted by HCl or NaOH solutions. It was found that the pH effect for all Ln^III solutions was the same, and as Fig. 2 shows (La^III as an example), the remarkable increments in CL intensity were at pH values of 5.0–6.5. A lower pH affects the luminol–lysozyme CL reaction and a higher pH promotes the hydrolysis of La^III, leading to a decrease in ΔI. Considering the sensitivity of La^III determination, finally, a pH of 6.0 was selected in this work.

Fig. 2 The pH effect of 10.0 nmol L⁻¹ La^III on the CL intensity of the luminol–lysozyme reaction.

2.6. The operational stability of the FI-CL system

The operational stability of the FI-CL system was tested by injecting 100 μL luminol solution into the flow system, which was further merged with the mixed solution of Eu^III and lysozyme (1.0 and 10.0 nmol L⁻¹, respectively). Then, the relative CL intensity (ΔI = I₀ – I) was recorded to test the stability of the system. The experiment lasted for 5 days with the FI-CL system regularly used over 8 h per day, and the stability and precision of the FI-CL system for Eu^III determination are shown in Table 1. It was found that ΔI kept stable under the fluctuation of I₀, and RSDs were less than 3.0%, which showed that the system had very good stability.

Table 1 The stability test of the FI-CL system under 1.0 and 10.0 nmol L⁻¹ Eu^IIIa

Time day	ICL blank	RSD%	ICL 1.0 nmol L^−1	RSD%	ICL 10.0 nmol L^−1	RSD%
a Each result is the average of seven separate determinations.
1^st	352	1.6	335	2.5	308	2.7
2^nd	351	2.0	337	1.8	306	2.4
3^rd	353	1.9	338	2.2	304	2.5
4^th	350	2.1	339	2.6	305	2.3
5^th	354	2.3	336	2.0	307	2.5

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3. Results and discussion

3.1. The relative CL intensity–time profile

The relative CL intensity–time profile of luminol–lysozyme–Ln^III is shown in Fig. 3. The time to reach the maximum CL intensity (T_max) for the luminol–dissolved oxygen system (curve 7) was 4.4 s, and the T_max of the luminol–lysozyme (100 nmol L⁻¹) system (curve 1) was 4.0 s. The maximum CL intensity (I_max) was increased from 98 to 352. It was suggested that lysozyme reacted with the luminol, leading to the enhancement CL intensity of luminol. In the presence of La^III, Eu^III, Gd^III, Tb^III and Lu^III (5 nmol L⁻¹), the I_max of luminol–lysozyme CL system decreased from 352 to 295, 268, 256, 247 and 193 by 8.5, 23.9, 27.3, 29.8 and 45.2%, respectively (curves 2–6). The decrement of luminol–lysozyme CL intensity was proportional to the concentration of Ln^III, and the quantitative correlation equation could be obtained. It was indicated that the interaction between lysozyme and Ln^III should exist and resulted in the CL intensity of luminol–lysozyme being inhibited.

Fig. 3 Relative CL intensity–time profiles in different CL systems. Curve 1: CL intensity of luminol–lysozyme reaction; curve 2–6: CL intensity in the presence La^III, Eu^III, Gd^III, Tb^III and Lu^III of luminol–lysozyme reaction; curve 7: CL intensity of luminol. The concentration of luminol was 2.5 × 10⁻⁵ mol L⁻¹, the concentration of lysozyme was 1.0 × 10⁻⁷ mol L⁻¹, and the concentrations of La^III, Eu^III, Gd^III, Tb^III and Lu^III were 5.0 × 10⁻⁹ mol L⁻¹.

3.2. The quantitative correlation equations of Ln^III

Under the optimized conditions, the standard solutions of Ln^III series were tested by FI-CL using the luminol–lysozyme system. It was found that the decrement of CL intensity (ΔI) was proportional to the logarithm of the concentration of Ln^III over the range from 0.1 to 50 nmol L⁻¹ with correlation coefficient R from 0.9960 to 0.9986 and obeyed the quantitative correlation equation, ΔI = A ln C_Ln + B, in which the slope A was defined as the sensitivity factor. The linear equations of the Ln^III series are given in Table 2, and the calibration graphs of La^III, Sm^III, Yb^III and Lu^III are shown in Fig. 4. It can be seen that A(B) varied with increasing Z, among A_LL from 15.39 to 18.48 and A_HL from 22.20 to 29.01 with linear equations A_LL = 0.55Z − 15.96 and A_HL = 1.26Z − 60.70.

Table 2 Linear equations of lysozyme with Ln^III

Ln^III	ΔI = A ln CLn + B	R
La^III	ΔI = (15.39 ± 0.02)ln CLa + (30.54 ± 0.04)	0.9960
Ce^III	ΔI = (16.52 ± 0.02)ln CCe + (34.81 ± 0.03)	0.9971
Pr^III	ΔI = (17.46 ± 0.01)ln CPr + (36.08 ± 0.01)	0.9961
Nd^III	ΔI = (17.55 ± 0.02)ln CNd + (38.46 ± 0.03)	0.9966
Sm^III	ΔI = (18.41 ± 0.03)ln CSm + (39.66 ± 0.02)	0.9976
Eu^III	ΔI = (18.89 ± 0.02)ln CEu + (43.74 ± 0.01)	0.9971
Gd^III	ΔI = (18.48 ± 0.02)ln CGd + (44.04 ± 0.03)	0.9968
Tb^III	ΔI = (22.20 ± 0.03)ln CTb + (53.34 ± 0.02)	0.9969
Dy^III	ΔI = (23.19 ± 0.01)ln CDy + (55.16 ± 0.01)	0.9971
Ho^III	ΔI = (24.63 ± 0.01)ln CHo + (57.51 ± 0.03)	0.9986
Er^III	ΔI = (25.65 ± 0.02)ln CEr + (59.14 ± 0.02)	0.9961
Tm^III	ΔI = (26.77 ± 0.02)ln CTm + (61.18 ± 0.03)	0.9975
Yb^III	ΔI = (27.41 ± 0.01)ln CYb + (63.29 ± 0.01)	0.9985
Lu^III	ΔI = (29.01 ± 0.01)ln CLu + (65.05 ± 0.01)	0.9981

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Fig. 4 The calibration graphs of La^III, Sm^III, Yb^III and Lu^III.

It is well known that the Ln^III series can be divided into two subgroups by the severity of the Gd break,^30,31 which is approximated in the existing diad theory.^32,33 By plotting A against Z, good linear relationships for LL and HL except at the Gd^III break at f⁷ are presented in Fig. 5. It was clear that Gd^III was a break point, which agreed with the lanthanide contraction and the Gd break effect. The correlation equations of A with some physical parameters (ionic radius γ_±, hydration enthalpy ΔH_hyd and standard redox potential E^o) of Ln^III are listed in Table 3, and the relationship curves of A vs. Z, γ_±, ΔH_hyd and E^o are shown in Fig. 6. It can be seen that A vs. |ΔH_hyd| (the absolute value of ΔH_hyd) was the best trend (the same A as a function Z), and few lines intersected at the point Gd, which indicated that HL had a bigger slope value than LL, and may be attributed to the increased polarizability caused by lanthanide contraction.^34–36

Fig. 5 Plots of sensitive factor A against atomic number Z for the LL and HL with linear equations.

Table 3 Correlations of A with the physical parameters^a for LL and HL

Parameters	LL		HL
	Linear equations	R	Linear equations	R
a Ionic radius γ± (Å), hydration enthalpy ΔHhyd (kJ mol^−1) and standard redox potential E^o (V).
A vs. γ±	A = −34.6γ± + 51.8	0.9741	A = −100.0γ± + 113.5	0.9923
A vs. |ΔHhyd|	A = 1.7 (|ΔHhyd|) – 39.7	0.9518	A = 4.2 (|ΔHhyd|) – 128.4	0.9750
A vs. |E^o|	A = −33.7 (|E^o|) + 99.9	0.9864	A = −55.3 (|E^o|) + 152.7	0.9705

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Fig. 6 The linear relationships of A vs. physical parameters of Ln^III.

3.3. The interaction parameters of lysozyme with Ln^III

Utilizing the homemade FI-CL model lg[(I₀ − I)/I] = n lg [Ln] + lg K,³⁷ the binding constants K and the number of binding sites n could be obtained from the plot of lg[(I₀ − I)/I] against lg[Ln], and the results are listed in Table 4. The binding constants K were 10⁵ to 10⁶ L mol⁻¹ level, suggesting that there existed a high binding affinity of Ln^III to lysozyme, which agreed well with the binding constants K obtained by fluorescence,^38–40 and the number of binding sites n were approximately equal to 1.0 and confirmed a 1 : 1 complex. It was found that the binding constants K and number of binding sites n increased with increasing Z, and the binding ability of Ln^III to lysozyme increased with increasing Z, which matched with the order of sensitivity in the presence of lanthanide contraction. As shown in Fig. 7, it was clear that Gd^III was a break point and the HL possessed relatively high binding ability compared with the LL, which was in good agreement with a large number of classical experimental results.^41–43

Table 4 Binding parameters of lysozyme with Ln^III at 288/300/310 K

Ln^III	K × 10^5/L mol^−1	n	R
La^III	1.26 ± 0.02/1.10 ± 0.01/1.05 ± 0.02	0.69 ± 0.01	0.9897/0.9876/0.9833
Ce^III	1.99 ± 0.01/1.70 ± 0.01/1.47 ± 0.01	0.70 ± 0.01	0.9898/0.9906/0.9892
Pr^III	2.68 ± 0.01/2.21 ± 0.01/1.91 ± 0.01	0.73 ± 0.01	0.9952/0.9948/0.9901
Nd^III	3.60 ± 0.01/2.97 ± 0.01/2.46 ± 0.01	0.75 ± 0.01	0.9990/0.9942/0.9916
Sm^III	4.37 ± 0.01/3.31 ± 0.01/3.10 ± 0.01	0.78 ± 0.02	0.9855/0.9926/0.9892
Eu^III	5.40 ± 0.01/4.23 ± 0.01/3.55 ± 0.01	0.80 ± 0.01	0.9854/0.9936/0.9838
Gd^III	5.63 ± 0.01/4.46 ± 0.01/3.67 ± 0.01	0.81 ± 0.01	0.9929/0.9974/0.9884
Tb^III	10.09 ± 0.01/7.14 ± 0.01/5.43 ± 0.02	0.92 ± 0.01	0.9882/0.9856/0.9896
Dy^III	13.33 ± 0.02/9.40 ± 0.01/7.01 ± 0.02	0.94 ± 0.02	0.9843/0.9872/0.9914
Ho^III	17.10 ± 0.01/11.86 ± 0.01/8.76 ± 0.01	0.97 ± 0.01	0.9952/0.9929/0.9879
Er^III	21.57 ± 0.01/14.36 ± 0.01/10.71 ± 0.01	1.00 ± 0.01	0.9917/0.9848/0.9945
Tm^III	24.92 ± 0.01/16.36 ± 0.01/12.04 ± 0.01	1.02 ± 0.01	0.9857/0.9927/0.9966
Yb^III	31.65 ± 0.01/20.56 ± 0.02/14.70 ± 0.01	1.05 ± 0.02	0.9987/0.9946/0.9905
Lu^III	39.47 ± 0.01/24.75 ± 0.02/17.69 ± 0.01	1.08 ± 0.02	0.9849/0.9884/0.9962

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Fig. 7 Plots of the number of binding sites n against atomic number Z of Ln^III.

3.4. The thermodynamic parameters of lysozyme with Ln^III

According to the obtained binding constants of lysozyme with Ln^III, the thermodynamic parameters (ΔH, ΔS and ΔG) at different temperatures (288, 300 and 310 K) were calculated using the Van't Hoff equation⁴⁴ and summarized in Table 5. It can be seen that ΔG < 0, ΔH < 0 and ΔS > 0. The results testified the formation complex of lysozyme with Ln^III was a spontaneous exothermic reaction, and the main binding force was electrostatic interaction.⁴⁵ As shown in Fig. 8, the absolute value of ΔG increased with the increasing of atomic number Z and HL increased more obviously than LL, which was consistent with the effect of lanthanide contraction and the diad theory. The binding ability and the affinity of lysozyme with Ln^III varied in the following sequence: La^III to Lu^III increased successively. With ion radii decreasing systematically, the electrostatic force of lysozyme with Ln^III and higher binding ability for HL was achieved. It was also found that with increasing temperature, the absolute value of ΔG increased and the spontaneous reaction was more easily initiated, which indicated that high temperature was favorable for reaction.

Table 5 Thermodynamic parameters of lysozyme with Ln^III

Ln^III	T/K	ΔG/kJ mol^−1	ΔH/kJ mol^−1	ΔS/J mol^−1 K^−1
La^III	288	−28.13 ± 0.01	−8.55 ± 0.02	68.03 ± 0.01
	300	−28.96 ± 0.02
	310	−29.63 ± 0.01
Ce^III	288	−29.21 ± 0.02	−10.10 ± 0.02	66.47 ± 0.01
	300	−30.04 ± 0.02
	310	−30.67 ± 0.01
Pr^III	288	−29.92 ± 0.01	−11.39 ± 0.01	64.33 ± 0.02
	300	−30.69 ± 0.01
	310	−31.34 ± 0.02
Nd^III	288	−30.64 ± 0.01	−12.82 ± 0.01	62.03 ± 0.01
	300	−31.43 ± 0.01
	310	−31.99 ± 0.01
Sm^III	288	−31.10 ± 0.02	−13.39 ± 0.01	61.03 ± 0.01
	300	−31.70 ± 0.01
	310	−32.47 ± 0.01
Eu^III	288	−31.61 ± 0.02	−14.23 ± 0.02	60.27 ± 0.01
	300	−32.31 ± 0.01
	310	−32.93 ± 0.01
Gd^III	288	−31.71 ± 0.02	−14.39 ± 0.01	60.20 ± 0.01
	300	−32.45 ± 0.01
	310	−33.03 ± 0.02
Tb^III	288	−33.10 ± 0.01	−20.90 ± 0.01	42.40 ± 0.01
	300	−33.62 ± 0.02
	310	−34.03 ± 0.01
Dy^III	288	−33.77 ± 0.01	−21.62 ± 0.02	42.27 ± 0.01
	300	−34.30 ± 0.01
	310	−34.69 ± 0.02
Ho^III	288	−34.36 ± 0.01	−22.52 ± 0.02	41.20 ± 0.01
	300	−34.88 ± 0.01
	310	−35.27 ± 0.01
Er^III	288	−34.92 ± 0.02	−23.65 ± 0.01	39.03 ± 0.01
	300	−35.36 ± 0.01
	310	−35.78 ± 0.01
Tm^III	288	−35.27 ± 0.01	−24.58 ± 0.01	37.03 ± 0.01
	300	−35.69 ± 0.02
	310	−36.09 ± 0.01
Yb^III	288	−35.84 ± 0.01	−25.86 ± 0.02	34.63 ± 0.01
	300	−36.26 ± 0.01
	310	−36.60 ± 0.01
Lu^III	288	−36.37 ± 0.02	−27.11 ± 0.01	32.03 ± 0.01
	300	−36.72 ± 0.01
	310	−37.08 ± 0.01

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Fig. 8 Plots of the absolute value of Gibbs free energy |ΔG| against atomic number Z of Ln^III at 288, 300 and 310 K.

3.5. The possible mechanism of luminol–lysozyme–Ln^III

The possible mechanism of luminol–lysozyme–Ln^III was investigated by FI-CL, fluorescence and molecule docking methods.

The possible mechanism of luminol to lysozyme: As Fig. 3 shows, the T_max of the maximum CL intensity of luminol-dissolved oxygen system shifts from 4.4 s to 4.0 s in the presence of lysozyme with the corresponding intensity changing 3.6-fold, which demonstrated that lysozyme could accelerate the electron transfer rate of excited 3-aminophthalate and enhance the CL intensity of the luminol-dissolved oxygen system. It was found that the fluorescence intensity of 0.5 μmol L⁻¹ luminol (λ_ex/λ_em = 350 nm/425 nm) was decreased in the presence of lysozyme with its concentrations ranging from 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ mol L⁻¹. Using the Stern–Volmer quenching equation,⁴⁶ the regression equation of F₀/F = 2.28 × 10⁵C_lysozyme + 1.04 (R = 0.9949) was obtained. The established equation for using a small molecule as the FL probe⁴⁷ could give the binding constant K (4.2 × 10⁴ L mol⁻¹) and the number of binding sites n (2.18). By molecular docking, it was revealed that luminol could enter the cleft and bind to the active sites of Glu35 and Trp62 on lysozyme with binding distances of 2.2 Å and 2.4 Å.

The possible mechanism of Ln^III to lysozyme: by the homemade FI-CL model,³⁷ the obtained binding parameters (K and n) of Ln^III to lysozyme were at the 10⁵ to 10⁶ L mol⁻¹ level and around 1.0, which indicates that a 1 : 1 lysozyme–Ln^III complex was formed with relatively high binding affinity. It has been reported that some metal ions (e.g. Cu^II, Mn^II, Pb^II, Hg^II and Co^II) could bind to the active site Asp52 of lysozyme,^48–50 and it was suggested that Ln^III should enter the Asp52 site of lysozyme. The thermodynamic parameters at different temperatures are listed in Table 5; the data support the finding that the reaction of Ln^III binding to lysozyme was a spontaneous process with electrostatic interaction.

The possible mechanisms of luminol–lysozyme–Ln^III can be explained as follows: lysozyme is a globular protein with the active sites of Glu35, Trp62 and Asp52; as shown in Fig. 9, luminol binds to Glu35 and Trp62 on lysozyme to enhance the CL intensity of luminol and produce the effect of complexation enhancement of CL (CEC); and Ln^III should be able to enter Asp52 on lysozyme, which could produce the effect of complexation quenching of CL (CQC).

Fig. 9 The possible mechanism luminol–lysozyme–Ln^III reaction process. The result was obtained by AutoDock 4.2 (The Scripps Research Institute) pump waste lysozyme mixing tube valve luminol/NaOH carrier Ln^III flow cell detector PC.

3.6. Determination of La^III in samples

The five samples were supplied by Dr Zhao's lab in our college and were complex admixtures of La with some organic ligands, and the results for the recoveries ranged from 96.4 to 102.5%, with RSDs less than 2.0%, as listed in Table 6. The same samples were also determined by ICP-AES, and it was found that the results obtained by the proposed CL method agreed well with the ICP-AES results. Comparing the two methods, it can be seen that the proposed CL method was more sensitive than ICP-AES.

Table 6 Results of determination of La^III in samples^a

Sample no.	Added/found ng mL^−1	RSD%	Recovery%	FI-CL μg mL^−1	ICP-AES μg mL^−1
a The average of five determinations.b Not detected.
1	0.0/50.8	0.9	98.6	50.30 ± 0.03	50.2 ± 1.6
	50.0/100.7	1.0
2	0.0/15.5	1.3	101.5	15.08 ± 0.01	15.2 ± 1.9
	15.0/30.2	0.9
3	0.0/7.1	1.4	97.8	7.05 ± 0.03	ND^b
	7.0/13.9	1.3
4	0.0/3.1	1.8	96.4	2.98 ± 0.01	ND^b
	3.0/6.0	1.2
5	0.0/0.5	1.4	102.5	0.51 ± 0.01	ND^b
	0.5/1.0	0.9

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4. Conclusion

The binding abilities of lysozyme to Ln^III were studied by a FI-CL method for the first time, and the results showed that the reaction of lysozyme with Ln^III was a spontaneous process mainly induced by an electrostatic effect, and the binding affinity increased with increasing Z of Ln^III. It was found that A varied as the atomic number Z increased monotonically, and the good correlations for LL and HL in [Xe]4fⁿ configurations were discussed and matched with the lanthanide contraction and the Gd break effect.

Declaration of interest

The authors declare there is no conflict of interest.

Acknowledgements

The authors gratefully acknowledge the financial support from the National Nature Science Foundation of China (no. 21275118) and the Open Funds from the Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, China.

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