Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Binding of copper to lysozyme: Spectroscopic, isothermal titration calorimetry and molecular docking studies
Graphical abstract
Copper could spontaneously bind lysozyme through hydrophobic forces with a positive ΔH, a favorable ΔS, and a negative ΔG. Binding of copper to lysozyme caused static quenching of the fluorescence, the change of the microenvironment of tryptophan residues and conformational changes of lysozyme. Besides, the lysozyme activity was also inhibited by the addition of copper with catalytic residues Glu 35 and Asp 52 locating at the binding sites. Thus, the direct interaction between copper and lysozyme might induce the conformational and functional alterations of lysozyme.
Introduction
Copper is an essential micronutrient that is vital to a wide range of metabolic processes [1]. It is widely used in military weapons, machinery manufacturing, dental products, intrauterine devices and cosmetics [2]. Although copper is a required element, exposure to elevated concentrations of copper becomes toxic. Copper poisoning may result in hepatocellular necrosis, acute tubular necrosis lethargy and anorexia in the early stages [2], [3]. The massive utilizations of copper have aroused considerable concerns regarding its biological roles and potential health impact [4], [5].
Lysozyme is abundant in various biological fluids and tissues, including skin, saliva, tears, liver blood and lymphatic tissues of humans and other animals [6], [7].It is a glycoside hydrolase consisting of 129 amino acid residues, which contains four disulfide bond, six tryptophan (Trp), three tyrosine (Tyr) and three phenylalanine (Phe) residues [6]. Previous research has shown that lysozyme is associated with innate immune system and can protect the cells from death by lysing the cytoderm of bacteria [8], [9]. Lysozyme also plays a vital role in physiological functions such as anti-tumor effects, anti-inflammatory, anti-viral and detumescence [10], [11], [12].
Studies have reported that excessive copper could lead to irreversible inactivation of lysozyme and extensive changes in the physical properties in weakly alkaline solution [13].Studies also have investigated the interaction between copper and hen egg-white lysozyme at different copper concentrations by electrospray ionization mass spectrometry to determine the binding capacity [14]. Thus, the direct interaction between copper and lysozyme might induce the conformational and functional alterations of lysozyme. The potential toxicity and toxic mechanisms of copper on lysozyme is significant for understanding heavy metal toxicity at molecular level. Although the interaction between copper and lysozyme has been explored by enzymatic, spectroscopy microscopy and spectroscopic techniques [15], [16], no research data were available on detailed thermodynamic descriptions of the interaction. Isothermal titration calorimetry (ITC) technique was used in this study to determine binding and thermodynamic parameters. Molecular docking studies were carried out with MOE software to illustrate the binding mode between lysozyme and copper and figure out the underlying mechanism. What's more, the fluorescence quenching mechanism, the microenvironment changes of Trp and Tyr residues and the binding sites in lysozyme have not been systematically determined in previous studies. Time-resolved fluorescence measurements was used to explore the fluorescence quenching mechanism (static or dynamic quenching).
In this work, we investigated the interacting mechanism of copper with lysozyme by multiple fluorescence measurements, UV–visible absorption, circular dichroism (CD) spectroscopy, time-resolved fluorescence measurements, ITC, molecular docking study and enzyme activity assay. Fluorescence quenching mechanism, conformational and functional changes of lysozyme, thermodynamic parameters, binding constants and modes were discussed in details. This study helps to understand comprehensively the effects of copper on lysozyme activity and toxic mechanism of copper on lysozyme.
Section snippets
Materials
Lysozyme (from chicken egg white) was purchased from Amersco (USA) and was dissolved in ultrapure water to form a 1.0 × 10− 4 M solution (preserved at 4 °C). A1 × 10− 2 M CuCl2 solution was prepared by dissolving 0.175 g CuCl2·2H2O (Tianjin Kermel Chemical Reagent Research Institute) in 100 mL using ultrapure water. 0.2 M acetic acid (HAc) and 0.2 M sodium acetate (NaAc) were used to prepare 0.2 M HAc-NaAc buffer solutions (pH = 5.5).
All the reagents were analytical-reagent grade unless otherwise noted and
Influence of copper on the fluorescence intensity of lysozyme
Fluorescence measurements were utilized to explore the alteration of lysozyme fluorescence with the copper addition. The absorption of proteins during excitation and emission processes resulted in the inner filter effect of fluorescence value [19]. For the sake of eliminating the possible inner filter effect (IFE), preliminary experiments were conducted. In this work, the sum of the absorption at 278 nm (excitation wavelength) and 338 nm (emission wavelength) could cause < 5% error, so IFE could
Conclusions
In this study, we investigated the interacting mechanism of copper with lysozyme by multiple spectroscopic measurements, ITC techniques, molecular docking study and enzyme activity assay. The experimental results showed that static quenching was the predominant quenching mechanism of the interaction between lysozyme and copper. In addition, copper induced the conformational changes of lysozyme and the microenvironment changes of Trp residues in lysozyme. ITC results revealed that copper could
Acknowledgments
This work is supported by NSFC (20875055, 21277081, 21477067), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education, Ministry of Education of China (708058, 20130131110016), Science and Technology Development Plan of Shandong Province (2014GSF117027) are also acknowledged.
References (43)
Copper and genomic stability in mammals
Mutat. Res. Fundam. Mol. Mech. Mutagen.
(2001)- et al.
Copper toxicity, oxidative stress, and antioxidant nutrients
Toxicology
(2003) Copper toxicity in Alzheimer's disease: cognitive loss from ingestion of inorganic copper
J. Trace Elem. Med. Biol.
(2012)- et al.
Lysozyme enhanced europium–metacycline complex fluorescence: a new spectrofluorimetric method for the determination of lysozyme
Anal. Chim. Acta
(2004) - et al.
Interaction of anthraquinone dyes with lysozyme: evidences from spectroscopic and docking studies
J. Hazard. Mater.
(2010) - et al.
Properties of lysozyme/low methoxyl (LM) pectin complexes for antimicrobial edible food packaging
J. Food Eng.
(2014) - et al.
Iron and citrate interactions with hen egg white lysozyme
Food Chem.
(2000) - et al.
Irreversible inactivation of lysozyme by copper
Arch. Biochem. Biophys.
(1956) - et al.
Fibrillation of hen egg white lysozyme triggers reduction of copper (II)
Int. J. Biol. Macromol.
(2012) - et al.
The effect of sarafloxacin on Cu/ZnSOD structure and activity
Spectrochim. Acta A Mol. Biomol. Spectrosc.
(2015)
Recent progress and perspectives on the toxicity of carbon nanotubes at organism, organ, cell, and biomacromolecule levels
Environ. Int.
Interactions of lead (II) acetate with the enzyme lysozyme: a spectroscopic investigation
J. Lumin.
Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation
Int. J. Biochem. Cell Biol.
Spectroscopic investigations on the effect of N-acetyl-L-cysteine-capped CdTe quantum dots on catalase
Spectrochim. Acta A Mol. Biomol. Spectrosc.
Spectroscopic studies on the interaction between novel polyvinylthiol-functionalized silver nanoparticles with lysozyme
Spectrochim. Acta A Mol. Biomol. Spectrosc.
Molecular mechanism on cadmium-induced activity changes of catalase and superoxide dismutase
Int. J. Biol. Macromol.
Interaction of oridonin with human serum albumin by isothermal titration calorimetry and spectroscopic techniques
Chem. Biol. Interact.
Investigation on the interaction of the toxicant, gentian violet, with bovine hemoglobin
Food Chem. Toxicol.
Binding of dihydromyricetin to human hemoglobin: fluorescence and circular dichroism studies
Spectrochim. Acta A Mol. Biomol. Spectrosc.
A spectrofluorometric study of tryptophan 108 in hen egg-white lysozyme
FEBS Lett.
Binding of divalent copper ions to aspartic acid residue 52 in hen egg-white lysozyme
J. Mol. Biol.
Cited by (73)
Molecular mechanisms of polystyrene nanoplastics and alpha-amylase interactions and their binding model: A multidimensional analysis
2024, Science of the Total EnvironmentMolecular mechanisms of nano-sized polystyrene plastics induced cytotoxicity and immunotoxicity in Eisenia fetida
2024, Journal of Hazardous MaterialspH-dependent interactions of biologically important metal ions with hen egg white lysozyme based on its hydration properties: Thermodynamic and mechanistic insights
2024, International Journal of Biological MacromoleculesInsights into the potential toxicity of Zn(II) to catalase and their binding mechanisms
2024, Journal of Molecular LiquidsLysozyme binding with amikacin and levofloxacin studied by tritium probe, fluorescence spectroscopy and molecular docking
2024, Archives of Biochemistry and BiophysicsElucidating binding mechanisms of naringenin by alpha-chymotrypsin: Insights into non-binding interactions and complex formation
2023, International Journal of Biological Macromolecules