Evaluation and Minimization of Uncertainty in ITC Binding Measurements: Heat Error,Concentration Error,Saturation, and Stoichiometry

BackgroundIsothermal titration calorimetry (ITC) is uniquely useful for characterizing binding thermodynamics, because it straightforwardly provides both the binding enthalpy and free energy. However, the precision of the results depends on the experimental setup and how thermodynamic results are obtained from the raw data.MethodsExperiments and Monte Carlo analysis are used to study how uncertainties in injection heat and concentration propagate to binding enthalpies in various scenarios. We identify regimes in which it is preferable to fix the stoichiometry parameter, N, and evaluate the reliability of uncertainties provided by the least squares method.ResultsThe noise in the injection heat is mainly proportional in character, with ~ 1% and ~ 3% uncertainty at 27C and 65C, respectively; concentration errors are ~ 1%. Simulations of experiments based on these uncertainties delineate how experimental design and curve fitting methods influence the uncertainty in the final results.ConclusionsIn most cases, experimental uncertainty is minimized by using more injections and by fixing N at its known value. With appropriate technique, the uncertainty in measured binding enthalpies can be kept below ~ 2% under many conditions, including low C values.General SignificanceWe quantify uncertainties in ITC data due to heat and concentration error, and identify practices to minimize these uncertainties. The resulting guidelines are important when ITC data are used quantitatively, such as to test computer simulations of binding. Reproducibility and further study are supported by free distribution of the new software developed here.     

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.     

Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1–3 with model membranes investigated by isothermal titration calorimetry

Human-β-defensins HBD-1–3 are important components of the innate immune system. Synthetic peptides Phd-1–3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1–3, have antimicrobial activity. The interaction of Phd-1–3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25 °C and 37 °C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1–3 at 37 °C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH ~ ? 1.3 to ? 2.8 kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy–Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1–3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1–3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.     

The thermodynamics of protein interactions with essential first row transition metals

BackgroundThe binding of metal ions to proteins is a crucial process required for their catalytic activity, structural stability and/or functional regulation. Isothermal titration calorimetry provides a wealth of fundamental information which when combined with structural data allow for a much deeper understanding of the underlying molecular mechanism.Scope of reviewA rigorous understanding of any molecular interaction requires in part an in-depth quantification of its thermodynamic properties. Here, we provide an overview of recent studies that have used ITC to quantify the interaction of essential first row transition metals with relevant proteins and highlight major findings from these thermodynamic studies.General significanceThe thermodynamic characterization of metal ion–protein interactions is one important step to understanding the role that metal ions play in living systems. Such characterization has important implications not only to elucidating proteins' structure-function relationships and biological properties but also in the biotechnology sector, medicine and drug design particularly since a number of metal ions are involved in several neurodegenerative diseases.Major conclusionsIsothermal titration calorimetry measurements can provide complete thermodynamic profiles of any molecular interaction through the simultaneous determination of the reaction binding stoichiometry, binding affinity as well as the enthalpic and entropic contributions to the free energy change thus enabling a more in-depth understanding of the nature of these interactions. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Study of inclusion complexes of cycloamylose with surfactants by isothermal titration calorimetry

Useful materials can be made from cycloamylose (CA) and the functional properties of CA could be improved by complexation with surfactants. Isothermal titration calorimetry (ITC) was used to investigate interactions between CA and surfactants in buffered solutions. Three surfactants with C₁₂ non-polar tail groups and charged [anionic: sodium dodecyl sulfate (SDS); cationic: dodecyl trimethylammonium bromide (DTAB)] or non-charged headgroups [non-ionic: polyoxyethylene 23 lauryl ether (Brij35)] were used in this study. The effects of temperature, pH, and salt concentration were also studied. All three surfactants bound to CA; however, Brij35 binding to CA was negligible. Enthalpy changes associated with binding of surfactants to CA were exothermic except for interactions measured at 50 °C. There was no effect of pH on surfactant demicellization or CA binding. Salt concentration affected surfactant demicellization, but the amount of SDS bound to CA at saturation was unaffected by salt. When the titration curves obtained for CA with SDS and DTAB were fitted, it could be analyzed using a model based on a single set of identical sites.     

Microtubule assembly-derived by dimerization of TPPP/p25. Evaluation of thermodynamic parameters for multiple equilibrium system from ITC data

BackgroundThe disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system. In this paper the role of dimerization in its microtubule-related functions is established, and an approach is proposed to evaluate thermodynamic constants for multiple equilibrium systems from ITC measurements.MethodsFor structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay. Numerical simulation of the multiple equilibrium was performed with Mathematica software.ResultsThe dimerization of TPPP/p25 is promoted by elevation of the protein concentration and by GTP addition. The dimeric form displaying enhanced tubulin polymerization promoting activity is stabilized by disulfide bond or chemical cross-linking. The GTP binding to the dimeric form (K_d-GTP = 200 μM) is tighter with one order of magnitude than to the monomeric one leading to the enrichment of the dimers. A mathematical model elaborated for the multiple equilibrium of the TPPP/p25-GTP system was validated by fitting the GTP-dependent changes of ellipticity and fluorescence signal in the course of TPPP/p25 titrations. The evaluation of the equilibrium constants rendered it possible to determine the thermodynamic parameters of the association of different TPPP/p25 forms with GTP from ITC measurements.Conclusions/General SignificanceThe dimerization of TPPP/p25 with favorable physiological functional potency is proposed to play significant role in the fine tuning of TPPP/p25-mediated microtubule assembly; the unfolded monomers might be involved in the formation of pathological inclusions characteristic for Parkinson's disease and other synucleinopathies.     

Calorimetric and spectroscopic investigations of the binding of metallated porphyrins to G-quadruplex DNA

BackgroundThe human telomere contains tandem repeat of (TTAGG) capable of forming a higher order DNA structure known as G-quadruplex. Porphyrin molecules such as TMPyP4 bind and stabilize G-quadruplex structure.MethodsIsothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectroscopy (ESI/MS), were used to investigate the interactions between TMPyP4 and the Co(III), Ni(II), Cu(II), and Zn(II) complexes of TMPyP4 (e.g. Co(III)-TMPyP4) and a model human telomere G-quadruplex (hTel22) at or near physiologic ionic strength ([Na⁺] or [K⁺] ≈ 0.15 M).ResultsThe apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 all formed complexes having a saturation stoichiometry of 4:1, moles of ligand per mole of DNA. Binding of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 is described by a “four-independent-sites model”. The two highest-affinity sites exhibit a K in the range of 10⁸ to 10¹⁰ M^− 1 with the two lower-affinity sites exhibiting a K in the range of 10⁴ to 10⁵ M^− 1. Binding of Co(III)-TMPyP4, and Zn(II)-TMPyP4, is best described by a “two-independent-sites model” in which only the end-stacking binding mode is observed with a K in the range of 10⁴ to 10⁵ M^− 1.ConclusionsIn the case of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4, the thermodynamic signatures for the two binding modes are consistent with an “end stacking” mechanism for the higher affinity binding mode and an “intercalation” mechanism for the lower affinity binding mode. In the case of Co(III)-TMPyP4 and Zn(II)-TMPyP4, both the lower affinity for the “end-stacking” mode and the loss of the intercalative mode for forming the 2:1 complexes with hTel22 are attributed to the preferred metal coordination geometry and the presence of axial ligands.General significanceThe preferred coordination geometry around the metal center strongly influences the energetics of the interactions between the metallated-TMPyP4 and the model human telomeric G-quadruplex. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

5-Aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of Hsp90 chaperone

A series of 5-aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles were synthesized and their binding to several constructs of human Hsp90 chaperone measured by isothermal titration calorimetry (ITC). The most potent compound bound Hsp90 with the dissociation constant of about 5 nM.     

Biomolecule–nanoparticle interactions: Elucidation of the thermodynamics by isothermal titration calorimetry

BackgroundNanomaterials (NMs) are often exposed to a broad range of biomolecules of different abundances. Biomolecule sorption driven by various interfacial forces determines the surface structure and composition of NMs, subsequently governs their functionality and the reactivity of the adsorbed biomolecules. Isothermal titration calorimetry (ITC) is a nondestructive technique that quantifies thermodynamic parameters through in-situ measurement of the heat absorption or release associated with an interaction.Scope of reviewThis review highlights the recent applications of ITC in understanding the thermodynamics of interactions between various nanoparticles (NPs) and biomolecules. Different aspects of a typical ITC experiment that are crucial for obtaining accurate and meaningful data, as well as the strengths, weaknesses, and challenges of ITC applications to NP research were discussed.Major conclusionsITC reveals the driving forces behind biomolecule–NP interactions and the effects of the physicochemical properties of both NPs and biomolecules by quantifying the crucial thermodynamics parameters (e.g., binding stoichiometry, ΔH, ΔS, and ΔG). Complimentary techniques would strengthen the interpretation of ITC results for a more holistic understanding of biomolecule–NP interactions.General significanceThe thermodynamic information revealed by ITC and its complimentary characterizations is important for understanding biomolecule–NP interactions that are fundamental to the biomedical and environmental applications of NMs and their toxicological effects. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Comparative serum albumin interactions and antitumor effects of Au(III) and Ga(III) ions

In the present study, interactions of Au(III) and Ga(III) ions on human serum albumin (HSA) were studied comparatively via spectroscopic and thermal analysis methods: UV–vis absorbance spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and isothermal titration calorimetry (ITC). The potential antitumor effects of these ions were studied on MCF-7 cells via Alamar blue assay. It was found that both Au(III) and Ga(III) ions can interact with HSA, however; Au(III) ions interact with HSA more favorably and with a higher affinity. FT-IR second derivative analysis results demonstrated that, high concentrations of both metal ions led to a considerable decrease in the α-helix content of HSA; while Au(III) led to around 5% of decrease in the α-helix content at 200 μM, it was around 1% for Ga(III) at the same concentration. Calorimetric analysis gave the binding kinetics of metal–HSA interactions; while the binding affinity (K_a) of Au(III)–HSA binding was around 3.87 × 10⁵ M⁻¹, it was around 9.68 × 10³ M⁻¹ for Ga(III)–HSA binding. Spectroscopy studies overall suggest that both metal ions have significant effects on the chemical structure of HSA, including the secondary structure alterations. Antitumor activity studies on MCF7 tumor cell line with both metal ions revealed that, Au(III) ions have a higher antiproliferative activity compared to Ga(III) ions.     

An easy-to-use tool for planning and modeling a calorimetric titration

Isothermal titration calorimetry (ITC) is widely employed to measure thermodynamic properties of binding interactions between two macromolecules or a macromolecule and a small ligand. No labeling of interacting species is required for ITC, but this advantage is offset by potentially material-consuming experimental optimization complicated by an indirect readout of an ITC titration. Here we present a simple, practical, and portable spreadsheet-based tool for planning and modeling an ITC titration experiment accompanied by basic guidelines.     

Application of ITC in foods: A powerful tool for understanding the gastrointestinal fate of lipophilic compounds

BackgroundIsothermal titration calorimetry (ITC) is a biophysical technique widely used to study molecular interactions in biological and non-biological systems. It can provide important information about molecular interactions (such as binding constant, number of binding sites, free energy, enthalpy, and entropy) simply by measuring the heat absorbed or released during an interaction between two liquid solutions.Scope of the reviewIn this review, we present an overview of ITC applications in food science, with particular focus on understanding the fate of lipids within the human gastrointestinal tract. In this area, ITC can be used to study micellization of bile salts, inclusion complex formation, the interaction of surface-active molecules with proteins, carbohydrates and lipids, and the interactions of lipid droplets.Major conclusionsITC is an extremely powerful tool for measuring molecular interactions in food systems, and can provide valuable information about many types of interactions involving food components such as proteins, carbohydrates, lipids, surfactants, and minerals. For systems at equilibrium, ITC can provide fundamental thermodynamic parameters that can be used to establish the physiochemical origin of molecular interactions.General significanceIt is expected that ITC will continue to be utilized as a means of providing fundamental information about complex materials such as those found in foods. This knowledge may be used to create functional foods designed to behave in the gastrointestinal tract in a manner that will improve human health and well-being. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX

BackgroundHuman carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding.MethodsThe observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry.ResultsThe pK_a of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was − 24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM.ConclusionsThe intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship.General significanceIt is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations.     

Microwave pretreatment of lignocellulosic material in cholinium ionic liquid for efficient enzymatic saccharification

We demonstrated that the enzymatic hydrolysis of cellulose after microwave pretreatment of lignocellulosic material in ionic liquids (ILs) is drastically enhanced compared with that after conventional thermal pretreatment in ILs. Three types of cholinium ILs, choline formate (ChFor), choline acetate (ChOAc), and choline propionate (ChPro), were examined. The cellulose saccharification percentage was approximately 20% for kenaf powders pretreated in ChFor, ChOAc, and ChPro by conventional heating at 110 °C for 20 min. In contrast, approximately 60–90% of cellulose was hydrolyzed to glucose after microwave pretreatment in the same ILs at 110 °C for 20 min.     

Isothermal titration calorimetry for drug design: Precision of the enthalpy and binding constant measurements and comparison of the instruments

Isothermal titration calorimetry (ITC) is one of the most robust label- and immobilization-free techniques used to measure protein – small molecule interactions in drug design for the simultaneous determination of the binding affinity (ΔG) and the enthalpy (ΔH), both of which are important parameters for structure-thermodynamics correlations. It is important to evaluate the precision of the method and of various ITC instrument models by performing a single well-characterized reaction. The binding between carbonic anhydrase II and acetazolamide was measured by four ITC instruments – PEAQ-ITC, iTC200, VP-ITC, and MCS-ITC and the standard deviation of ΔG and ΔH was determined. Furthermore, the limit of an approach to reduce the protein concentration was studied for a high-affinity reaction (K_d = 0.3 nM), too tight to be measured by direct (non-displacement) ITC. Chemical validation of the enthalpy measurements is discussed.     

Benzenesulfonamides with pyrimidine moiety as inhibitors of human carbonic anhydrases I,II, VI,VII, XII,and XIII

Two groups of benzenesulfonamide derivatives, bearing pyrimidine moieties, were designed and synthesized as inhibitors of carbonic anhydrases (CA). Their binding affinities to six recombinant human CA isoforms I, II, VI, VII, XII, and XIII were determined by the thermal shift assay (TSA). The binding of several inhibitors was measured by isothermal titration calorimetry (ITC). Direct demonstration of compound inhibition was achieved by determining the inhibition constant by stopped-flow CO₂ hydration assay. The most potent compounds demonstrated selectivity towards isoform I and affinities of 0.5 nM. The crystal structures of selected compounds in complex with CA II, XII, and XIII were determined to atomic resolution. Compounds described here were compared with previously published pyrimidinebenzenesulfonamides.¹ Systematic structure–activity analysis of 40 compound interactions with six isoforms yields clues for the design of compounds with greater affinities and selectivities towards target CA isoforms.     

Isothermal titration calorimetry for chiral chemistry

One of the most powerful techniques that are currently available to measure thermodynamic parameters such as enthalpy (ΔH), Gibbs free energy (ΔG), entropy changes (ΔS), and binding affinity in chemical reactions is isothermal titration calorimetry (ITC). Recent advances in instrumentation have facilitated the development of ITC as a very essential analytical tool in biology and chemistry. In this article, we will focus on a review of the literature on the application of ITC for the study of chiral systems and chiral interactions. We present studies in which the ITC technique is used to study chiral interactions, for instance in chiral solutions, chiral organometallic complexes, guest‐host chiral binding interactions, and biological macromolecules. Finally, we put strong emphasis on the most recent application of ITC for the study of chirality in nanosystems and at the nanoscale.     

Thermodynamics of substrate binding to the metal site in homoprotocatechuate 2,3-dioxygenase: Using ITC under anaerobic conditions to study enzyme–substrate interactions

BackgroundExtradiol dioxygenases are a family of nonheme iron (and sometimes manganese) enzymes that catalyze an O₂-dependent ring-opening reaction in a biodegradation pathway of aromatic compounds. Here we characterize the thermodynamics of two substrates binding in homoprotocatechuate 2,3-dioxygenase (HPCD) prior to the O₂ activation step.MethodsThis study uses microcalorimetry under an inert atmosphere to measure thermodynamic parameters associated with catechol binding to nonheme metal centers in HPCD. Several stopped-flow rapid mixing experiments were used to support the calorimetry experiments.ResultsThe equilibria constant for 4-nitrocatechol and homoprotocatechuate binding to the iron(II) and manganese(II) forms of HPCD range from 2 × 10⁴ to 1 × 10⁶, suggesting there are distinctive differences in how the enzyme–substrate complexes are stabilized. Further experiments in multiple buffers allowed us to correct the experimental ΔH for substrate ionization and to fully derive the pH and buffer independent thermodynamic parameters for substrate binding to HPCD. Fewer protons are released from the iron(II) dependent processes than their manganese(II) counterparts.ConclusionsCondition independent thermodynamic parameters for 4-nitrocatechol and homoprotocatechuate binding to HPCD are highly consistent with each other, suggesting these enzyme–substrate complexes are more similar than once thought, and the ionization state of metal coordinated waters may be playing a role in tuning redox potential and in governing reactivity.General significanceSubstrate binding to HPCD is a complex set of equilibria that includes ionization of substrate and water release, yet it is also the key step in O₂ activation. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies

Isothermal titration calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equilibrium constants and thermodynamics of molecular binding events. Thus, important information about the interaction of metal ions with biological macromolecules can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodynamics of metal-protein interactions.     

Association analysis of single nucleotide polymorphisms of proinflammatory cytokine and their receptors genes with rheumatoid arthritis in northwest Chinese Han population

ObjectiveTo analyze the relationship of genetic polymorphisms in IL1β, IL6, TNF-α genes and their receptors genes with rheumatoid arthritis (RA) for northwest Han Chinese. This study also explores whether there are gene–gene interactions among these genetic polymorphisms.MethodsA total of 452 patients with RA and 373 matched healthy controls were enrolled to carry out a case-control study for 16 SNPs of IL1B-511 C > T, IL1B-31 T > C, IL1B+3954 C > T, IL1RN T > C, IL6-597 G > A, IL6-572 G > C, IL6-174 G > C, IL6R-183 G > A, IL6R exon2 T > A, IL6R exon1 A > C, TNFA-863 C > A, TNFA-857 C > T, TNFA-308 G > A, TNFA-238 G > A, TNFR1-383 A > C and TNFR2 T676G T > G from seven genes. Genotyping for the SNPs was conducted on the RotorGene 6000 PCR platform using in-house high resolution melting (HRM) approaches. Detection correctness was validated through direct sequencing. Generalized multifactor dimensionality reduction (GMDR) analysis was applied to discover likely gene–gene interaction model among the SNPs.ResultsThe results showed that the genotype distributions of TNFA-308, TNFA-857 and TNFA-863 are significantly different between case and control groups (P = 0.016, P = 0.048 and P = 0.016, respectively). Carriers of TNFA-857 mutant allele conferred risk to RA (OR = 1.525, 95% CI = 1.157–2.009) while those of TNFA-308 and TNFA-863 mutant alleles conferred protection to RA (OR = 0.459, 95% CI = 0.286–0.739; OR = 0.490, 95% CI = 0.329–0.732). GMDR analysis for the SNPs indicated that gene–gene interaction existed among IL1B-31, IL1RN, IL6-572, IL6R-183, IL6R-exon1 and TNFA-857. Thirteen of all genotypes of the six SNPs combination were discovered to have significant distribution difference between RA group and the control.ConclusionsThis study demonstrated that PCR-HRM assay is a highly efficient SNP genotyping method especially for the detection of large-scale samples. The SNPs of TNFA-308 and TNFA-863 are closely associated with RA susceptibility and that gene–gene interactions may occur among the six SNPs of IL1B-31, IL1RN, IL6-572, IL6R-183, IL6R-exon1 and TNFA-857 in RA patients from northwest Chinese Han population, especially these SNPs’ combination genotypes CT/TT/CC/GG/AC/CC, CT/TT/GC/AA/AC/CT and CT/CT/CC/GA/AC/CC to show high risk of RA susceptibility in our study.