RESUMO
Spectroscopic studies combined with computational analysis indicate the inherent conformational flexibility of the ß-carbolin derivative evodiamine (EVD) featured with diverse pharmacological activities. Qualitative evaluation of the circular dichroism (CD) spectra of EVD enantiomers complemented with quantum chemical calculations reveals a chiral exciton signature that can be assigned to the folded, L-shaped conformation of the molecule. Changes of the exciton couplet measured in different solvents and the near-UV CD profile upon binding to human serum albumin (HSA) refer to the structural adaptability of EVD. The enantioselectivity of EVD-HSA interaction is demonstrated showing the binding preference of the (R)-enantiomer. Comparison of experimental and calculated CD spectra of various conformers of EVD as well as the results of molecular docking data suggest that the (R)-antipode is accomodated within the IIA subdomain of HSA in ridge-tile conformation. Rutaecarpine (RTC), the close congener of EVD, forms much tighter association complexes both with HSA and α1-acid glycoprotein. In contrast to EVD, the nearly planar geometry of the indoloquinazoline ring system of RTC allows its stacked dimeric binding to the HSA.
Assuntos
Alcaloides Indólicos/química , Quinazolinas/química , Albumina Sérica/química , Sítios de Ligação , Dicroísmo Circular , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Espectrofotometria Ultravioleta , EstereoisomerismoRESUMO
Harmane and norharmane are representative members of the large group of natural ß-carboline alkaloids featured with diverse pharmacological activities. In blood, these agents are transported by human serum albumin (HSA) which has a profound impact on the pharmacokinetic and pharmacodynamic properties of many therapeutic drugs and xenobiotics. By combination of various spectroscopic methods, the present contribution is aimed to elucidate how nonesterified fatty acids (FAs), the primary endogenous ligands of HSA, affect the binding properties of harmane and norharmane. Analysis of induced circular dichroism (CD) and fluorescence spectroscopic data indicates the inclusion of the neutral form of both molecules into the binding pocket of subdomain IIIA, which hosts two FA binding sites, too. The induced CD and UV absorption spectra of harmane and norharmane exhibit peculiar changes upon addition of FAs, suggesting the formation of ternary complexes in which the lipid ligands significantly alter the binding mode of the alkaloids via cooperative allosteric mechanism. To our knowledge, it is the first instance of the demonstration of drug-FA cobinding at site IIIA. In line with these results, molecular docking calculations showed two distinct binding positions of norharmane within subdomain IIIA. The profound increase in the affinity constants of ß-carbolines estimated in the presence of FAs predicts that the unbound, pharmacologically active serum fraction of these compounds strongly depends on the actual lipid binding profile of HSA.
Assuntos
Alcaloides/química , Carbolinas/química , Ácidos Graxos/química , Harmina/análogos & derivados , Albumina Sérica/química , Sítios de Ligação , Harmina/química , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Ligação ProteicaRESUMO
We report for the first time on neuronal signaling for the evaluation of interactions between native plasmamembrane and polyamidoamine (PAMAM) dendrimers. Generation 5 polycationic (G5-NH(2)), novel ß-D-glucopyranose-conjugated G5-NH(2) and generation 4.5 polyanionic (G4.5-COONa) polyamidoamine (PAMAM) dendrimers (1-0.0001 mg/ml) were applied in acute brain slices. Functional toxicity assessments-validated by fluorescence imaging of dead cells-were performed by employing electrophysiological indicators of plasma membrane breakdown and synaptic transmission relapse. Irreversible membrane depolarization and decrease of membrane resistance predicted substantial functional neurotoxicity of unmodified G5-NH(2), but not of the G4.5-COONa PAMAM dendrimers. Model calculations suggested that freely moving protonated NH(2) groups of terminal monomeric units of PAMAM dendrimers may be able directly destroy the membrane or inhibit important K(+) channel function via contacting the positively charged NH(2). In accordance, conjugation of surface amino groups by ß-D-glucopyranose units reduced functional neurotoxicity that may hold great potential for biomedical applications.
Assuntos
Dendrímeros/toxicidade , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Transmissão Sináptica/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dendrímeros/química , Corantes Fluorescentes/análise , Glucose/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Simulação de Dinâmica Molecular , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Neurotoxinas/química , Ressonância Magnética Nuclear Biomolecular , Ratos , Ratos WistarRESUMO
High-affinity human serum albumin (HSA) binding of the C3-substituted antimalarial 2-hydroxy-1,4-naphthoquinone derivative atovaquone (ATQ) has been demonstrated and studied by circular dichroism (CD), UV/VIS absorption, fluorescence spectroscopy and affinity chromatography methods. The analysis of induced CD data generated upon HSA binding of ATQ revealed two high-affinity binding sites (K(a) ≈ 2 × 10(6) M(-1)). CD interaction studies and displacement of specific fluorescent and radioactive marker ligands indicated the contribution of both principal drug binding sites of HSA to complexation of ATQ, and also suggested the possibility of simultaneous binding of ATQ and some other drugs (e.g. warfarin, phenylbutazone, diazepam). Comparison of UV/VIS spectra of ATQ measured in aqueous solutions indicated the prevalence of the anionic species formed by dissociation of the 2-hydroxyl group. HSA binding of related natural hydroxynaphthoquinones, lapachol and lawsone also induces similar CD spectra. The much weaker binding affinity of lawsone (K(a) ≈ 10(4) M(-1)) bearing no C3 substituent highlights the importance of hydrophobic interactions in the strong HSA binding of ATQ and lapachol. Since neither drug exhibited significant binding to serum α(1)-acid glycoprotein, HSA must be the principal plasma protein for the binding and transportation of 2-hydroxy-1,4-naphthoquinone-type compounds which are ionized at physiological pH values.
Assuntos
Antimaláricos/química , Antimaláricos/metabolismo , Atovaquona/análogos & derivados , Atovaquona/metabolismo , Albumina Sérica/metabolismo , Cromatografia de Afinidade , Dicroísmo Circular , Humanos , Modelos Moleculares , Ligação Proteica , Albumina Sérica/química , Espectrometria de Fluorescência , EspectrofotometriaRESUMO
BACKGROUND: alpha(1)-Acid glycoprotein (AGP) plays a decisive role in the serum protein binding of several drugs.Genetic variants of AGP have different ligand binding properties. The binding of deramciclane (DER), a chiral anxiolytic agent, has been studied on A and F1/S genetic variants of AGP. METHODS: The effects of DER and reference drugs on the binding of specific fluorescent and circular dichroism (CD) probes of AGP were determined. Dicumarol (DIC) binding was measured by CD and equilibrium dialysis. RESULTS: DER effectively displaced probes bound to variant A, while it was less effective at displacing probes bound to variant F1/S. DER increased the binding and inverted the induced CD spectrum of DIC in the solution of variant F1/S. This phenomenon could not be brought about by the enantiomer of DER. CONCLUSION: DER has high-affinity binding (K(a)> or =2x10(6) M(-1)) to variant A, while its binding to the variant F1/S is about thirty times weaker. During simultaneous binding of DER and DIC to variant F1/S a ternary complex having about four times higher affinity is formed, in which the opposite chiral conformation of DIC is favored. GENERAL SIGNIFICANCE: The binding interactions found prove that AGP can simultaneously accommodate different ligand molecules. Even weakly bound ligands can provoke unexpected allosteric protein binding interactions.
Assuntos
Ansiolíticos/metabolismo , Canfanos/metabolismo , Variação Genética , Orosomucoide/genética , Regulação Alostérica , Substituição de Aminoácidos , Ansiolíticos/química , Ligação Competitiva , Canfanos/química , Dicroísmo Circular , Humanos , Cinética , Modelos Moleculares , Orosomucoide/metabolismo , Ligação Proteica , Albumina Sérica/metabolismo , Espectrometria de FluorescênciaRESUMO
There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.
Assuntos
Marketing , Orosomucoide/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Albumina Sérica/metabolismo , Animais , Humanos , Orosomucoide/química , Ligação Proteica , Inibidores de Proteínas Quinases/química , Albumina Sérica/químicaRESUMO
Human alpha(1)-acid glycoprotein (AAG) is an acute phase component of the plasma, binding numerous drugs and natural compounds with high-affinity. Using circular dichroism (CD) spectroscopy, strong AAG binding of organogold complexes was found, the molecular size and chemical structure of which differ from known AAG binding agents. The 16-membered Au(2)P(4)C(8)O(2) macrocycles interconvert rapidly between two helical forms and produce enantiomeric conformations which are in dynamic equilibrium in solution. AAG binds preferentially one of the chiral conformers as indicated by strong Cotton effects generated by intramolecular exciton coupling between the pairs of hetercyclic chromophores. Lipophilic nature of the guest molecules suggests the dominant contribution of hydrophobic interactions in the AAG binding. Comparison of the main genetic variants of AAG revealed that both the 'F1/S' and 'A' variants bind with high-affinity the gold(I) macrocycles (K(a) approximately 10(6) M(-1)). CD/fluorescence displacement, and fluorescence quenching experiments indicated inclusion of the compounds into the central beta-barrel cavity of AAG of which exact tertiary structure is yet unknown. Molecular dimensions of the gold(I) macrocycles (13 x 14 x 14 A) indicate that the principal ligand binding cavity of both the 'F1/S' and 'A' variants must be larger compared to the models published to date. Based on these findings, a novel homology model of AAG 'F1' variant was constructed using the human neutrophil gelatinase-associated lipocalin as a template. The organogold complexes were successfully docked into the central cavity of this model.
Assuntos
Sondas Moleculares , Compostos Organoáuricos/química , Orosomucoide/química , Dicroísmo Circular , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Espectrofotometria UltravioletaRESUMO
Human plasma protein binding of six antimalarial agents of quinoline and acridine types was investigated by using spectroscopic techniques, affinity chromatography, ultrafiltration and HPLC methods. Induced circular dichroism (ICD) spectra showed binding of amodiaquine (AMQ), primaquine (PRQ), tafenoquine (TFQ), and quinacrine (QR) to alpha(1)-acid glycoprotein (AAG), the serum level of which greatly increases in Plasmodium infections. Association constant (K(a)) values of about 10(5)-10(6) M(-1) could be determined. Analysis of the ICD and UV spectra of the drug-AAG complexes suggested the inclusion of the ligands into the central hydrophobic cavity of the protein. Using the purified forms of the two main genetic variants of AAG, ICD data indicated the selective binding of AMQ and PRQ to the 'F1/S', while QR to the 'A' variant. Results of fluorescence experiments supported the AAG binding of these drugs and provided further insights into the binding details of TFQ and QR. Fluorescence and CD displacement experiments showed the high-affinity AAG binding of mefloquine (K(a) approximately 10(6) M(-1)). For this drug, inverse binding stereoselectivities were found with the 'F1/S' and 'A' genetic variants of AAG. HSA association constants estimated from affinity chromatography results lag behind (10(3)-10(5) M(-1)) the similar values derived for AAG. In case of chloroquine, no significant binding interaction was found either with AAG or HSA. Pharmacological aspects of the results are discussed.
Assuntos
Antimaláricos/síntese química , Orosomucoide/química , Antimaláricos/química , Sítios de Ligação , Cromatografia de Afinidade , Dicroísmo Circular , Humanos , Estrutura Molecular , Ligação Proteica , Albumina Sérica/química , Espectrofotometria , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Diazepam, a 1,4-benzodiazepine lacking chiral centre, exists in an equimolar mixture of two chiral conformers. Induced circular dichroism spectra for the binding of diazepam and its 3,3-dimethyl substituted analogues to alpha1-acid glycoprotein (AGP) revealed that opposite to human serum albumin, AGP preferably binds the P-conformers. Accordingly, slightly favoured binding of (R)-enantiomers of 3-alkyl derivatives having P-conformation was found. In case of 3-acyloxy derivatives, however, AGP preferably binds the (S)-enantiomers. Studies with the separated genetic variants of AGP proved similar binding affinities, but markedly different conformation selectivities. For diazepam bound by the F1-S variant, a P/M selectivity of about 2 could be estimated.
Assuntos
Diazepam/análogos & derivados , Diazepam/química , Orosomucoide/química , Orosomucoide/metabolismo , Dicroísmo Circular , Variação Genética/genética , Humanos , Conformação Molecular , Orosomucoide/genética , Ligação Proteica , Albumina Sérica/químicaRESUMO
Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to alpha1-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of Ka=1.7(+/-0.2)x10(6)M(-1) and n=0.94 could be determined for the binding on the F1-S variant at 37 degrees . Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (nKa approximately 3 x 10(4)M(-1)) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP.
Assuntos
Orosomucoide/efeitos dos fármacos , Orosomucoide/genética , Piperazinas/química , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Laranja de Acridina/química , Laranja de Acridina/metabolismo , Benzamidas , Sítios de Ligação , Dicroísmo Circular , Humanos , Mesilato de Imatinib , Imipramina/química , Imipramina/metabolismo , Naftalenossulfonatos/química , Naftalenossulfonatos/metabolismo , Orosomucoide/química , Piperazinas/metabolismo , Polimorfismo Genético , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Pirimidinas/metabolismo , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
Coumarin-type anticoagulants, warfarin, phenprocoumon and acenocoumarol, were tested for their stereoselective binding to the human orosomucoid (ORM; AGP) genetic variants ORM 1 and ORM 2. Direct binding studies with racemic ligands were carried out by the ultrafiltration method; the concentrations of free enantiomers were determined by capillary electrophoresis. The binding of pure enantiomers was investigated with quinaldine red fluorescence displacement measurements. Our results demonstrated that all investigated compounds bind stronger to ORM 1 variant than to ORM 2. ORM 1 and human native AGP preferred the binding of (S)-enantiomers of warfarin and acenocoumarol, while no enantioselectivity was observed in phenprocoumon binding. Acenocoumarol possessed the highest enantioselectivity in AGP binding due to the weak binding of its (R)-enantiomer. Furthermore, a new homology model of AGP was built and the models of ORM 1 and ORM 2 suggested that difference in binding to AGP genetic variants is caused by steric factors.
Assuntos
Cumarínicos/química , Cumarínicos/metabolismo , Orosomucoide/metabolismo , Acenocumarol/química , Acenocumarol/metabolismo , Anticoagulantes/química , Anticoagulantes/metabolismo , Variação Genética , Humanos , Ligantes , Modelos Moleculares , Mutação , Orosomucoide/genética , Femprocumona/química , Femprocumona/metabolismo , Ligação Proteica/genética , Quinaldinas/química , Estereoisomerismo , Varfarina/química , Varfarina/metabolismoRESUMO
The effects of (R)- and (S)-ibuprofen on the binding of benzodiazepines to human serum albumin (HSA) were examined by biointeraction chromatography. The displacement of benzodiazepines from HSA by (R)- and (S)-ibuprofen was found to involve negative allosteric interactions (or possible direct competition) for most (R)-benzodiazepines. However, (S)-benzodiazepines gave positive or negative allosteric effects and direct competition when displaced by (R)- or (S)-ibuprofen. Association equilibrium constants and coupling constants measured for these effects indicated that they involved two classes of ibuprofen binding regions (i.e., low- and high-affinity sites). Based on these results, a model was proposed to explain the binding of benzodiazepines to HSA and their interactions with ibuprofen. This model gave good agreement with previous reports examining the binding of benzodiazepines to HSA.
Assuntos
Benzodiazepinas/química , Ibuprofeno/química , Albumina Sérica/metabolismo , Regulação Alostérica , Cromatografia de Afinidade , Humanos , Cinética , Lorazepam/análogos & derivados , Lorazepam/química , Modelos Químicos , Oxazepam/análogos & derivados , Oxazepam/química , Albumina Sérica/química , TermodinâmicaRESUMO
The tetrahydrochloride salt of astaxanthin di-L-lysinate (lys(2)AST) is a highly water-dispersible astaxanthin-amino acid conjugate, with an aqueous dispersibility of > or = 181.6 mg/mL. The statistical mixture of stereoisomers has been well characterized as an aqueous-phase superoxide anion scavenger, effective at micromolar (microM) concentrations. In the current study, the aqueous aggregation behavior and in vitro plasma protein binding [with fatty-acid-free human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP)] were investigated with a suite of techniques, including circular dichroism (CD) and UV-vis spectroscopy, ultrafiltration, competitive ligand displacement, and fluorescence quenching. Induced CD bands obtained in Ringer buffer solution of HSA demonstrated high affinity monomeric binding of the compound at low ligand per protein (L/P) ratios (in aqueous solution alone the carotenoid molecules formed card-pack aggregates). The binding constant ( approximately 10(6)M(-1)) and the binding stoichiometry (approximately 0.2 per albumin molecule) were calculated from CD titration data. CD displacement and ultrafiltration experiments performed with marker ligands of HSA indicated that the ligand binding occurred at a site distinct from the main drug binding sites of HSA (i.e., Sites I and II). At intermediate L/P ratios, both monomeric and aggregated ("chirally complexed") binding occurred simultaneously at distinct sites of the protein. At high L/P ratios, chiral complexation predominantly occurred on the asymmetric protein template. The tentative location of the chirally-complexed aggregation on the HSA template was identified as the large interdomain cleft of HSA, where carotenoid derivatives have been found to bind previously. Only weak binding to AGP was observed. These results suggest that parenteral use of this highly potent, water-dispersible astaxanthin-amino acid conjugate will result in plasma protein association, and plasma protein binding at sites unlikely to displace fatty acids and drugs bound at well-characterized binding sites on the albumin molecule.
Assuntos
Dipeptídeos/farmacologia , Orosomucoide/química , Albumina Sérica/química , beta Caroteno/análogos & derivados , beta Caroteno/farmacologia , Soluções Tampão , Dicroísmo Circular , Dipeptídeos/síntese química , Humanos , Ligantes , Ligação Proteica , Soluções , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Estereoisomerismo , Água , Xantofilas , beta Caroteno/síntese químicaRESUMO
Human alpha1-acid glycoprotein displays genetic polymorphism. Different drug binding properties of the two main genetic products (F1-S and A variants) have been demonstrated. In search for specific circular dichroism (CD) probes, dicumarol and acridine orange were found to specifically bind to the F1-S and A variants, respectively. Dicumarol binding to the F1-S variant produced induced Cotton effects originating from the favored chiral conformation of the bound label. Acridine orange gave induced biphasic Cotton effects due to chiral intermolecular exciton interaction between label molecules bound to the A variant. Displacement of the CD probes by specific marker ligands was demonstrated. The induced CD spectrum of dicumarol was found to change sign in the presence of imipramine, as a manifestation of high-affinity ternary complex formation on the F1-S variant.
Assuntos
Dicroísmo Circular/métodos , Variação Genética , Orosomucoide/metabolismo , Laranja de Acridina/farmacologia , Sítios de Ligação , Dicumarol/farmacologia , Corantes Fluorescentes/farmacologia , Humanos , Ligantes , Orosomucoide/química , Orosomucoide/genética , Relação Estrutura-Atividade , Desacopladores/farmacologiaRESUMO
Pharmacological and pharmacodynamic properties of biologically active natural and synthetic compounds are crucially determined via their binding to proteins of the human body. Several spectroscopic techniques are available to study these mainly non-covalent interactions. Circular dichroism (CD) spectroscopy, being sensitive to the chirality of ligand molecules induced by the asymmetric protein environment, has widely and successfully been applied for many decades. Chiral conformation of the ligand due to conformational adaptation to its binding site, or interaction between ligand molecules held in chiral arrangement relative to each other by the protein sites, results in one or more induced CD bands with different shape, sign and intensity. These extrinsic Cotton effects present in light absorbing region of the optically active or inactive ligand molecules give qualitative and quantitative information of the binding process. It can provide valuable data on the stereochemistry, number, location and nature of the binding sites. This paper is aimed to survey briefly the literature and the results of recent investigations undertaken in this field.
Assuntos
Dicroísmo Circular , Ligação Proteica , Proteínas/química , Animais , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Conformação ProteicaRESUMO
The binding of bimoclomol enantiomers to human plasma, its components, as well as to plasma from monkey, dog, rat, and mouse was investigated by ultrafiltration and equilibrium dialysis. The considerably stronger binding of the (-)-(S)-enantiomer found in human plasma is due to the alpha(1)-acid glycoprotein (AAG) component. The binding parameters for AAG (n(R)K(R) = 1.3 x 10(4) M(-1) and n(S)K(S) = 1.0 x 10(5) M(-1)) revealed high enantioselectivity, while the binding to human serum albumin was found to be weak (nK = 5 x 10(3) M(-1)) and not stereoselective. (-)-(S)-Bimoclomol was extensively displaced in the presence of specific marker ligands for the "FIS" subfraction of human AAG. Comparative binding studies indicated considerable differences between plasma of the five species investigated.
Assuntos
Proteínas Sanguíneas/metabolismo , Imidas/metabolismo , Piridinas/metabolismo , Animais , Cães , Humanos , Macaca mulatta , Masculino , Camundongos , Orosomucoide/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Albumina Sérica/metabolismo , Especificidade da Espécie , EstereoisomerismoRESUMO
Kinetic and equilibrium binding studies were performed on the interaction of warfarin enantiomers with human serum albumin (HSA) in the absence and presence of lorazepam acetate (LoAc) enantiomers. Binding kinetics were followed by recording changes in the fluorescence of warfarin upon binding to HSA using the stopped-flow technique. The binding of (R)-warfarin displayed an exponentially increasing fluorescence, satisfying the two-step mechanism reported previously for the racemate, i.e., a diffusion controlled pre-equilibrium is followed by a slower rearrangement of the complex. In the case of (S)-warfarin, the signal was biphasic: a fast fluorescence enhancement was followed by a slow decline. The different kinetic features indicate that the equilibrium conformations of the [(S)-warfarin-HSA] and [(R)-warfarin-HSA] complexes are achieved via different mechanisms. The phenomenon was seen in buffers of different pH and compositions. Equilibrium binding measurements indicated significantly lower molar intrinsic fluorescence for the (S)-warfarin complex, suggesting differences in the microenvironments of the bound enantiomers. In the presence of (S)-LoAc, the allosterically enhanced binding of (S)-warfarin manifested itself in accelerated relaxation kinetics. In accordance with the low molar intrinsic fluorescence determined for the stable ternary complex, the amplitude of the decline in fluorescence became larger.