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Human serum albumin (HSA) displays several metal binding sites, participating to essential and toxic metal ions disposal and transport. The major Zn(II) binding site, called Site A, is located at the I/II domain interface, with residues His67, Asn99, His247, and Asp249 contributing with five donor atoms to the metal ion coordination. Additionally, one water molecule takes part of the octahedral coordination geometry. The occurrence of the metal-coordinated water molecule allows the investigation of the metal complex geometry by water H-1-NMR relaxation, provided that the diamagnetic Zn(II) is replaced by the paramagnetic Mn(II). Here, the H-1-NMR relaxometric study of Mn(II) binding to HSA is reported. Mn(II) binding to HSA is modulated by Zn(II), pH, and myristate through competitive inhibition and allosteric mechanisms. The body of results indicates that the primary binding site of Zn(II) corresponds to the secondary binding site of Mn(II), i.e. the multimetal binding site A. Excess Zn(II) completely displaces Mn(II) from its primary site suggesting that the primary Mn(II) site corresponds to the secondary Zn(II) site. This uncharacterized site is functionally-linked to FA1; moreover, metal ion binding is modulated by myristate and pH. Noteworthy, water H-1-NMR relaxometry allowed a detailed analysis of thermodynamic properties of HSA-metal ion complexes. (C) 2012 Elsevier Inc. All rights reserved

Human serum albumin (HSA), the most abundant protein in plasma, is a monomeric multi-domain macromolecule, representing the main determinant of plasma oncotic pressure and the main modulator of fluid distribution between body compartments. HSA displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds. Indeed, HSA represents the main carrier for fatty acids, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays (pseudo-)enzymatic properties. HSA is a valuable biomarker of many diseases, including cancer, rheumatoid arthritis, ischemia, post-menopausal obesity, severe acute graft-versus-host disease, and diseases that need monitoring of the glycemic control. Moreover, HSA is widely used clinically to treat several diseases, including hypovolemia, shock, burns, surgical blood loss, trauma, hemorrhage, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, nutrition support, resuscitation, and hypoalbuminemia. Recently, biotechnological applications of HSA, including implantable biomaterials, surgical adhesives and sealants, biochromatography, ligand trapping, and fusion proteins, have been reported. Here, genetic, biochemical, biomedical, and biotechnological aspects of HSA are reviewed.

""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.""

Heme endows human serum albumin (HSA) with heme-protein-like reactivity and spectroscopic properties. Here, the kinetics and thermodynamics of reductive nitrosylation of ferric human serum heme-albumin [HSA-heme-Fe(III)] are reported. All data were obtained at 20 degrees C. At pH 5.5, HSA-heme-Fe(III) binds nitrogen monoxide (NO) reversibly, leading to the formation of nitrosylated HSA-heme-Fe(III) [HSA-heme-Fe(III)-NO]. By contrast, at pHor= 6.5, the addition of NO to HSA-heme-Fe(III) leads to the transient formation of HSA-heme-Fe(III)-NO in equilibrium with HSA-heme-Fe(II)-NO(+). Then, HSA-heme-Fe(II)-NO(+) undergoes nucleophilic attack by OH(-) to yield ferrous human serum heme-albumin [HSA-heme-Fe(II)]. HSA-heme-Fe(II) further reacts with NO to give nitrosylated HSA-heme-Fe(II) [HSA-heme-Fe(II)-NO]. The rate-limiting step for reductive nitrosylation of HSA-heme-Fe(III) is represented by the OH(-)-mediated reduction of HSA-heme-Fe(II)-NO(+) to HSA-heme-Fe(II). The value of the second-order rate constant for OH(-)-mediated reduction of HSA-heme-Fe(II)-NO(+) to HSA-heme-Fe(II) is 4.4 x 10(3) M(-1) s(-1). The present results highlight the role of HSA-heme-Fe in scavenging reactive nitrogen species.

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO(3)(-) catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 degrees C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO(2); the values of the second order catalytic rate constant (k(on)) are 4.1 x 10(5) and 4.5 x 10(5) m(-1) s(-1), respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of k(on) (pK(a) = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO(2). The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO(2), ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 x 10(-4) and 9.7 x 10(-4) m. Under conditions where [ibuprofen] is >>L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.

Lactoferrin (Lf) is a mammalian iron binding protein present in external secretions and in polymorphonuclear leukocytes. Its role in host defense mechanisms related to the non-immune defense system has been definitively established. Lf has two identical iron-binding sites, far from each other (44.3 Angstrom) and magnetically non-interacting. Fe(III) ions are six-coordinated, with four donor atoms provided by protein sidechains (two Tyr, one His, one Asp) and two oxygen atoms from a bridged HCO3-. This set of ligands provides an ideal coordination scheme for stable and reversible iron binding. Nuclear magnetic relaxation dispersion (NMRD) profiles of Lf are consistent with a closest distance for a single water hydrogen atom of 3.1 Angstrom. By looking at the X-ray structure of Lf (PDB ID code: I BLF) we can locate two water oxygens at 3.95 and 4.27 Angstrom from each Fe(III), respectively. Temperature dependence data suggest that an important contribution to the overall paramagnetic contribution to the solvent water relaxation rate arises from one or more second sphere water molecules in slow exchange with the bulk. A decreasing value of the exchange rate is obtained, ranging from 1.2 to 0.7 mus in the observed temperature range (25-65 degreesC), with an activation enthalpy of 7.3 +/- 0.8 kJ mol(-1). The low exchange rate obtained from NMRD data can be explained by the observation that both water molecules are bound to several polar groups of the protein backbone and side chains. By increasing the pH from 6.5 to 12 two distinct titrations are observed, consistent with sequential removal of both water molecules. (C) 2004 Elsevier Inc. All rights reserved.

Human serum heme-albumin (HSA-heme-Fe) displays heme-based ligand binding and (pseudo-)enzymatic properties. Here, the effect of the prototypical drug warfarin on kinetics and thermodynamics of NO binding to ferric and ferrous HSA-heme-Fe (HSA-heme-Fe(III) and HSA-heme-Fe(II), respectively) and on the NO-mediated reductive nitrosylation of the heme-Fe atom is reported; data were obtained between pH 5.5 and 9.5 at 20.0 °C. Since warfarin is a common drug, its effect on the reactivity of HSA-heme-Fe represents a relevant issue in the pharmacological therapy management. The inhibition of NO binding to HSA-heme-Fe(III) and HSA-heme-Fe(II) as well as of the NO-mediated reductive nitrosylation of the heme-Fe(III) atom by warfarin has been ascribed to drug binding to the fatty acid binding site 2 (FA2), shifting allosterically the penta-to-six coordination equilibrium of the heme-Fe atom toward the low reactive species showing the six-coordinated metal center by His146 and Tyr161 residues. These data: (i) support the role of HSA-heme-Fe in trapping NO, (ii) highlight the modulation of the heme-Fe-based reactivity by drugs, and (iii) could be relevant for the modulation of HSA functions by drugs in vivo.

Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

Imatinib, an inhibitor of the Bcr-Abl tyrosine kinase, is approximately 95% bound to plasma proteins, alpha 1-acid glycoprotein (AGP) being the primary carrier. However, human serum albumin (HSA) may represent the secondary carrier of imatinib in pathological states characterized by low AGP levels, such as pancreatic cancer, hepatic cirrhosis, hepatitis, hyperthyroidism, nephrotic syndrome, malnutrition, and cachexia. Here, thermodynamics of imatinib binding to full-length HSA and its recombinant Asp1-Glu382 truncated form (containing only the FA1, FA2, FA6, and FA7 binding sites; trHSA), in the absence and presence of ferric heme (heme-Fe(III)), and the thermodynamics of heme-Fe(III) binding to RSA and trHSA, in the absence and presence of imatinib, has been investigated. Moreover, the effect of imatinib on kinetics of peroxynitrite detoxification by ferric human serum heme-albumin (HSA-heme-Fe(III)) and ferric truncated human serum heme-albumin (trHSA-heme-Fe(III)) has been explored. All data were obtained at pH 7.0, and 20.0 degrees C and 37.0 degrees C. Imatinib binding to the FA7 site of HSA and trHSA inhibits allosterically heme-Fe(III) association to the FA1 site and vice versa, according to linked functions. Moreover, imatinib binding to the secondary FA2 site of HSA-heme-Fe(III) inhibits allosterically peroxynitrite detoxification. Docking simulations and local structural comparison with other imatinib-binding proteins support functional data indicating the preferential binding of imatinib to the FA1 and FA7 sites of HSA, and to the FA2 and FA7 sites of HSA-heme-Fe(III). Present results highlight the allosteric coupling of the FA1, FA2, and FA7 sites of HSA, and may be relevant in modulating ligand binding and reactivity properties of HSA in vivo.

Human serum albumin (HSA) displays esterase activity reflecting multiple irreversible chemical modifications rather than turnover. Here, kinetics of the pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate (NphOAc) are reported. Under conditions where [HSA] >= 5x [NphOAc] and [NphOAc] 5x [HSA], the HSA-catalyzed hydrolysis of NphOAc is a first-order process for more than 95% of its course. From the dependence of the apparent rate constants k(app) and k(obs) on [HSA] and [NphOAc], respectively, values of K-s, k(+2), and k(+2)/K-s were determined. Values of K-s, k(+2), and k(+2/K), obtained at [HSA] 5 x [NphOAc] and [NphOAc],>= 5 x [HSA] are in good agreement, the deacylation step being rate limiting in catalysis. The pH-dependence of k(+2)/K-s, k(+2), and K, reflects the acidic pK(a) shift of the Tyr411 catalytic residue from 9.0 +/- 0.1 in the substrate-free HSA to 8.1 +/- 0.1 in the HSA:NphOAc complex. Accordingly, diazepam inhibits competitively the HSA-catalyzed hydrolysis of NphOAc by binding to Tyr411

Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 degrees C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S=5/2 manifold. Values of correlation times for tensor fluctuation (tau(v)) and chemical exchange of water molecules (tau(M)) show the expected temperature dependence, with activation enthalpies of -1.94 and -2.46+/-0.2 kJ mol(-1), respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with DeltaH=68+/-28 kJ mol(-1) and DeltaS=200+/-80 J mol(-1) K(-1). These results highlight the role of the water solvent in heme-HSA structure-function relationships.

Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.

Human serum albumin (HSA), the most abundant protein in plasma, displays several functions including heme transfer from high- and low-density lipoproteins to hemopexin; therefore, the HSA-heme complex acquires transiently heme-based (pseudo-)enzymatic properties. In particular, ferric human serum heme-albumin (HSA-heme) and ferrous nitrosylated HSA-heme inactivate peroxynitrite, and ferrous HSA-heme catalyzes the conversion of nitrite to nitrogen monoxide. The (pseudo-)enzymatic properties of HSA-heme are modulated allosterically by endogenous and exogenous ligands, such as drugs. The modulation of ligand binding to plasma proteins is relevant not only under physiological conditions but also in the pharmacological therapy management. Here, drug-dependent HSA-heme properties are reviewed from the functional and structural viewpoints. In particular, the drug-dependent five- to six-coordination transition of the heme-Fe atom is at the root of the allosteric modulation of the HSA-heme reactivity.

Human serum albumin (HSA) has an extraordinary ligand-binding capacity, and transports Fe(III)heme and medium- and long-chain fatty acids. In human immunodeficiency virus-infected patients the administered drugs bind to HSA and act as allosteric effectors. Here, the binding of Fe(III)heme to HSA in the presence of three representative anti-HIV drugs and myristate is investigated. Values of the dissociation equilibrium constant K(d) for Fe(III)heme binding to HSA were determined at different myristate concentrations, in the absence and presence of anti-HIV drugs. Nuclear magnetic relaxation dispersion profiles of HSA-Fe(III)heme were measured, at different myristate concentrations, in the absence and presence of anti-HIV drugs. Structural bases for anti-HIV drug binding to HSA are provided by automatic docking simulation. Abacavir and nevirapine bind to HSA with K(d) values of 1 x 10(-6) and 2 x 10(-6) M, respectively. Therefore, at concentrations used in therapy (in the 1-5 x 10(-6) M range) abacavir and nevirapine bind to HSA and increase the affinity of heme for HSA. In the presence of abacavir or nevirapine, the affinity is not lowered by myristate. FA7 should therefore be intended as a secondary binding site for abacavir and nevirapine. Binding of atazanavir is limited by the large size of the drug, although preferential binding may be envisaged to a site positively coupled with FA1 and FA2, and negatively coupled to FA7. As a whole, these results provide a foundation for the comprehension of the complex network of links modulating HSA-binding properties.

We hypothesize that the structure of the heme binding site of paralogous albuminoids alpha-fetoprotein and serum albumin has evolved from the ancestor vitamin D binding protein through the 'phylogenetic intermediate' afamin, the most recently discovered albuminoid. Heme binding to plasma proteins should serve not only as a buffer for heme homeostasis, avoiding heme binding to lipoproteins with the consequent oxidative stress, but also for heme transfer to the liver, complementing the function of hemopexin.

Human serum albumin (HSA): (i) controls the plasma oncotic pressure, (ii) modulates fluid distribution between the body compartments, (iii) represents the depot and carrier of endogenous and exogenous compounds, (iv) increases the apparent solubility and lifetime of hydrophobic compounds, (v) affects pharmacokinetics of many drugs, (vi) inactivates toxic compounds, (vii) induces chemical modifications of some ligands, (viii) displays antioxidant properties, and (ix) shows enzymatic properties. Under physiological and pathological conditions, HSA has a pivotal role in heme scavenging transferring the metal-macrocycle from high- and low-density lipoproteins to hemopexin, thus acquiring globin-like reactivity. Here, the heme-based catalytic properties of HSA are reviewed and the structural bases of drug-dependent allosteric regulation are highlighted.

Retinoids are a class of chemicals derived from vitamin A metabolism, playing important and diverse functions. Vitamin A, also named all-trans-retinol (all-trans-ROL), is coverted into two classes of biologically active retinoids, i.e. 11-cis-retinoids and acidic retinoids. Among acidic retinoids, all-trans-retinoic acid (all-trans-RA) and 9-cis-retinoic acid (9-cis-RA) represent the main metabolic products. Specific and aspecific proteins solubilize, protect, and detoxify retinoids in the extracellular environment. The retinoid binding protein 4 (RBP4), the epididymal retinoid-binding protein (ERBP), and the inter photoreceptor matrix retinoid-binding protein (IRBP) play a central role in ROL transport, whereas lipocalin-type prostaglandin D synthase (also named beta-trace) and human serum albumin (HSA) transport preferentially all-trans-RA. Here, the modulatory effect of all-trans-RA and all-trans-ROL on ferric heme (heme-Fe(III)) binding to HSA is reported. All-trans-RA and all-trans-ROL binding to the FA1 site of HSA competitively inhibit heme-Fe(III) association. Docking simulations and local structural comparison of HSA with all-trans-RA- and all-trans-ROL-binding proteins support functional data indicating the preferential binding of all-trans-RA and all-trans-ROL to the FA1 site of HSA. Present results may be relevant in vivo, in fact HSA could act as a secondary carrier of retinoids in human diseases associated with reduced levels of RBP4 and IRBP.

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K= (1.1 ± 0.1)× 10(-4) M, k on= (5.3 ± 0.6)× 10(3) M(-1) s(-1) and k off= (4.6 ± 0.5)× 10(-1) s(-1); and D= (1.2 ± 0.2)× 10(-3) M, d on= (1.3 ± 0.4)× 10(3) M(-1) s(-1), and d off=1.5 ± 0.4 s(-1), respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.

Serum albumin, α-fetoprotein, afamin (also named α-albumin and vitamin E binding protein), and vitamin D binding protein are members of the albuminoid superfamily. Albuminoids are plasma proteins characterized by a marked ability for ligand binding and transport. Here, a focused phylogenetic analysis of sequence evolution by maximum likelihood of fatty acid binding sites FA1–FA7 of mammalian albuminoids reveals that the FA1, FA2, and FA3+FA4 sites in serum albumins have evolved from the most recent common ancestor through an intermediate that has originated the α-fetoprotein and afamin clades. The same topology has been observed for the whole protein sequences, for the sequences of all the fatty acid binding sites (FA1–FA7) taken together, and for the allosteric core corresponding to residues 1–303 of human serum albumin. The quantitative divergence analysis indicates that the ligand binding cleft corresponding to the FA2 site could be the main determinant of allosteric properties of serum albumins only. In fact, this binding cleft is structurally not effective in vitamin D binding proteins, whereas key residues that serve to allocate the allosteric effectors are not present in afamins and α-fetoproteins. © 2013 IUBMB Life, 65(6):544–549, 2013

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $$ {\text{NO}}_{2}^{ - } $$ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k on) is 1.3 M−1 s−1 at pH 7.4 and 20 °C. Values of k on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10−4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $$ {\text{NO}}_{2}^{ - } $$ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.

Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II).

Given the ability of human serum albumin (HSA) to bind hydrophobic ligands, the binding mode of α-tocopherol, the most representative member of the vitamin E family, is reported. α-Tocopherol binds to HSA with K = (7.0 ± 3.0) × 10−6 M (pH 7.2, 25.0°C). Competitive and allosteric modulation of α-tocopherol binding to full-length and truncated (Asp1-Glu382) HSA by endogenous and exogenous ligands suggests that it accommodates preferentially in the FA3–FA4 site. As HSA is taken up into cells, colocalizes with the α-tocopherol transfer protein, and contributes to ligand secretion via ABCA1, it might participate in the distribution of α-tocopherol between plasma, cells, and tissues. © 2013 BioFactors, 2013

Serum albumin (SA) is a circulating protein providing a depot and carrier for many endogenous and exogenous compounds. At least seven major binding sites have been identified by structural and functional investigations mainly in human SA. SA is conserved in vertebrates, with at least 49 entries in protein sequence databases. The multiple sequence analysis of this set of entries leads to the definition of a cladistic tree for the molecular evolution of SA orthologs in vertebrates, thus showing the clustering of the considered species, with lamprey SAs (Lethenteron japonicum and Petromyzon marinus) in a separate outgroup. Sequence analysis aimed at searching conserved domains revealed that most SA sequences are made up by three repeated domains (about 600 residues), as extensively characterized for human SA. On the contrary, lamprey SAs are giant proteins (about 1400 residues) comprising seven repeated domains. The phylogenetic analysis of the SA family reveals a stringent correlation with the taxonomic classification of the species available in sequence databases. A focused inspection of the sequences of ligand binding sites in SA revealed that in all sites most residues involved in ligand binding are conserved, although the versatility towards different ligands could be peculiar of higher organisms. Moreover, the analysis of molecular links between the different sites suggests that allosteric modulation mechanisms could be restricted to higher vertebrates.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.

Cannabis is the most commonly used illicit drug worldwide. Cannabis users also appear to use other psychoactive drugs more frequently than noncannabis users. Here, Δ9- tetrahydrocannabinol (THC) and diazepam binding to human serum albumin (HSA) and HSA-heme is reported. THC binds to two different binding sites of HSA (Kd1 ≤ 10−7 M and Kd2= 10−3 M) without affecting diazepam binding (Kd = 1.2 × 10−5 M). THC binding to the high-affinity site accounts for the low free fraction of the drug in plasma. Moreover, THC increases the affinity of heme for HSA. Accordingly, the affinity of THC for HSA-heme is higher than that for HSA. THC could bind to FA2 and FA7 sites, as substantiated by docking simulations; nevertheless, the observed allosteric effect(s) suggests that the primary binding site of THC is the FA2 cleft that positively modulates heme affinity. Possibly, the HSA conformational transition(s) induced by THC binding could account for drug delivery to the liver through receptor- mediated endocytosis. © 2011 IUBMB IUBMB Life, 63(6): 446–451, 2011

Ferrous human serum heme-albumin (HSA-heme-Fe(II)) displays globin-like properties. Here, the effect of ibuprofen and warfarin on kinetics of HSA-heme-Fe(II) nitrosylation is reported. Values of the second-order rate constant for HSA-heme-Fe(II) nitrosylation (k(on)) decrease from 6.3 × 10(6)M(-1)s(-1) in the absence of drugs, to 4.1 × 10(5)M(-1)s(-1) and 4.8 × 10(5)M(-1)s(-1), in the presence of saturating amounts of ibuprofen and warfarin, respectively, at pH 7.0 and 20.0°C. From the dependence of k(on) on the drug concentration, values of the dissociation equilibrium constant for ibuprofen and warfarin binding to HSA-heme-Fe(II) (i.e., K=3.2 × 10(-3)M and 2.6 × 10(-4)M, respectively) were determined. The observed allosteric effects could indeed reflect ibuprofen and warfarin binding to the regulatory fatty acid binding site FA2, which brings about an alteration of heme coordination, slowing down HSA-heme-Fe(II) nitrosylation. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.

Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K 0 = (3.9 ± 0.4) × 10−4 and (1.3 ± 0.1) × 10−5 M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K h = (4.3 ± 0.4) × 10−3 and (1.2 ± 0.1) × 10−4 M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H 0 = (1.9 ± 0.2) × 10−8 M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H d = (2.1 ± 0.2) × 10−7 M]. In the absence and presence of CO2, the values of the second-order rate constant (l on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 105 and 4.3 × 105 M−1 s−1, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO2. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3 × 10−6 and 3.9 × 10−5 M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.

Heme endows human serum albumin (HSA) with globin-like reactivity and spectroscopic properties. Here, the effect of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, tolbutamide, and warfarin on peroxynitrite isomerization to NO(3) (-) by ferric HSA-heme (HSA-heme-Fe(III)) is reported. Drugs binding to Sudlow's site I impair dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III). The allosteric modulation of HSA-heme-Fe(III)-mediated peroxynitrite isomerization by drugs has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow's site I or protrudes into the heme cleft (i.e., the fatty acid site 1, FA1), depending on ligand occupancy of either sites.

Binding of drugs to plasma proteins is an important determinant for their efficacy because it modulates drug availability to the intended target. Co-administered drugs may bind to the same protein site or to different functionally linked clefts following competitive and allosteric mechanisms. Here, we report a thermodynamic and computational characterization of the binding mode of apomorphine and benserazide, two therapeutic agents co-administered in the treatment of Parkinson's disease, to human serum albumin (HSA). Apomorphine binds to HSA with a simple equilibrium (K(d) = 3.1 x 10(-6) M). Conversely, benserazide binds to HSA with two independent equilibria (K(d1)< or = 10(-6) M and K(d2) = 5.0 x 10(-5) M). Values of K(d) and K(d2) increase to 1.5 x 10(-5) M and 5.0 x 10(-4) M, respectively, in the presence of heme. Accordingly, the K(d) value for heme binding to HSA increases from 5.0 x 10(-7) M to 4.8 x 10(-6) M and 9.2 x 10(-7) M, in the presence of saturating amounts of apomorphine and benserazide, respectively. The K(d1) value for benserazide binding to HSA is not affected by heme binding, whereas apomorphine and benserazide inhibit warfarin binding to HSA, and vice versa. Therefore, apomorphine and the second benserazide molecule bind to the warfarin site, allosterically linked to the heme site. Simulated docking of apomorphine and benserazide into the warfarin site provides favorable values of intermolecular energy (-23.0 kJ mol(-1) and -15.2 kJ mol(-1), respectively). Considering the apomorphine, benserazide, and HSA-heme plasma levels and the possible co-administration of warfarin, these results appear relevant in the management of patients affected by Parkinson's disease.

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO(3)(-) catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 degrees C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO(2); the values of the second order catalytic rate constant (k(on)) are 4.1 x 10(5) and 4.5 x 10(5) m(-1) s(-1), respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of k(on) (pK(a) = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO(2). The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO(2), ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 x 10(-4) and 9.7 x 10(-4) m. Under conditions where [ibuprofen] isL, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.

Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme–HSA has been investigated by 1H-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate (thus fully saturating all available fatty acid binding sites in serum heme–albumin), 1.0 M guanidinium chloride induces some unfolding of heme–HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K d = (1.2 ± 0.1) × 10−6 M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K d = (3.4 ± 0.1) × 10−5 M]. Further addition of guanidinium chloride (up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme–HSA in the presence of myristate.

Human serum albumin (HSA) is the most prominent protein in plasma, but it is also found in tissues and secretions throughout the body. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme and drugs. HSA has been used as a model multidomain protein to investigate how interdomain interactions affect the global folding/unfolding process. Here, we report on the reversible chemical denaturation of heme-HSA involving three different conformational states (F, N, and B, occurring at pH 4.0, 7.0, and 9.0, respectively) and on the effect of prototypic drugs ibuprofen and warfarin on thermodynamics of the reversible unfolding process. Chaotropic unfolding of heme-HSA in the F, N, and B conformations is governed by different thermodynamic regimes, with the B form showing an entropic stabilization of the structure that compensates an enthalpic destabilization, and the F form easily unfolding under entropic control. Warfarin and ibuprofen binding stabilizes heme-HSA in both N and B states.

Human serum albumin displays time-dependent heme-based catalytic properties,1 representing a case for ‘chronosteric effects'.2 In fact, HSA has a pivotal role in heme transfer from high- and low-density lipoproteins to hemopexin. After endocytosis of the hemopexin-heme complex into the hepatic parenchymal cells through the CD91 receptor, hemopexin releases the heme, which undergoes degradation. Then, hemopexin is released intact into the bloodstream.3, 4 The three-domain organization of HSA is at the root of its extraordinary ligand-binding capacity and allosteric control. The most relevant clefts hosting ligands are the so-called fatty acid (FA) binding sites (named FA1 to FA9). Bacterial protein-recognition cleft(s), thyroxine-binding pockets and metal ion-recognition sites also participate to HSA actions.4 Noteworthy, the HSA structure and reactivity is affected not only reversibly by pH and ligands (e.g., heme, FAs and drugs), but also irreversibly by chemical modifications, which in turn confer antigenicity properties.4 Ferrous human serum heme-albumin (HSA-heme-Fe(II)) binds reversibly to NO and CO. Although the heme-Fe atom of HSA-heme-Fe(II) is rapidly oxidized by O2, HSA-heme-Fe(II) mutants bearing residues pivotal for O2 recognition have been proposed not only as red blood cell substitutes, but also as O2-therapeutic agents.1, 4, 5 Moreover, HSA-heme-Fe(II) catalyzes the nitrite conversion to nitrogen monoxide under acidosis and anaerobic conditions,6 HSA-heme-Fe(II)-NO reacts with O2 and peroxynitryte leading to the formation of NO3−,7, 8 and ferric HSA-heme-Fe (HSA-heme-Fe(III)) catalyzes the conversion of peroxynitryte to NO3−,9 and displays weak catalase and peroxidase activities.10 The heme-based catalytic properties of HSA are allosterically modulated by drugs (Figure 1).1 Domains I and II have a major role in the allosteric modulation of ligand-binding and reactivity properties of HSA, the FA1, FA2, FA6 and FA7 sites being functionally linked. Allosteric modulators (e.g., drugs) of the heme-based catalytic properties of HSA-heme affect the coordination state of the heme-Fe atom. In ligand-free active HSA-heme, the heme-Fe atom displays a four- or five-coordinated heme-Fe atom, whereas inactive HSA-heme shows a six-coordinated heme-Fe atom. Upon drug binding to HSA-heme (most probably to the FA2 site), the re-orientation of the Glu131-Arg145 α-helix and the axial coordination of the heme-Fe atom by His146 and Tyr161 occur; as a consequence, the unreactive six-coordinated HSA-heme species becomes predominant.1, 11, 12 Figure 1 Human serum albumin displays time-dependent heme-based catalytic properties, which are allosterically modulated by drugs As a whole, the allosteric modulation of heme-based reactivity properties of HSA-heme by drugs represents a pivotal issue in the pharmacological therapy management, heme-binding switching HSA from a plasmatic carrier to a transient metal-enzyme.

Human serum albumin (HSA), the most prominent protein in plasma, binds different classes of ligands at multiple sites. HSA provides a depot for many compounds, affects pharmacokinetics of many drugs, holds some ligands in a strained orientation providing their metabolic modification, renders potential toxins harmless transporting them to disposal sites, accounts for most of the antioxidant capacity of human serum, and acts as a NO-carrier. The globular domain structural organization of monomeric HSA is at the root of its allosteric properties which are reminiscent of those of multimeric proteins. Here, structural, functional, biotechnological, and biomedical aspects of ligand binding to HSA are summarized.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its extraordinary ligand binding capacity. The three homologous domains of HSA (labeled I, II, and III), each in turn composed of two subdomains (named A and B), give rise to the three-dimensional structure of HSA. This flexible structural organization allows the protein structure to adapt to a variety of ligands. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA similar to a multimeric protein. Although kinetic and thermodynamic parameters for ligand binding to HSA calculated by quantitative structure-activity relationship models are in excellent agreement with those obtained in vitro, cooperative and allosteric equilibria between different binding sites and competition between drugs or between drugs and endogenous ligands make difficult the interpretation of HSA binding properties in vivo. Binding of exogenous and endogenous ligands to HSA appears to be relevant in drug therapy and management. Here, the allosteric modulation of drug binding to HSA is briefly reviewed.

Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity. HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids. Here, we report myristate binding to the N and B conformational states of Mn(III)heme–HSA (i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal. Myristate binding to a secondary site FAx, allosterically coupled to the heme site, not only increases optical absorbance of Mn(III)heme-bound HSA by a factor of approximately three, but also increases the Mn(III)heme affinity for the fatty acid binding site FA1 by 10–500-fold. Cooperative binding appears to occur at FAx and accessory myristate binding sites. The conformational changes of the Mn(III)heme–HSA tertiary structure allosterically induced by myristate are associated with a noticeable change in both optical absorbance and NMR spectroscopic properties of Mn(III)heme–HSA, allowing the Mn(III)-coordinated water molecule to exchange with the solvent bulk. At pH = 10.0 both myristate affinity for FAx and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme binds to HSA with higher affinity than at pH 7.0 even in the absence of myristate, and the metal-coordinated water molecule is displaced. As a whole, these results suggest that FA binding promotes conformational changes reminiscent of N to B state HSA transition, and appear of general significance for a deeper understanding of the allosteric modulation of ligand binding properties of HSA.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional capacity to bind ligands (e.g. heme and drugs). Here, binding of the anti-HIV drugs abacavir, atazanavir, didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, and zidovudine to HSA and ferric heme-HSA is reported. Ferric heme binding to HSA in the absence and presence of anti-HIV drugs was also investigated. The association equilibrium constant and second-order rate constant for the binding of anti-HIV drugs to Sudlow's site I of ferric heme-HSA are lower by one order of magnitude than those for the binding of anti-HIV drugs to HSA. Accordingly, the association equilibrium constant and the second-order rate constant for heme binding to HSA are decreased by one order of magnitude in the presence of anti-HIV drugs. In contrast, the first-order rate constant for ligand dissociation from HSA is insensitive to anti-HIV drugs and ferric heme. These findings represent clear-cut evidence for the allosteric inhibition of anti-HIV drug binding to HSA by the heme. In turn, anti-HIV drugs allosterically impair heme binding to HSA. Therefore, Sudlow's site I and the heme cleft must be functionally linked.

Lactoferrin (Lf) is a mammalian iron binding protein present in external secretions and in polymorphonuclear leukocytes. Its role in host defense mechanisms related to the non-immune defense system has been definitively established. Lf has two identical iron-binding sites, far from each other (44.3 A) and magnetically non-interacting. Fe(III) ions are six-coordinated, with four donor atoms provided by protein sidechains (two Tyr, one His, one Asp) and two oxygen atoms from a bridged HCO(3)(-). This set of ligands provides an ideal coordination scheme for stable and reversible iron binding. Nuclear magnetic relaxation dispersion (NMRD) profiles of Lf are consistent with a closest distance for a single water hydrogen atom of 3.1 A. By looking at the X-ray structure of Lf (PDB ID code: 1BLF) we can locate two water oxygens at 3.95 and 4.27 A from each Fe(III), respectively. Temperature dependence data suggest that an important contribution to the overall paramagnetic contribution to the solvent water relaxation rate arises from one or more second sphere water molecules in slow exchange with the bulk. A decreasing value of the exchange rate is obtained, ranging from 1.2 to 0.7 micros in the observed temperature range (25-65 degrees C), with an activation enthalpy of 7.3+/-0.8 kJ mol(-1). The low exchange rate obtained from NMRD data can be explained by the observation that both water molecules are bound to several polar groups of the protein backbone and side chains. By increasing the pH from 6.5 to 12 two distinct titrations are observed, consistent with sequential removal of both water molecules.