Antibodies Use Heme as a Cofactor to Extend Their Pathogen Elimination Activity and to Acquire New Effector Functions
2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês
10.1074/jbc.m702751200
ISSN1083-351X
AutoresJordan D. Dimitrov, Lubka T. Roumenina, Virjinia Doltchinkova, Nikolina Mihaylova, Sébastien Lacroix‐Desmazes, Srini V. Kaveri, Tchavdar L. Vassilev,
Tópico(s)Neonatal Health and Biochemistry
ResumoVarious pathological processes are accompanied by release of high amounts of free heme into the circulation. We demonstrated by kinetic, thermodynamic, and spectroscopic analyses that antibodies have an intrinsic ability to bind heme. This binding resulted in a decrease in the conformational freedom of the antibody paratopes and in a change in the nature of the noncovalent forces responsible for the antigen binding. The antibodies use the molecular imprint of the heme molecule to interact with an enlarged panel of structurally unrelated epitopes. Upon heme binding, monoclonal as well as pooled immunoglobulin G gained an ability to interact with previously unrecognized bacterial antigens and intact bacteria. IgG-heme complexes had an enhanced ability to trigger complement-mediated bacterial killing. It was also shown that heme, bound to immunoglobulins, acted as a cofactor in redox reactions. The potentiation of the antibacterial activity of IgG after contact with heme may represent a novel and inducible innate-type defense mechanism against invading pathogens. Various pathological processes are accompanied by release of high amounts of free heme into the circulation. We demonstrated by kinetic, thermodynamic, and spectroscopic analyses that antibodies have an intrinsic ability to bind heme. This binding resulted in a decrease in the conformational freedom of the antibody paratopes and in a change in the nature of the noncovalent forces responsible for the antigen binding. The antibodies use the molecular imprint of the heme molecule to interact with an enlarged panel of structurally unrelated epitopes. Upon heme binding, monoclonal as well as pooled immunoglobulin G gained an ability to interact with previously unrecognized bacterial antigens and intact bacteria. IgG-heme complexes had an enhanced ability to trigger complement-mediated bacterial killing. It was also shown that heme, bound to immunoglobulins, acted as a cofactor in redox reactions. The potentiation of the antibacterial activity of IgG after contact with heme may represent a novel and inducible innate-type defense mechanism against invading pathogens. Heme is a pivotal molecule for prokaryote and eukaryote organisms. As a prosthetic group of different proteins, it performs versatile biological functions, e.g. oxygen transport and storage, oxygen activation, and electron transport. The characteristics that make heme a key component of the aerobic metabolism (easy acceptance or donation of electrons by the iron ion) also make it inherently dangerous. Heme is redox-active and when liberated from hemoproteins may induce or aggravate the oxidative stress (1Darley-Usmar V. Halliwell B. Pharm. Res. (N. Y.). 1996; 13: 649-662Crossref PubMed Scopus (298) Google Scholar, 2Kumar S. Bandyopadhyay U. Toxicol. Lett. 2005; 157: 175-188Crossref PubMed Scopus (618) Google Scholar). 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Chem. 1983; 258: 12069-12072Abstract Full Text PDF PubMed Google Scholar). Free heme is also implicated in the catalytic nitration of tyrosine residues of proteins in a number of pathological conditions (14Bian K. Gao Z. Weisbrodt N. Murad F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5712-5717Crossref PubMed Scopus (182) Google Scholar, 15Thomas D.D. Espey M.G. Vitek M.P. Miranda K.M. Wink D.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12691-12696Crossref PubMed Scopus (179) Google Scholar). In addition to its pro-oxidative potential, it has been found that free heme also possesses potent pro-inflammatory activity. In vivo it induces increase in the vascular permeability, an increase in the expression of the adhesion molecules on the endothelial cells, and leukocyte recruitment to the organs (6Wagener F.A. Volk H.D. Willis D. Abraham N.G. Soares M.P. Adema G.J. Figdor C.G. Pharmacol. Rev. 2003; 55: 551-571Crossref PubMed Scopus (474) Google Scholar, 16Wagener F.A. Eggert A. 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In addition, heme possesses potent mitogenic activity for human peripheral blood mononuclear cells and increases the expression of the pro-inflammatory cytokines tumor necrosis factor-α and interferon-γ from these cells (19Stenzel K.H. Rubin A.L. Novogrodsky A. J. Immunol. 1981; 127: 2469-2473PubMed Google Scholar, 20Novogrodsky A. Suthanthiran M. Stenzel K.H. J. Immunol. 1989; 143: 3981-3987PubMed Google Scholar). Because of its pro-oxidative and pro-inflammatory properties, free heme is implicated in the pathogenesis of many disease conditions such as acute renal failure after hemolysis, tissue injury induced by ischemia/reperfusion, as well as chronic inflammation that could be observed in some hemolytic diseases (6Wagener F.A. Volk H.D. Willis D. Abraham N.G. Soares M.P. Adema G.J. Figdor C.G. Pharmacol. Rev. 2003; 55: 551-571Crossref PubMed Scopus (474) Google Scholar, 7Balla J. Vercellotti G.M. Nath K. Yachie A. Nagy E. Eaton J.W. Balla G. Nephrol. Dial. Transplant. 2003; 5: 8-12Crossref Google Scholar, 21Tracz M.J. Alam J. Nath K.A. J. Am. Soc. Nephrol. 2007; 18: 414-420Crossref PubMed Scopus (249) Google Scholar). Immunoglobulins are the main serum glycoproteins responsible for detection and destruction of pathogens or their products. In disease conditions accompanied by the release of large amounts of heme from hemoproteins Igs could be exposed to high concentrations of this molecule. This may influence their biological functions. Indeed, it has been observed recently that the in vitro exposure of polyclonal antibodies from healthy individuals to heme resulted in the appearance of new strong reactivities toward various autoantigens (22McIntyre J.A. Thromb. Res. 2004; 114: 579-587Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23McIntyre J.A. Wagenknecht D.R. Faulk W.P. J. Autoimmun. 2005; 24: 311-317Crossref PubMed Scopus (62) Google Scholar, 24McIntyre J.A. Wagenknecht D.R. Faulk W.P. Autoimmun. Rev. 2006; 5: 76-83Crossref PubMed Scopus (58) Google Scholar, 25Dimitrov J.D. Roumenina L.T. Doltchinkova V.R. Vassilev T.L. Scand. J. Immunol. 2007; 65: 230-239Crossref PubMed Scopus (29) Google Scholar). The molecular mechanisms responsible for the effects on heme on the antigen binding activity of Igs are not understood. It is also not clear what is the biological significance of the interaction of the heme with the Igs. In an attempt to address these questions and because of the importance of such studies for understanding the pathophysiological mechanisms of diseases accompanied by release of heme, this study was undertaken. Our results demonstrated that an exposure of antibodies to heme resulted in a substantial increase in their binding to bacterial antigens. Kinetic, thermodynamic, and spectroscopic analyses revealed that antibodies have an intrinsic property to bind heme and to use it for acquiring promiscuous antigen binding. It was also found that this caused more efficient pathogen elimination by complement. The heme-mediated broadening of the antibody repertoire may provide an inducible innate-like defense against invading pathogens. However, it may also contribute to the pathogenesis of inflammation and autoimmunity. Immunoglobulin Preparations—As sources of polyclonal IgG (pIgG) 4The abbreviations used are:pIgGnormal human polyclonal IgGROSreactive oxygen speciesICindigo carmineELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineCFUcolony-forming unit22′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) human therapeutic polyclonal intravenous immunoglobulin preparations Intraglobin F (Biotest AG, Dreieich, Germany) and Sandoglobulin (Novartis, Pharmaceuticals, NJ) were used. Preparation containing Fc fragments from polyclonal human IgG was a kind gift from Dr. Marianne Debré (Hospital Necker, Paris, France) (26Debre M. Bonnet M.C. Fridman W.H. Carosella E. Philippe N. Reinert P. Vilmer E. Kaplan C. Teillaud J.L. Griscelli C. Lancet. 1993; 342: 945-949Abstract PubMed Scopus (302) Google Scholar). F(ab′)2 fragments from polyclonal IgG were obtained by pepsin hydrolysis and chromatography on protein G-Sepharose (Amersham Biosciences). Z2 hybridoma that produced a mouse IgG2b antibody with specificity for mouse IgG2a (27Fazekas G. Rajnavolgyi E. Kurucz I. Sintar E. Kiss K. Laszlo G. Gergely J. Eur. J. Immunol. 1990; 20: 2719-2729Crossref PubMed Scopus (25) Google Scholar) and the IP2-11-1 hybridoma producing a mouse IgG2a were kindly provided by Dr. Eva Rajnavolgyi (University of Lorand Eötvös, Budapest, Hungary). The growth of hybridomas and purification of antibodies were performed as described in Ref. 28Dimitrov J.D. Ivanovska N.D. Lacroix-Desmazes S. Doltchinkova V.R. Kaveri S.V. Vassilev T.L. J. Biol. Chem. 2006; 281: 439-446Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar. All chemicals, except where indicated, were from Sigma. Exposure of immunoglobulins to hematin is described in the supplemental Experimental Procedures. normal human polyclonal IgG reactive oxygen species indigo carmine enzyme-linked immunosorbent assay phosphate-buffered saline colony-forming unit 2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) Evaluation of Immunoreactivities of Native and Hematin-exposed IgG—The ability of native and hematin-exposed IgG to bind to bacterial antigens or myosin was assessed by immunoblot analyses or by ELISA as described previously for ferrous ion-treated IgG (28Dimitrov J.D. Ivanovska N.D. Lacroix-Desmazes S. Doltchinkova V.R. Kaveri S.V. Vassilev T.L. J. Biol. Chem. 2006; 281: 439-446Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The binding to intact bacteria was evaluated as in Ref. 29Wentworth Jr., P. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Crossref PubMed Scopus (304) Google Scholar. More details for the procedures are given in the supplemental Experimental Procedures. Surface Plasmon Resonance Analysis—The kinetic constants of the interactions between native or hematin-exposed monoclonal antibodies were determined by using a surface plasmon resonance BIAcore 2000 (Biacore AB, Uppsala, Sweden). Purified mouse IgG2a (clone IP2-11-1) was immobilized on research grade CM5 sensor chip (Biacore) using amino-coupling kit as described by the manufacturer (Biacore). After that, the monoclonal IgG2a antibody solutions in 5 mm maleate, pH 4, with final concentrations of 25, 10, or 5 μg/ml, were injected with a flow rate of 10 μl/min and a contact time of 7 min on the surfaces of three independent flow cells. All the experiments were performed using HEPES-buffered saline (0.01 m HEPES, pH 7.4, containing 0.15 m NaCl, 3 mm EDTA, and 0.005% polysorbate) as running buffer and sample dilution buffer. All solutions were filtered through 0.22-μm filters and degassed under vacuum. Concentrations ranging from 37.5 to 600 nm of the native or 5 μm hematin-exposed Z2 antibody were injected on the immobilized monoclonal IgG2a with flow rate of 10 μl/min. Association phase was monitored for 10 min and then the chip surfaces were exposed to running buffer for 10 min to monitor the dissociation phase. For the regeneration of the chip surface, a3 m solution of potassium thiocyanate (Sigma) was used. All kinetic measurements were performed at 5, 10, 15, 20, 25, and 30 °C. The binding to the surface of the control flow cell was always subtracted from the binding to the antigen-coated cells. The details of surface plasmon resonance measurements of the interactions of 77IP52H7 antibody with FVIII are given in the supplemental Experimental Procedures. BIAevaluation version 4.1 software (Biacore) was used for the calculation of the kinetic rate constants of association and of dissociation. Analysis was performed by global analysis of the experimental data using the kinetic models, included in the software, fitting the data with lowest value of χ2. The values of the equilibrium dissociation constant were determined as the result of the ratio between the kinetic rate constants (KD = koff/kon). The evaluation of the thermodynamic parameters of the interactions of Z2 antibody was described under the supplemental Experimental Procedures. Determination of the Salt Concentration Dependence of the Kinetic Rate Constants—The salt concentration dependence of the kinetic rate constants of native and of hematin-exposed Z2 were determined by using BIAcore. For this, the kinetic measurements were performed consecutively in variants of phosphate buffer (5 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.4, and 0.005% Tween 20) differing by the NaCl concentrations(0, 50, 100, 200, 400, or 800 mm). The native and hematin-exposed Z2 antibodies were diluted in various buffers to concentrations ranging from 18.75 to 300 nm and injected on the immobilized monoclonal IgG2a with a flow rate of 20 μl/min. Association times of 5 min and dissociation times of 10 min were monitored. For the regeneration of the chip surface, a 3 m solution of KSCN was used. All measurements were performed at 25 °C. UV-visible Absorption Spectroscopy—UV-visible spectroscopic assays for the binding of heme to polyclonal IgG were taken in PBS, pH 7.4, or in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl with Ultraspec 1000 spectrophotometer (Amersham Biosciences). A stock solution (2.5 mm) of hematin in 0.1 m NaOH was added to the buffer containing IgG (0, 0.125, 0.25, 0.5, or 1 μm) to a final concentration of 2 μm. After hematin addition, the samples were homogenized and allowed to stand for 5 min at room temperature. The absorption spectra of the solutions were then recorded in wavelength range from 350 to 650 nm. In some cases potassium cyanide or imidazole were added (from 25 mm stock solutions) to buffers containing hematin or IgG-hematin. The absorption spectra of IgG alone in the range 350–650 nm were also recorded. It was found not to be significantly different from the background absorbance of the buffer alone. For titration of binding of heme to IgG, aliquots from a hematin stock solution were added to 1 μm solution of IgG and to reference cuvette containing buffer only. After each addition, samples in two cuvettes were homogenized and incubated for 5 min in dark at 25 °C. Then difference absorption spectra were recorded. The IgG heme binding curve was build by plotting the difference in absorbance (Aheme-IgG – Aheme) at 390 nm versus molar concentrations of heme. For determination of heme binding to Fc-γ or to F(ab′)2 fragments from human pIgG, a stock solution (2.5 mm) of hematin was added to the PBS containing 50 μm F(ab′)2 fragments or 100 μm Fc-γ fragments to a final concentration of 20 μm. Assessment of Peroxidase Activity—For measurement of the peroxidase-like activity of the complexes of hematin with pIgG, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used as the chromogenic substrate. ABTS was added to 0.1 m phosphate citrate buffer, pH 5, to a final concentration of 1 mm. Samples under study were added to the reaction buffer at quantities resulting in final hematin concentrations of 0.2 or 2 μm. The hydrogen peroxide to a concentration of 6 mm was then added. The peroxidase activities were followed by an increase in absorbance at 414 nm. The measurements were performed in a 1-cm cuvette at 20-s time intervals for at least 600 s. For preparation of the samples, hematin from 2.5 mm stock solution in 0.1 m NaOH was added to PBS alone or to PBS containing pIgG (50 or 100 μm), Fc-γ (50 μm), or F(ab′)2 (50 μm) to final concentrations 10 or 50 μm. In some cases, 10 mm NaN3 was also added. After incubation for 30 min at 25 °C, samples were tested for their peroxidase activity as described above. Oxidation of Indigo Carmine by a Metal-catalyzed Oxidation System—Fe(II) ions (from ferrous sulfate) and EDTA were both added in PBS (150 mm NaCl, 5 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.4) to final concentrations of 100 μm. The reaction buffer also contained 300 μm indigo carmine. Also added to the reaction buffer were pIgG to a final concentration of 10 μm, hematin to a final concentration of 1 μm, or pIgG preincubated with hematin (100 μm pIgG preincubated with 10 μm for 1 h at 25 °C) to final concentrations of 10 μm pIgG and 1 μm hematin. The Fenton reaction was started by addition of hydrogen peroxide to concentration of 1 mm. After 10 min of incubation at 25 °C, the absorption at 610 nm was measured. In some cases reaction buffer contained 200 units/ml CuZn-superoxide dismutase from bovine erythrocytes or 40 units/ml crystalline bovine liver catalase. The redox activity of the pIgG-hematin complex preincubated with 1000-fold molar excess (in relation to hematin) of KCN or NaN3 was evaluated by using the same assay. Bactericidal Activity by Hydrogen Peroxide—In a typical experiment, a culture of ampicillin-resistant Escherichia coli (HB101 strain) (in log phase growth, A600 = 0.2–0.3) was repeatedly pelleted (three times at 3000 rpm) and resuspended in PBS, pH 7.4. The PBS suspended cells were then added to sterile Eppendorf tubes. Pooled human IgG (final concentration 100 μm) was pretreated with hematin at final concentrations of 0, 1, 5, 10, 25, 50, and 100 μm. Control preparations of hematin with the same final concentration but without IgG were also prepared. After 1 h, four series of E. coli were incubated as follows: with IgG (10 μm final concentration), pretreated with increasing hematin concentrations; IgG, pretreated with increasing hematin concentrations in the presence of 0.5 mm H2O2 (this concentration was estimated to cause 50% killing); PBS with increasing hematin concentrations (0–10 μm); PBS with increasing hematin concentrations (0–10 μm) in the presence of 0.5 mm H2O2 for 1 h at 37 °C. Viability of bacteria was assessed by recovery of colony-forming units (CFUs) on agar plates after overnight incubation at 37 °C. Each experiment was performed at least in duplicate. Data are given as percent of the viability of E. coli without any treatment. Bacterial Killing by Complement—E. coli was incubated for 1 h at 37 °C with hematin (0, 10, 25, 50, and 100 μm)-pretreated pIgG. Subsequently, guinea pig complement (1:100, 1:40, or 1:20 dilutions, or PBS) was added, and again bacteria were incubated for 1 h at 37 °C. Viability was determined by recovery of CFUs on agar plates after 12 h of incubation at 37 °C. Each experiment was performed in at least duplicate. As a control, the experiment was performed with two different preparations of pIgG, and the results were comparable. Data are given as percent of the viability of E. coli without any treatment. Exposure of Polyclonal IgG to Heme Resulted in Increased Antibody Binding to Foreign Antigens and to Myosin—We first addressed the question whether the heme exposure could influence the ability of antibodies to recognize foreign antigens. Normal human pIgG was briefly incubated in the presence of increasing concentrations of hematin, and its ability to interact with bacterial proteins was then evaluated by an immunoblot technique. A significant hematin dose-dependent increase in the binding of pIgG to these antigens was observed (Fig. 1A). Moreover, we tested the ability of hematin-exposed pIgG to bind to intact E. coli cells, immobilized on a solid matrix. A considerable increase in the binding of heme-exposed pIgG to bacteria was detected (Fig. 1B). Interestingly, the appearance of new binding specificities of pIgG for E. coli antigens was detected after exposure to concentrations of heme that were lower than those reached in vivo under certain pathological conditions (3Muller-Eberhard U. Javid J. Liem H.H. Hanstein A. Hanna M. Blood. 1968; 32: 811-815Crossref PubMed Google Scholar). The binding of secondary antibodies to native or heme-exposed pIgG, immobilized on ELISA plates, did not differ significantly (data not shown). This observation ruled out the possibility that heme treatment of IgG results in enhanced recognition by secondary antibodies. Furthermore, we studied the interaction of heme-exposed pIgG with myosin. A significant increase in the binding of pIgG to this self-protein was detected after exposure to heme (Fig. 1D). Antibodies from native pIgG interacted only negligibly with myosin, whereas the pIgG exposure to even 1 μm hematin resulted in an ∼2-fold increase in the binding. In addition, by using label-free real time interaction measurements, a significant increase in the binding of heme-exposed pIgG or of heme-exposed F(ab′)2 fragments (obtained from pIgG) to human complement factor H was observed (data not shown). To rule out possibility that increased antigen binding activity of IgG is caused by heme-induced IgG multimer formation, size-exclusion chromatography was performed. The exposure of pIgG to hematin, at concentrations that considerably influence antigen binding behavior (2, 10, 20, and 40 μm), did not result in a significant increase in the proportion of IgG dimers or multimers (supplemental Fig. S1A). Moreover, the reactivity of the monomer fraction isolated from hematin-exposed IgG with human factor VIII was considerably higher than that of the monomer fraction from native pIgG (Fig. S1B), as seen in the case of unfractionated IgG (Fig. 1). Kinetic and Thermodynamic Analyses of the Interactions of Heme-exposed IgG—To elucidate the molecular mechanisms responsible for the effects of heme on the antigen-binding properties of antibodies, kinetic and thermodynamic analyses were performed. The characteristics of the interactions of the mouse monoclonal IgG2b antibody Z2 to its cognate antigenmouse IgG2a were deciphered by surface plasmon resonance. The exposure of Z2 to hematin resulted in the appearance of new antigen binding specificities (Fig. 1C). This allows us to use Z2 as a suitable model system for studies on the biophysical mechanisms of heme-induced changes in antigen binding behavior of antibodies. Comparison of the interaction profiles of the native with those of hematin-exposed Z2 antibody revealed a qualitative difference in the interactions with the same antigen between the two forms of the antibody (supplemental Fig. S2A). In contrast, the exposure to hematin of monoclonal antibody 77IP52H7 resulted only in small difference in the interaction profiles (supplemental Fig. S2B). These data clearly indicate that heme does not influence the antigen-binding properties of all antibodies and rule out nonspecific effects of heme on immunoglobulin molecules. The studies of the interactions of Z2 allowed us to determine the binding affinity for the target antigen. The value of the equilibrium dissociation constant (KD) measured at 25 °C was 140 nm (±12, n = 3) for the native antibody. The binding affinity of Z2 was increased more than twice after exposure to hematin (to KD value of 56 nm (±10, n = 3)). We then evaluated the effect of temperature on the KD. For the native form of Z2, the correlation between the interaction temperatures and the change in the KD values was not significant (Fig. 2A). In contrast, the values of KD characterizing the binding of the heme-exposed antibody showed significant dependence on the change in the temperature. Interestingly, with increasing temperatures from 5 to 25 °C, 2-fold elevation of the antigen binding affinity of the antibody was observed (Fig. 2A). The value of the equilibrium dissociation constant depends on the values of the kinetic rate constants that describe the dissociation and the association phases of the interaction process. The comparison of the kinetic rate constants of the native and of heme-exposed Z2, measured at 25 °C, revealed that the increased antigen binding affinity after contact with heme was because of both an increase of the association rate constant (kon) and a decrease of dissociation rate constant (koff). Thus, the value of kon for native antibody was 3.60 × 104 m–1 s–1 (±0.24 × 104, n = 3), whereas it was 5.50 × 104 m–1 s–1 (±0.026 × 104, n = 3) for the heme-exposed antibody. The koff values of 5.10 × 10–3 s–1 (±0.126 × 10–3, n = 3) and of 3.11 × 10–3 s–1 (±0.0156 × 10–3, n = 3), respectively, were measured. Furthermore, the temperature dependences of the kinetic rate constants were evaluated. On Fig. 2B Arrhenius plots depict the temperature dependences of the kon and of koff constants, characterizing the interaction of the native and heme-exposed Z2. The rate of association for both forms of the antibody was weakly dependent on temperature change. However, although an increase of the temperature resulted in a decrease of the rate of association of native Z2, a reverse temperature dependence of kon was observed for heme-exposed Z2 (Fig. 2B). It is interesting to note that the rates of association at low temperatures (10 and 15 °C) were slightly slower for heme-exposed Z2 than those for the native one. At higher temperatures the opposite tendency was seen. In contrast to the results for kon, the temperature sensitivity of the dissociation kinetic rate constant was similar for the native and heme-treated antibodies (Fig. 2B). Moreover, at all studied temperatures, the values of koff of the hematin-treated antibody were lower than those of native one. The kinetic measurements performed on surfaces with different quantities of the target antigen or by using different flow rates gave similar values of the kinetic parameters. This rules out a skewing of the kinetic data by effects of the mass transport or by other rebinding artifacts. The activation energies derived from the Arrhenius plots were used to evaluate the changes in the thermodynamic parameters that characterize association, dissociation, and equilibrium phases of the interactions of the native and of hematin-exposed Z2 (Fig. 2C). The change in the entropy term (TΔS) for the association step was with a negative value for the interactions of both forms of the antibody. However, considerable differences were observed in the extent of the change of this parameter. Thus, the unfavorable TΔS of –75.90 ± 1.10 kJ mol–1 for the association of native Z2 was significantly attenuated to –36.34 ± 2.23 kJ mol–1 in the case of the hematin-exposed antibody (Fig. 2C). Interestingly, the change in the enthalpy (ΔH) differed qualitatively between both forms of Z2. Although the association ΔH for the native antibody was favorable with a value of –28.96 ± 0.4 kJ mol–1, the change of the same parameter for the heme-treated antibody was unfavorable with a value of 9.58 ± 0.6 kJ mol–1. No significant difference in the change of the Gibbs free energy (ΔG) of association was found between two forms of the Z2. During the dissociation phase, neither of the thermodynamic parameters differed between native and heme-exposed antibody. The value of the changes in enthalpy at equilibrium was favorable for the native antibody and highly unfavorable for the heme-exposed one (–9.66 ± 1.45 and 27.62 ± 4.46 kJ mol–1, respectively). A favorable TΔS at equilibrium was observed to be characteristic for the interactions of both forms of the antibody (TΔS = 36.84 ± 5.60 kJ mol–1, in the case of native antibody, and TΔS = 68.96 ± 11.14 kJ mol–1, in the case of heme-exposed one). This result indicates that the binding of IgG2a by Z2 is essentially an entropy-driven process. No significa
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