Insights into the interactions between tetracycline, its degradation products and bovine serum albumin
© The Author(s) 2016
Received: 3 October 2015
Accepted: 13 May 2016
Published: 13 July 2016
Tetracyclines (TCs) are the most widely used antibiotics in the world. Because antibiotics have low bioavailability and are difficult to completely remove using current sewage treatment facilities, residual TCs and their degradation products in the environment, animal and plant foodstuffs and personal care products may enter the body through the food chain, thus causing unpredictable effects on human health. We studied bovine serum albumin (BSA) (a functional protein) as a target of tetracycline-induced toxicity by examining its interactions with TC, anhydrotetracycline (ATC) and epitetracycline (ETC), based on a fluorescence spectroscopy and molecular docking method under simulated physiological conditions. The interaction mechanism was elucidated at the molecular level. The results show that TC, ATC and ETC bind at site II of BSA and interact mainly through hydrogen bonding interactions and van der Waals interactions. The binding affinities can be ranked in the order ATC > TC > ETC.
KeywordsTetracycline Degradation products BSA Spectroscopic method Molecular docking
As one of the most important medical findings of the twentieth century, antibiotics not only make great contributions to the treatment of human and animal bacterial infections, but have been used globally at subclinical doses in the animal breeding industry as feed additives for a long time (Gu and Karthikeyan 2005; Hvistendahl 2012). Specifically, tetracyclines (TCs) are a large group of antibiotics widespread used in human and veterinary medicine and account for approximately 29 % of total antibiotic use (Khetan and Collins 2007; Wammer et al. 2011). TCs are broad-spectrum antibiotics synthesized or semi-synthesized from actinomycetes, and are widely used in livestock and poultry breeding and aquaculture as veterinary and feed additives (Bowman et al. 2011), ranking second in the global production and application of veterinary drugs. Generally, TCs cannot be completely absorbed and metabolized after ingestion by animals but are excreted in feces and urine as prototype and active metabolites (Hu et al. 2011). Because antibiotics also cannot be completely removed through existing sewage treatment processes, they ultimately reach the environment and accumulate through surface runoff scour percolation, effluents from sewage treatment plants and the deposition on land of manure from livestock and poultry (Spongberg and Witter 2008). TCs have now been detected in many countries and in various environmental systems, including surface water (Kim and Carlson 2007), ground water (Krapac et al. 2005) and soil (Kulshrestha et al. 2004), as well as in animals and plants (Ji et al. 2010).
TCs entering the environment can produce corresponding metabolites by epimerism, dehydration, proton transfer and other methods under the actions of environmental biological and non-biological factors (Jia et al. 2009). Mackie et al. (2006) detected dehydrated degradation products of tetracycline, chlortetracycline and oxytetracycline in the ground water under soil that had been subjected to long-term fertilization with manure. Residues in honey of TCs used to prevent foulbrood infection in bees readily degrade to epimers during processing and storage. Liu et al. (2011) verified the existence of epi-degradation products from tetracycline and oxytetracycline (at 4.9 and 3.8 µg kg−1, respectively). Dehydration products of TCs can be detected in personal care products such as facial cleansers and bath foams. TC residues and their catabolites that accumulate in the environment, animal- and plant-based foods and personal care products are likely to be transferred into the human body through the food chain, resulting in negative impacts on human health. Current research shows that catabolites of TCs usually have relatively decreased activities but possibly increased toxicities than the parent compound (Halling-Sørensen et al. 2002). The epimers of TCs can cause strong toxic effects clinically in animals and human beings (Daghrir and Drogui 2013). Halling-Sørensen et al. (2002) also found that the degradation products of some TCs had decreased activities but stronger toxic effects than their parent antibiotics towards drug-resistance bacteria and soil bacteria in the environment.
Serum albumin, the most abundant protein in plasma, has important carrier functionality, is one of the components of the blood buffer system and is the major component responsible for maintaining colloidal osmotic pressure in blood. Its ability to combine with various endogenous and exogenous pollutants also makes it important in maintaining the free activity concentration of such substances in plasma and affects their transport, distribution, storage and metabolic processes inside the organism. The interactions between TCs and biomacromolecules such as serum proteins have been reported in the literature (Chi and Liu 2011; Khan et al. 2002), but research into the influence of TC degradation products on macromolecular structures and functions has been rarely reported to date. Based on fluorescence spectroscopy and molecular docking technology under simulated physiological conditions, this paper focuses on interactions of tetracycline (TC) and its degradation products anhydrotetracycline (ATC) and epitetracycline (ETC) with serum albumin. In particular, we examine the effects of such substances on protein conformation and the modes of action between the three drugs and serum protein from the molecular perspective. The results provide insights into the processes of distribution, transfer and transportation of TC and its degradation products to toxic endpoints in the human body and associated health risks. In addition, the results provide data regarding safe dosages and risk assessment for such substances.
One milliliter of Tris–HCl buffer solution at 0.2 mol L−1 and pH 7.40, 1 mL of sodium chloride solution at 1 mol L−1 and 1 mL of standard BSA solution at 2.0 × 10−5 mol L−1 were added in sequence into 10 mL colorimetric tubes. These were then gently mixed and held for 10 min. Using a micro syringe, a certain volume of drug was added to each colorimetric tube and diluted with distilled water to 10 mL. The final concentrations were 0, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6 and 4.0 × 10−5 mol L−1 respectively. All solutions were then placed at constant temperatures of 293, 298, 304 and 310 K for 60 min until binding equilibrium was reached. The fluorospectrophotometer F-4600 was then used to measure the emission spectra over the wavelength range 300–400 nm of the test solutions after obtaining reaction equilibrium at constant temperature. The maximum emission wavelength and fluorescence intensity were recorded. The excitation wavelength was 285 nm, the slit width was 5 nm and the scanning speed was 240 nm min−1. The synchronous fluorescence spectra of sample solutions were scanned over the wavelength range 300–400 nm under ∆λ = 15 nm and ∆λ = 60 nm constant steps.
To define the type of quenching, it was initially assumed to be dynamic quenching, and the fluorescent data were then analyzed using the Stem–Volmer equation.
In Eq. (1), KA represents the binding constant, n represents the number of micromolecules and protein binding-site, which is actually the concentration of free micromolecules because the concentration of micromolecules is lower than that of the protein and the combination through non-covalent bonds is relatively weak. Thus, we regard [Q] as the total concentration of micromolecules.
When the temperature does not vary markedly, the reaction enthalpies (ΔH) can be regarded as constant, and the enthalpy and entropy changes of reaction can be calculated using the van’t Hoff equation. The free energy change can be further calculated using the formula ΔG = ΔH − TΔS.
The crystal structure of BSA in the system BSA-TC/ATC/ETC was downloaded from the Protein Database (PDB ID: 4JK4, 2.65 Å). Water molecules were removed, nonpolar hydrogen atoms were merged, and rotatable bonds were also defined (Chi and Liu 2011). The MOE 2009 software (Chemical Computation Group, Montreal, Quebec, Canada) was applied to build and optimize the tertiary structures of these three small molecules at minimal energy. Molecular docking was performed using MVD 4.0 software. The binding pocket was designated as a sphere with radius 20 Å around the ligands. Thirty runs were carried out, producing thirty conformations for selection. The poses which that had the highest scores were chosen for the molecular dockings.
Results and discussion
Fluorescence quenching constants of TC and its degradation products on BSA
Kq (L mol s−1)
Ksv (L mol−1)
6.61 × 1012
6.61 × 104
6.27 × 1012
6.27 × 104
6.05 × 1012
6.05 × 104
5.70 × 1012
5.70 × 104
2.70 × 1013
2.70 × 105
2.61 × 1013
2.61 × 105
2.36 × 1013
2.36 × 105
2.10 × 1013
2.10 × 105
4.31 × 1012
4.31 × 104
4.09 × 1012
4.09 × 104
3.71 × 1012
3.71 × 104
3.49 × 1012
3.49 × 104
It can be seen from the data in Table 1 that the quenching constant K decreased with increasing temperature, and the order of magnitude of Kq reached 1012, which is 2–3 orders of magnitude higher than the maximum diffusion and collisional quenching constant (2 × 1010 L mol−1 s−1) of biomacromolecules in aqueous solution (Pan et al. 2011).
Binding constants, binding-site numbers and relative thermodynamic parameters of TC and its degradation products with BSA
KA (L mol−1)
∆G (KJ mol−1)
∆H (KJ mol−1)
∆S (J mol−1 K−1)
5.13 × 104
4.50 × 104
3.60 × 104
3.39 × 104
1.87 × 105
1.45 × 105
1.36 × 105
1.30 × 105
2.95 × 104
2.44 × 104
2.30 × 104
2.21 × 104
Interaction energies between TC/ATC/ETC and responsive amino acid residues in molecular docking
Interaction energy (kJ mol−1)
TC and its degradation products (ATC and ETC) interact with BSA. The binding affinities were in the order ATC > TC > ETC. The drugs bind mainly at site II of BSA and the three drugs interact with BSA mainly through hydrogen bonding and van der Waals interactions. TC and ETC enhance the hydrophobicity of the microenvironment of BSA tryptophan residues. In contrast, the action of ATC enhanced the polarity of the fluorophore microenvironment of BSA tryptophan and tyrosine residues and decreased its hydrophobicity. The ATC has a stronger affinity towards BSA, tending to cause more significant conformational changes in BSA.
XT and MM designed experiments, XT, MM and KZ carried out experiments, DX and JX analyzed experimental results and wrote the manuscript. All authors read and approved the final manuscript.
The work was financially supported by the National Undergraduate Training Program of China for Innovation and Entrepreneurship (201411842002) and the Natural Science Foundation of Zhejiang Province of China (LY13B070010).
The authors declare that they have no competing interests.
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