Stereoselective disposition of tiaprofenic acid enantiomers in rats

The pharmacokinetics and metabolic chiral inversion of the S(+)‐ and R(−)‐enantiomers of tiaprofenic acid (S‐TIA, R‐TIA) were assessed in vivo in rats, and in addition the biochemistry of inversion was investigated in vitro in rat liver homogenates. Drug enantiomer concentrations in plasma were investigated following administration of S‐TIA and R‐TIA (i.p. 3 and 9 mg/kg) over 24 hr. Plasma concentrations of TIA enantiomers were determined by stereospecific HPLC analysis. After administration of R‐TIA it was found that 1) there was a time delay of peak S‐TIA plasma concentrations, 2) S‐TIA concentrations exceeded R‐TIA concentrations from ∼2 hr after dosing, 3) C_max and AUC_(0‐∞) for S‐TIA were greater than for R‐TIA following administration of S‐TIA, and 4) inversion was bidirectional but favored inversion of R‐TIA to S‐TIA. Bidirectional inversion was also observed when TIA enantiomers were incubated with liver homogenates up to 24 hr. However, the rate of inversion favored transformation of the R‐enantiomer to the S‐enantiomer. In conclusion, stereoselective pharmacokinetics of R‐ and S‐TIA were observed in rats and bidirectional inversion in rat liver homogenates has been demonstrated for the first time. Chiral inversion of TIA may involve metabolic routes different from those associated with inversion of other 2‐arylpropionic acids such as ibuprofen. Chirality 11:103–108, 1999. © 1999 Wiley‐Liss, Inc.     

Chromatographic analysis of allosteric effects between ibuprofen and benzodiazepines on human serum albumin

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.     

Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

Binding of nonsteroidal anti‐inflammatory drugs to Aβ fibril

Nonsteroidal anti‐inflammatory drugs are considered as potential therapeutic agents against Alzheimer's disease. Using replica exchange molecular dynamics and atomistic implicit solvent model, we studied the mechanisms of binding of naproxen and ibuprofen to the Aβ fibril derived from solid‐state NMR measurements. The binding temperature of naproxen is found to be almost 40 K higher than of ibuprofen implicating higher binding affinity of naproxen. The key factor, which enhances naproxen binding, is strong interactions between ligands bound to the surface of the fibril. The naphthalene ring in naproxen appears to provide a dominant contribution to ligand‐ligand interactions. In contrast, ligand‐fibril interactions cannot explain differences in the binding affinities of naproxen and ibuprofen. The concave fibril edge with the groove is identified as the primary binding location for both ligands. We show that confinement of the ligands to the groove facilitates ligand‐ligand interactions that lowers the energy of the ligands bound to the concave edge compared with those bound to the convex edge. Our simulations appear to provide microscopic rationale for the differing binding affinities of naproxen and ibuprofen observed experimentally. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Author index of volume 26, 2008

The stereospecific esterification of ibuprofen by Candida rugosa lipase (CRL) with 1-butanol was optimised. The yield of the butyl ester was nearly quantitative with an enantiomeric excess of 95% and E 100. Enzyme desiccation over P₂O₅ had a pronounced effect on the esterification yield and its role in stereospecificity is discussed. Molecular modelling and energy-minimisation studies were also performed to understand the stereospecific binding of substrates to the active site of CRL. The docking of the substrate S(+) ibuprofen to the active site of CRL was performed based on the three-dimensional structure of CRL (PDB entry 1CRL). The results show that only the active S(+) enantiomer of ibuprofen is able to form direct contacts with the reactive hydroxyl group (Ser209) and imidazole base (His449) at the active site, whereas with the R enantiomer the functional group COOH points away from the (His449) base of CRL. This is in accordance with experimental results which show that the stereospecifity of CRL is towards S(-) ibuprofen.     

Preparation and configuration of racemic and optically active analgesic cycloaminoalkylnaphthalenes

Cycloaminoalkylnaphthalene 3 shows interesting opioid‐like analgesic properties. It possesses two chiral centers and can exist as two racemic pairs and four diastereomers. Since the binding of opioids with receptors is stereoselective, it was important to have the two racemic pairs as well as the four diastereomers. In this paper the synthesis of the (2R,3S/2S,3R) racemate and the (2R,3S) and (2S,3R) enantiomers of the 1,2‐dimethyl‐3‐[2‐(6‐hydroxynaphthyl)]‐3‐hydroxypyrrolidine 3 is considered and the determination of absolute configuration is described. The (2R,3S/2S,3R)‐ 3 racemate and the (2R,3S)‐ 3 and (2S,3R)‐ 3 enantiomers were prepared by reaction of the racemic and optically active 1,2‐dimethyl‐3‐pyrrolidone 2, respectively, with the lithiation product obtained from 2‐bromo‐6‐tetrahydropyranyloxy‐naphthalene 1 and acidic hydrolysis. The above‐mentioned enantiomers of 3 were also obtained by optical resolution via fractional crystallization of the salts with d ‐ and l ‐tartaric acids. The configuration of the optically active compounds was determined by X‐ray analysis of a crystal of (−)‐(2S,3R)‐ 3 · HCl · H₂O. The pharmacological test HPT showed that (−)‐(2S,3R)‐ 3 · HCl · H₂O enantiomer is able to induce opioid‐like analgesia with a relative potency 1.5 times that of (2R,3S/2S,3R)‐ 3 and ∼1.5 times that of morphine. Chirality 11:21–28, 1999. © 1999 Wiley‐Liss, Inc.     

Purification and characterization of fertility‐associated antigen (FAA) in bovine seminal fluid

Heparin‐binding proteins (HBP) recognized by a monoclonal antibody (M1) are produced by male accessory sex glands and bind to distinct regions of ejaculated bull sperm. Immunoblots of sperm proteins probed with M1 identified HBP variants of approximately 31‐, 24‐, and 21.5‐kDa that were associated with increased fertility of bulls. The purpose of this study was to identify the 31‐kDa HBP known as fertility‐associated antigen (FAA). FAA was isolated by heparin‐affinity chromatography and reversed‐phase high performance liquid chromatography near homogeneity. Biochemical characterization indicated that FAA was an unglycosylated, basic protein. FAA protein was detected in seminal vesicle and prostate gland homogenates, and FAA extracted from sperm membranes by treatment with hypertonic media was identical biochemically to seminal fluid‐derived FAA. N‐terminal sequence analysis of purified FAA yielded a 26 amino acid sequence (L K I X S F N V R S F G E S K K A G F N A M R V I V) with 73% identity to a recently identified human deoxyribonuclease (DNase) I‐like protein. Two internal amino acid sequences generated from lys‐C digested FAA were 85% and 92% identical to the same DNase I‐like protein. In conclusion, we have identified a bovine seminal heparin‐binding protein that binds to sperm and is indicative of bull fertility as being similar to the family of DNase I‐like proteins. These data demonstrate the presence of a novel DNase I‐like protein in bull accessory sex glands and form the groundwork for the identification of a candidate genetic marker for fertility of bulls. Mol. Reprod. Dev. 54:145–153, 1999. © 1999 Wiley‐Liss, Inc.     

Bioactivity of Albumins Bound to Silver Nanoparticles

The last decade has witnessed a tremendous rise in the proposed applications of nanomaterials in the field of medicine due to their very attractive physiochemical properties and novel actions such as the ability to reach previously inaccessible targets such as brain. However biological activity of functional molecules bound to nanoparticles and its physiological consequences is still unclear and hence this area requires immediate attention. The functional properties of Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) bound to silver nanoparticles (~60 nm) have been studied under physiological environment. Esterase activity, binding of drugs (warfarin and ibuprofen), antioxidant activity and copper binding by albumins was evaluated. The catalytic efficiencies of HSA and BSA diminished upon binding to silver nanoparticles. Perturbation in binding of warfarin and ibuprofen, loss of free sulphydryls, antioxidant activity and enhancement of copper binding were observed in albumins bound to nanoparticles. These alterations in functional activity of nanoparticle bound albumins which will have important consequences should be taken into consideration while using nanoparticles for diagnostic and therapeutic purposes.     

Cucurbitacins from Ecballium elaterium juice increase the binding of bilirubin and ibuprofen to albumin in human plasma

Ecballium elaterium, a medicinal plant, whose fruit juice is used for the treatment of jaundice in folk medicine, has been reported as being capable of decreasing bilirubinemia in animals with jaundice [H.H. Elayan, M.N. Garaibeh, S.M. Zmeili, S.A. Salhab, Effects of Ecballium elaterium juice on serum bilirubin concentration in male rats, Int. J. Crude Drug Res. 27 (1989) 227-234]. The aim of this study is to identify the Ecballium elaterium components, which are able to modify the binding of bilirubin to albumin. The juice is fiber-free but contains proteins, lipids, sugars, and minerals. The extract of the juice, analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), contains cucurbitacins (Cuc) B, D, E, and I as well as several glycosylated compounds. Human plasma containing no or serial concentrations of Ecballium elaterium components were prepared and the direct bilirubin (DB) and total bilirubin (TB) were determined by the Jendrassik and Grof method. Our results showed that Cuc D, E, and B decreased the levels of DB and TB in plasma, while Cuc I, glycosyl derivatives, and proteins of the juice did not modify the bilirubin levels. The binding of domain specific ligands to HSA, bilirubin (domain IIA), and ibuprofen (domain IIIA), were studied in the absence and presence of Cuc D, E, and I, by fluorescence spectroscopy. The values of binding constant K(a) and binding site number n, determined by Scatchard method, increased for the both ligands only in the presence of Cuc E and D. Cuc I decreased slightly the K(a) of ibuprofen, suggesting an interaction with the domain IIIA of the protein. As a conclusion, Cuc E, D, and B produce rearrangement in the structure of albumin leading to increase the binding of domain specific ligands, ibuprofen and bilirubin.     

Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA

Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell‐specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte‐ and mesenchymal cell‐derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type‐specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included. J. Cell. Biochem. 76:341–351, 1999. © 1999 Wiley‐Liss, Inc.     

Molecular Dynamics Simulations of Ibuprofen Binding to Aβ Peptides

Using replica exchange molecular dynamics simulations and the implicit solvent model we probed binding of ibuprofen to Aβ_10-40 monomers and amyloid fibrils. We found that the concave (CV) fibril edge has significantly higher binding affinity for ibuprofen than the convex edge. Furthermore, binding of ibuprofen to Aβ monomers, as compared to fibrils, results in a smaller free energy gain. The difference in binding free energies is likely to be related to the presence of the groove on the CV fibril edge, in which ibuprofen tends to accumulate. The confinement effect of the groove promotes the formation of large low-energy ibuprofen clusters, which rarely occur on the surface of Aβ monomers. These observations led us to suggest that the ibuprofen binding mechanism for Aβ fibrils is different from that for monomers. In general, ibuprofen shows a preference to bind to those regions of Aβ monomers (amino terminal) and fibrils (the CV edge) that are also the primary aggregation interfaces. Based on our findings and on available experimental data, we propose a rationale for the ibuprofen antiaggregation effect.     

A method of chemiluminescence coupled with ultrafiltration for investigating the interaction between ibuprofen and human serum albumin

In acidic media, ibuprofen substantially enhanced the weak chemiluminescence (CL) produced by sodium sulfite and potassium permanganate. The increased signals were linearly correlated with ibuprofen concentrations ranging from 1.2 × 10^‐3 to 4.8 μM, with a detection limit of 4.8 × 10^‐4 μM. Two ultrafiltration (UF) membranes were used to construct a unit for trapping 0.15 and 0.75 μM human serum albumin (HSA) and coupled online with the CL system. At low HSA concentrations, the numbers of bound molecules per binding site were calculated to be 0.9 for Sudlow site I and 6.2 for Sudlow site II. The association constants on these binding sites were 5.9 × 10⁵ and 3.4 × 10⁴ M^‐1, respectively. Our CL–UF protocol presents a rapid and sensitive method for studies on drug–protein interaction. Copyright © 2012 John Wiley & Sons, Ltd.     

One-step method for plasma determination of ibuprofen by chemiluminescence-coupled ultrafiltration and application in a pharmacokinetic study

A simple one‐step method is established for plasma determination of ibuprofen and its pharmacokinetic study. The method involves simple sample pre‐treatment by dilution, rapid separation by ultrafiltration (UF) and online sensitive detection by chemiluminescence (CL) based on significant intensity enhancement of ibuprofen on the weak CL of potassium permanganate and sodium sulphite in an acidic system. The calibration curve for ibuprofen is linear in the range 0.1–50.0 µg/mL in rat plasma. Average recoveries of ibuprofen at 0.80, 12.0 and 40.0 µg/mL amounted to 98.0 ± 4.2%, 101.2 ± 3.6% and 99.3 ± 5.4%, respectively. Standard deviations of intra‐ and inter‐day measurement precision and accuracy are within ±10.0%. The detection limit for ibuprofen is 10.0 µg/L in plasma samples. Pharmacokinetic study of ibuprofen by the validated method shows that the mean plasma drug concentration–time course confirms to a classical two‐compartment open model with first‐order absorption. The proposed method will be an alternative for pre‐clinical pharmacokinetic study of ibuprofen and other non‐steroidal anti‐inflammatory drugs. Copyright © 2011 John Wiley & Sons, Ltd.     

Stereoselective disposition of ibuprofen and flurbiprofen in rats

(R)-2-Arylpropionates are often inverted to the pharmacologically active S-enantiomers in vivo, although there is significant interspecies variability in inversion. In order to provide a basis for determining the biochemical consequences of this unique process using rats as a model, it was important to establish the pharmacokinetic disposition of the enantiomers of ibuprofen, a drug well inverted in man and flurbiprofen, a drug apparently poorly inverted in man. Rats were dosed i.v. with a single dose of (R)- or (S)-ibuprofen (20 mg/kg), (R,S)-ibuprofen (40 mg/kg), (R)- or (S)-flurbiprofen (10 mg/kg), or (R,S)-flurbiprofen (20 mg/kg). Each treatment group consisted of six animals. Serial blood samples were withdrawn over a period of 6 h for ibuprofen and 10 h for flurbiprofen. These drugs were assayed in plasma by a stereospecific HPLC assay. The pharmacokinetics of the ibuprofen and flurbiprofen enantiomers were evaluated using a two-compartment open model with conversion of the R- to S-enantiomers in the central compartment. There was 50 +/- 4% inversion of (R)-ibuprofen, a figure similar to that observed in man and (R)-ibuprofen had a higher clearance (12.6 +/- 1.3 ml/min/kg) than (S)-ibuprofen (7.7 +/- 0.7 ml/min/kg; P less than 0.01). The clearance of (R)-flurbiprofen after racemate (2.3 +/- 0.1 ml/min/kg) was higher than its clearance when administered alone (1.7 +/- 0.2 ml/min/kg; P less than 0.01), indicating a pharmacokinetic interaction between the enantiomers (most probably at plasma protein binding sites). A corresponding difference was not observed for ibuprofen. There was a small amount of inversion of (R)-flurbiprofen as determined by area analysis (4.5 +/- 1.6%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of competitive binding of ibuprofen and salicylic acid with serum albumin by affinity capillary electrophoresis

Ibuprofen and salicylic acid, two typical non-steroidal anti-inflammatory drugs, are used commonly as analgesic drug in clinical medicine and sometimes are co-administered. When the drugs are co-administered, the drug-drug interactions may occur, and can lead to alter the safety and efficacy of drugs, resulting in variations in drug response of the co-administered drugs. Affinity capillary electrophoresis (ACE) was employed to investigate the competitive binding of ibuprofen and salicylic acid on serum albumin. Mobility ratio, derivatives from mobility shift method, was used to deduce the binding constant (K(b)). The binding constants of ibuprofen with HSA are 2.97×10? M?1 and 7.07×10? M?1, respectively; while for salicylic acid, the binding constant is 5.99×10? M?1. The competitive binding of the two drugs was investigated by addition of excessive ibuprofen into the solutions containing constant concentrations of salicylic acid and serum albumin. The results confirmed that ibuprofen and salicylic acid have different high-affinity binding site, but share some low-affinity binding sites on the serum albumin; and ibuprofen is able to partially replace salicylic acid from the preformed binary complexes of serum albumin and salicylic acid.     

Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study.

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-haem) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem-NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules--buried in close proximity to the haem binding pocket--induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1H-NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, are 5.4 +/- 1.1 x 10(-4) m and 2.1 +/- 0.4 x 10(-5) m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA-haem is lower than that reported for drug binding to haem-free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) m in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA-haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA.     

Study of the interaction between ofloxacin and human serum albumin by spectroscopic methods

The binding of ofloxacin (OFLX) to human serum albumin (HSA) was investigated by fluorescence and circular dichroism (CD) techniques. The binding parameters have been evaluated by a fluorescence quenching method. Competitive binding measurements were performed in the presence of warfarin and ibuprofen and suggest binding to the warfarin site I of HSA. The distance r between donor (HSA) and acceptor (OFLX) was estimated according to the Forster's theory of non‐radiatiative energy transfer. CD spectra revealed that the binding of OFLX to HSA induced conformational changes in HSA. Molecular docking was performed and shows that for the lowest energy complex OFLX is located in site I of HSA, which correlate to the competitive binding experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

Preparation of (2S,4R)‐2,4‐thiazolidinedicarboxylic acid by separation of diastereoisomeric salts with organic amines

L ‐Cysteine was condensed with glyoxylic acid monohydrate in acetic acid at 30°C to give (4R)‐2,4‐thiazolidinedicarboxylic acid [(4R)‐TDA] as a mixture of two diastereoisomers, (2R,4R)‐ and (2S,4R)‐TDA. An attempt was made to separate (2S,4R)‐TDA from the diastereoisomeric salts of (4R)‐TDA with 1‐propylamine, 2‐methyl‐2‐propylamine, benzylamine, and (R)‐ and (S)‐1‐phenylethylamines [(R)‐ and (S)‐PEA]. The salts of (2S,4R)‐TDA were preferentially crystallized as less soluble diastereoisomeric salts. When the salt with (R)‐PEA was employed, the separation was successfully achieved to afford optically pure (2S,4R)‐TDA in a yield of 41%, based on the starting amount of the diastereoisomeric mixture of (4R)‐TDA. Chirality 11:326–329, 1999. © 1999 Wiley‐Liss, Inc.     

Efficient synthesis and chiral separation of 11C-labeled ibuprofen assisted by DMSO for imaging of in vivo behavior of the individual isomers by positron emission tomography

The pharmacological mechanisms focusing on chiral isomer of ibuprofen are not fully understood. Only the (S)-isomer of ibuprofen inhibits cyclooxygenases, which mediates the generation of prostanoids and thromboxanes. Consequently, (S)-isomers represent a major promoter of the anti-inflammatory effect, and the effects of the (R)-isomers have not been widely discussed. However, more recently, the cyclooxygenase-independent pharmacological effects of ibuprofen have been elucidated. Pharmacokinetic studies with individual isomers of ibuprofen by positron emission tomography should aid our understanding of the pharmacological mechanisms of ibuprofen. The efficient (11)C-labeling of ibuprofen for chiral separation via the TBAF-promoted α-[(11)C]methylation was achieved by using DMSO rather than THF as the reaction solvent. The robust production of the radiochemically labile (11)C-labeled ibuprofen ester was realized by the protective effect of DMSO on radiolysis. After intravenous injection of each enantiomer of [(11)C]ibuprofen, significantly high radioactivity was observed in the joints of arthritis mice when compared to the levels observed in normal mice. However, the high accumulation was equivalent between the enantiomers, indicating that ibuprofen is accumulated in the arthritic joints regardless of the expression of cyclooxygenases.     

Ibuprofen binding to secondary sites allosterically modulates the spectroscopic and catalytic properties of human serum heme-albumin

The ibuprofen primary binding site FA3-FA4 is located in domain III of human serum albumin (HSA), the secondary clefts FA2 and FA6 being sited in domains I and II. Here, the thermodynamics of ibuprofen binding to recombinant Asp1-Glu382 truncated HSA (tHSA)-heme-Fe(III) and nitrosylated tHSA-heme-Fe(II), encompassing domains I and II only, is reported. Moreover, the allosteric effect of ibuprofen on the kinetics of tHSA-heme-Fe(III)-mediated peroxynitrite isomerization and nitrosylated tHSA-heme-Fe(II) denitrosylation has been investigated. The present data indicate, for the first time, that the allosteric modulation of tHSA-heme and HSA-heme reactivity by ibuprofen depends mainly on drug binding to the FA2 and FA6 secondary sites rather than drug association with the FA3-FA4 primary cleft. Thus, tHSA is a valuable model with which to investigate the allosteric linkage between the heme cleft FA1 and the ligand-binding pockets FA2 and FA6, all located in domains I and II of (t)HSA.