Mechanistic and conformational studies on the interaction of sulfamethazine with human immunoglobulin G by molecular modeling and multi‐spectroscopic approach in vitro

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ ? HIgG complex. Fluorescence spectroscopy, ultraviolet–visible (UV–vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?19.12 kJ · mol^?1 and 20.22 J · mol^?1 · K^?1, respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ ? HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three‐dimensional fluorescence spectroscopy, UV–vis absorption spectroscopy and CD spectroscopy. Copyright © 2014 John Wiley & Sons, Ltd.     

Multi-spectroscopic studies on the interaction between traditional Chinese herb,helicid with pepsin

Study on the binding properties of helicid by pepsin systematically using multi-spectroscopic techniques and molecular docking method, and these interactions comprise biological recognition at molecular level and backbone of biological significance in medicine concerned with the uses, effects, and modes of action of drugs. We investigated the mechanism of interaction between helicid and pepsin by using various spectroscopic techniques viz., fluorescence spectra, UV–Vis absorption spectra, circular dichroism (CD), 3D spectra, synchronous fluorescence spectra and molecular docking methods. The quenching mechanism associated with the helicid–pepsin interaction was determined by performing fluorescence measurements at different temperatures. From the experimental results show that helicid quenched the fluorescence intensity of pepsin via a combination of static and dynamic quenching process. The binding constants (K_a) at three temperatures (288, 298, and 308 K) were 7.940?×?10⁷, 2.082?×?10⁵ and 3.199?×?10⁵ L mol^?1, respectively, and the number of binding sites (n) were 1.44, 1.14, and 1.18, respectively. The n value is close to unity, which means that there is only one independent class of binding site on pepsin for helicid. Thermodynamic parameters at 298 K were calculated as follows: ΔH^o (??83.85 kJ mol^?1), ΔG^o (??33.279 kJ mol^?1), and ΔS^o (??169.72 J K^?1 mol^?1). Based on thermodynamic analysis, the interaction of helicid with pepsin is driven by enthalpy, and Van der Waals’ forces and hydrogen bonds are the main forces between helicid and pepsin. A molecular docking study further confirmed the binding mode obtained by the experimental studies. The conformational changes in the structure of pepsin was confirmed by 3D fluorescence spectra and circular dichroism.     

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (K_a), ΔH, ΔS and ΔG values to be 15.5×10⁴M^?1, ?116.2±18.1 Kcal mol^?1, ?366 cal mol^?1K^?1 and ?7.13 Kcal mol^?1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.     

Interaction of cyproheptadine hydrochloride with human serum albumin using spectroscopy and molecular modeling methods

The interaction between cyproheptadine hydrochloride (CYP) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT‐IR) and molecular modeling at a physiological pH (7.40). Fluorescence of HSA was quenched remarkably by CYP and the quenching mechanism was considered as static quenching since it formed a complex. The association constants K_a and number of binding sites n were calculated at different temperatures. According to Förster's theory of non‐radiation energy transfer, the distance r between donor (human serum albumin) and acceptor (cyproheptadine hydrochloride) was obtained. The effect of common ions on the binding constant was also investigated. The effect of CYP on the conformation of HSA was analyzed using FT‐IR, synchronous fluorescence spectroscopy and 3D fluorescence spectra. The thermodynamic parameters ΔH and ΔS were calculated to be ?14.37 kJ mol^?1 and 38.03 J mol^?1 K^?1, respectively, which suggested that hydrophobic forces played a major role in stabilizing the HSA‐CYP complex. In addition, examination of molecular modeling indicated that CYP could bind to site I of HSA and that hydrophobic interaction was the major acting force, which was in agreement with binding mode studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Spectroscopic Studies on the Interaction of Fluorine Containing Triazole with Bovine Serum Albumin

The binding of one fluorine including triazole (C₁₀H₉FN₄S, FTZ) to bovine serum albumin (BSA) was studied by spectroscopic techniques including fluorescence spectroscopy, UV–Vis absorption, and circular dichroism (CD) spectroscopy under simulative physiological conditions. Fluorescence data revealed that the fluorescence quenching of BSA by FTZ was the result of forming a complex of BSA–FTZ, and the binding constants (K _a) at three different temperatures (298, 304, and 310 K) were 1.516?×?10⁴, 1.627?×?10⁴, and 1.711?×?10⁴?mol L^?1, respectively, according to the modified Stern–Volmer equation. The thermodynamic parameters ΔH and ΔS were estimated to be 7.752 kJ mol^?1 and 125.217 J?mol^?1?K^?1, respectively, indicating that hydrophobic interaction played a major role in stabilizing the BSA–FTZ complex. It was observed that site I was the main binding site for FTZ to BSA from the competitive experiments. The distance r between donor (BSA) and acceptor (FTZ) was calculated to be 7.42 nm based on the Förster theory of non-radioactive energy transfer. Furthermore, the analysis of fluorescence data and CD data revealed that the conformation of BSA changed upon the interaction with FTZ.     

Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations

Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 10⁴ L·mol^?1. The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine–DNA. Copyright © 2015 John Wiley & Sons, Ltd.     

Binding of Paeonol to Human Serum Albumin: A Hybrid Spectroscopic Approach and Conformational Study

The purpose of this study was to elucidate the binding of paeonol to human serum albumin (HSA) through spectroscopic methods. The fluorescence quenching of HSA by paeonol was a result of the formation of the HSA–paeonol complex with low binding affinity (K = 4.45 × 10³ M^?1 at 298 K). Thermodynamic parameters (ΔG^○ = –2.08 × 10⁴ J·mol^?1, ΔS^○ = 77.9 J·mol^?1·K^?1, ΔH^○ = 2.41 × 10³ J·mol^?1, k_q = 9.67 × 10¹² M^?1·s^?1) revealed that paeonol mainly binds HSA through hydrophobic force following a static quenching mode. The binding distance was estimated to be 1.91 nm by fluorescence resonant energy transfer. The conformation of HSA was changed and aggregates were formed in the presence of paeonol, revealed by synchronous fluorescence, circular dichroism, Fourier transform infrared spectroscopy, three‐dimensional fluorescence spectroscopy, and resonance light scattering results.     

A multitechnique approach to probe the interaction of a therapeutic tyrosine kinase inhibitor nintedanib and bovine serum albumin

Drug and protein interaction provides a structural guideline in the rational drug designing and in the synthesis of new and improved drugs with greater efficacy. We have examined here the interaction tendency and mechanism of nintedanib (NTB), an anticancer drug (tyrosine kinase inhibitor) with bovine serum albumin (BSA), by spectroscopic techniques. The decline in Stern–Volmer quenching constants and binding constant with the temperature rise suggests that BSA forms a complex with NTB. Binding constant obtained by modified Stern–Volmer equation at 3 temperatures was realized to be of the order of ~10⁴?M^?1. Negative ΔG (~?5.93?kcal?mol^?1), ΔH (?3.74?kcal?mol^?1), and ΔS (?1.50?kcal?mol^?1) values exhibited a spontaneous and exothermic reaction between BSA and NTB. NTB molecule interacts with BSA by forming hydrogen bonds, as elucidated by fluorescence results. Moreover, a minor increment in the helical conformation of BSA upon its binding to NTB was observed by circular dichroism spectroscopy. The modification in protein’s symmetry and a decline in hydrodynamic radii were observed in the presence of NTB (from ~3.6 to ~3?nm) as obtained by the dynamic light scattering measurement results.     

Spectroscopic Investigation of the Interaction Between Copper (II) 2-oxo-propionic Acid Salicyloyl Hydrazone Complex and Bovine Serum Albumin

The interaction between copper (II) 2-oxo-propionic acid salicyloyl hydrazone (Cu^IIL) and bovine serum albumin (BSA) under physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-Vis absorption, and circular dichroism spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by Cu^IIL was the result of the formation of the BSA–Cu^IIL complex. The apparent binding constants (K _a) between Cu^IIL and BSA at four different temperatures were obtained according to the modified Stern–Volmer equation. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), for the reaction were calculated to be ?80.79 kJ mol^?1 and ?175.48 J mol^?1 K^?1 according to van’t Hoff equation. The results indicated that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. The binding distance (r) between Cu^IIL and the tryptophan residue of BSA was obtained to be 4.1 nm according to Förster’s nonradioactive energy transfer theory. The conformational investigation showed that the application of Cu^IIL increased the hydrophobicity of amino acid residues and decreased the α-helical content of BSA (from 62.71% to 37.31%), which confirmed some microenvironmental and conformational changes of BSA molecules.     

Evaluation of the effect of Tb(IV)-NR complex on herring sperm DNA genetic information by mean of spectroscopic

Abstract

The interaction between Tb(IV)-NR complex and herring sperm DNA in buffer solution of Tris-HCl was investigated with the use of acridine orange(AO) as a spectral probe. The binding modes and other information were provided by the UV–spectrophotometry and fluorescence spectroscopy. The thermodynamic functions expressed that the binding constants of Tb(IV)-NR complex with DNA was K^θ_298.15K = 4.03?×?10⁵?L·mol^?1, K^θ_310.15K =1.30?×?10⁷?L·mol^?1, and the Δ_rGθ m _298.15?K＝?3.20?×?10⁴ J·mol^?1. The scatchard equation suggested that the interaction mode between Tb(IV)-NR complex and herring sperm DNA is electrostatic and weak intercalation bindings. FTIR spectroscopy results also indicate that there is a specific interaction between the Tb(IV)-NR complex and the A and G bases of DNA.     

In vitro study on binding interaction of quinapril with bovine serum albumin (BSA) using multi-spectroscopic and molecular docking methods

The binding interaction between quinapril (QNPL) and bovine serum albumin (BSA) in vitro has been investigated using UV absorption spectroscopy, steady-state fluorescence spectroscopic, synchronous fluorescence spectroscopy, 3D fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and molecular docking methods for obtaining the binding information of QNPL with BSA. The experimental results confirm that the quenching mechanism of the intrinsic fluorescence of BSA induced by QNPL is static quenching based on the decrease in the quenching constants of BSA in the presence of QNPL with the increase in temperature and the quenching rates of BSA larger than 10¹⁰ L mol^?1 s^?1, indicating forming QNPL–BSA complex through the intermolecular binding interaction. The binding constant for the QNPL–BSA complex is in the order of 10⁵ M^?1, indicating there is stronger binding interaction of QNPL with BSA. The analysis of thermodynamic parameters together with molecular docking study reveal that the main binding forces in the binding process of QNPL with BSA are van der Waal’s forces and hydrogen bonding interaction. And, the binding interaction of BSA with QNPL is an enthalpy-driven process. Based on Förster resonance energy transfer, the binding distance between QNPL and BSA is calculated to be 2.76 nm. The results of the competitive binding experiments and molecular docking confirm that QNPL binds to sub-domain IIA (site I) of BSA. It is confirmed there is a slight change in the conformation of BSA after binding QNPL, but BSA still retains its secondary structure α-helicity.     

Hydrogen bonding-assisted interaction between amitriptyline hydrochloride and hemoglobin: spectroscopic and molecular dynamics studies

Herein, we have explored the interaction between amitriptyline hydrochloride (AMT) and hemoglobin (Hb), using steady-state and time-resolved fluorescence spectroscopy, UV–visible spectroscopy, and circular dichroism spectroscopy, in combination with molecular docking and molecular dynamic (MD) simulation methods. The steady-state fluorescence reveals the static quenching mechanism in the interaction system, which was further confirmed by UV–visible and time-resolved fluorescence spectroscopy. The binding constant, number of binding sites, and thermodynamic parameters viz. ΔG, ΔH, ΔS are also considered; result confirms that the binding of the AMT with Hb is a spontaneous process, involving hydrogen bonding and van der Waals interactions with a single binding site, as also confirmed by molecular docking study. Synchronous fluorescence, CD data, and MD simulation results contribute toward understanding the effect of AMT on Hb to interpret the conformational change in Hb upon binding in aqueous solution.     

Synthesis of hydroxyethyl starch 200/0.5-loaded albumin nanoparticles: biocompatibility and interaction mechanism

We aimed to synthesize hydroxyethyl starch (HES) 200/0.5-loaded bovine serum albumin nanoparticles (HBNs) and investigate the compatibility and binding mechanism in simulated physiological environments. Here, to elucidate the morphology, biocompatibility, and formation mechanism of HBNs, techniques such as scanning electron microscopy, haemolysis test, fluorescence, and circular dichroism spectroscopy were applied. The thermodynamic parameters at body temperature (ΔS° = −26.7 J·mol⁻¹·K⁻¹, ΔH° = −3.20 × 10⁴ J·mol⁻¹, and ΔG = −2.35 × 10⁴ J·mol⁻¹) showed a 1:1 binding stoichiometry, which was formed by hydrogen bonds and van der Waals interactions. In addition, the conformational analysis showed that the microenvironment of fluorophores was altered with the adaptational protein secondary structural changes. Energy transfer occurred from the fluorophores to HES with a high possibility. All these results provided accurate and complete primary data for demonstrating the interaction mechanisms of HES with BSA, which helps to understand its pharmaceutical effects in blood.     

Electronic and fluorescent studies on the interaction of DNA and BSA with a new ternary praseodymium complex containing 2,9-dimethyl 1,10-phenanthroline and antibacterial activities testing

In this study, fluorescence emission spectra, UV–vis absorption spectra, ethidium bromide (EB)-competition experiment, and iodide quenching experiment were used for the interaction study of the Fish salmon DNA (FS-DNA) with [Pr(dmp)2Cl3(OH2)] where dmp is 2,9-dimethyl 1,10-phenanthroline. The binding constant and the number of binding sites of the complex with FS-DNA were 6.09?±?0.04 M^?1 and 1.18, respectively. The free energy, enthalpy, and entropy changes (ΔG°, ΔH°, and ΔS°) in the binding process of the Pr(III) complex with FS-DNA were –8.02?kcal mol^?1, +39.44?kcal mol^?1, and +159.56?cal mol^?1 K^?1, respectively. Based on these results, the interaction process between FS-DNA with [Pr(dmp)2Cl3(OH2)] was spontaneous and the main binding interaction force was groove binding mode. Also, Fluorescence and electronic absorption spectroscopy were used in order to evaluate the binding characteristics, stoichiometry, and interaction mode of praseodymium(III) (Pr(III)) complex with bovine serum albumin (BSA). Title complex showed good binding propensity to BSA presenting moderately high Kb values. The fluorescence quenching of BSA by Pr(III) complex has been observed to be the static process. The positive ΔH° and ΔS° values showed that the hydrophobic interaction is the main force in the binding of Pr(III) complex and BSA. Eventually, the average aggregation number, <J>, of BSA potentially induced by title complex confirmed the 1:1 stoichiometry for title complex-BSA adducts. In vitro, antimicrobial activity of title complex was indicated that the complex is more active against both Escherichia coli and Enterococcus faecalis bacterial strains than Staphylococcus aureus, and Pseudomonas aeruginosa.

Communicated by Ramaswamy H. Sarma     

Thermodynamics of the slow–fast transition in bacteriophage T2L

We have measured the thermodynamic parameters of the slow-fast tail-fiber reorientation transition on T2L bateriophage. Proportions of the virus in each form were determined from peak-height measurements in sedimention-velocity runs and from average diffusion coefficients obtained by quasielastic laser light scattering. Computer simulation of sedimentation confirmed that there were no undetected intermediates in the transition, which was analyzed as a two-state process. Van't Hoff-type plots of the apparent equilibrium constant and of the pH midpoint of the transition as function of reciprocal temperature led to the following estimates of the thermodynamic parameters for the transition at pH 6.0 and 20°C: ΔH° = ?139 ± 18Kcal mol^?1, ΔS° = ?247 ± 46 cal K^?1 mol^?1, and ΔG° = ?66 ± 22 kcal mol^?1. Per mole of protons taken up in the transition, the analogous quantities were ?15.9 ± 1.7 kcal mol^?1, ?26.3 ± 2.2 cal K^?1 mol^?1, and ?8.22 ± 1.8 kcal mol^?1. The net number of protons taken up was about 8.5 ± 1.5. The large values of the thermodynamic functions are consistent with a highly cooperative reaction and with multiple interactions between the fibres and the remainder of the phage. The negative entropy of the transition is probably due to immobilization of the fibres.     

Multi-spectroscopic and voltammetric evidences for binding,conformational changes of bovine serum albumin with thiamine

The interaction between thiamine hydrochloride (TA) and bovine serum albumin (BSA) was investigated by fluorescence, FTIR, UV–vis spectroscopic and cyclic voltammetric techniques under optimised physiological condition. The fluorescence intensity of BSA is gradually decreased upon addition of TA due to the formation of a BSA–TA complex. The binding parameters were evaluated and their behaviour at different temperatures was analysed. The quenching constants (K_sv) obtained were 2.6 × 10⁴, 2.2 × 10⁴ and 2.0 × 10⁴ L mol^?1 at 288, 298 and 308 K, respectively. The binding mechanism was static-type quenching. The values of ΔH° and ΔS° were found to be 26.87 kJ mol^?1 and 21.3 J K^?1 mol^?1, and indicated that electrostatic interaction was the principal intermolecular force. The changes in the secondary structure of BSA upon interaction with TA were confirmed by synchronous and 3-D spectral results. Site probe studies reveal that TA is located in site I of BSA. The effects of some common metal ions on binding of BSA–TA complex were also investigated.     

The Interaction of Chromium(VI) with Urease in Solution

The interaction between K₂Cr₂O₇ and urease was investigated using fluorescence, UV-vis absorption, and circular dichroism (CD) spectroscopy. The experimental results showed that the fluorescence quenching of urease by K₂Cr₂O₇ was a result of the formation of K₂Cr₂O₇–urease complex. The apparent binding constant K _A between K₂Cr₂O₇ and urease at 295, 302, and 309 K were obtained to be 2.14?×?10⁴, 1.96?×?10⁴, and 1.92?×?10⁴ L mol^?1, respectively. The thermodynamic parameters, ΔH° and ΔS° were estimated to be ?5.90 kJ mol^?1, 43.67 J mol^?1 K^?1 according to the Van’t Hoff equation. The electrostatic interaction played a major role in stabilizing the complex. The distance r between donor (urease) and acceptor (K₂Cr₂O₇) was 5.08 nm. The effect of K₂Cr₂O₇ on the conformation of urease was analyzed using UV-vis absorption, CD, synchronous fluorescence spectroscopy, and three-dimensional fluorescence spectra, the environment around Trp and Tyr residues were altered.     

Multi-spectroscopic and molecular modelling approach to investigate the interaction of riboflavin with human serum albumin

Riboflavin (RF) plays an important role in various metabolic redox reactions in the form of flavin adenine dinucleotide and flavin mononucleotide. Human serum albumin (HSA) is an important protein involved in the transportation of drugs, hormones, fatty acid and other molecules which determine the biodistribution and physiological fate of these molecules. In this study, we have investigated the interaction of riboflavin RF with HSA under simulative physiological conditions using various biophysical, calorimetric and molecular docking techniques. Results demonstrate the formation of riboflavin–HSA complex with binding constant in the order of 10⁴ M^?1. Fluorescence spectroscopy confirms intermediate strength having a static mode of quenching with stoichiometry of 1:1. Experimental results suggest that the binding site of riboflavin mainly resides in sub-domain IIA of HSA and that ligand interaction increases the α-helical content of HSA. These parameters were further verified by isothermal titration calorimetry ITC which confirms the thermodynamic parameters obtained by fluorescence spectroscopy. Molecular docking was employed to suggest a binding model. Based on thermodynamic, spectroscopic and computational observations it can be concluded that HSA-riboflavin complex is mainly stabilized by various non-covalent forces with binding energy of ?7.2 kcal mol^?1.     

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40–60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10^?7 cm² s^?1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (K_i = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters – ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (?5.78 kcal mol^?1) and positive ΔS (11.01 cal mol^?1 deg^?1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (?9.19 kcal mol^?1) value implies a spontaneous CBC-papain interaction.     

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine and human serum albumin

The binding characteristics of the interaction between 3-(2-cyanoethyl) cytosine (CECT) and human serum albumin (HSA) were investigated using fluorescence, UV absorption spectroscopic and molecular modeling techniques under simulative physiological conditions. The intrinsic fluorescence intensity of HSA was decreased with the addition of CECT. The fluorescence data handled by Stern–Volmer equation proved that the quenching mechanism of the interaction between CECT and HSA was a static quenching procedure. The binding constants evaluated utilizing the Lineweaver–Burk equation at 17, 27 and 37?°C, were 2.340?×?10⁴, 2.093?×?10⁴ and 1.899?×?10⁴?L?mol^?1, respectively. The thermodynamic parameters were calculated according to van’t Hoff equations. Negative enthalpy (ΔH) and positive entropy (ΔS) values indicated that both hydrogen bond and hydrophobic force played a major role in the binding process of CECT to HSA, which was consistent with the results of the molecular modeling study. In addition, the effect of other ions on the binding constant of CECT-HSA was examined.