Determining molecular binding sites on human serum albumin by displacement of oleic acid

An NMR method was developed for determining binding sites of small molecules on human serum albumin (HSA) by competitive displacement of (13)C-labeled oleic acid. This method is based on the observation that in the crystal structure of HSA complexed with oleic acid, two principal drug-binding sites, Sudlow's sites I (warfarin) and II (ibuprofen), are also occupied by fatty acids. In two-dimensional [(1)H,(13)C]heteronuclear single quantum coherence NMR spectra, seven distinct resonances were observed for the (13)C-methyl-labeled oleic acid as a result of its binding to HSA. Resonances corresponding to the major drug-binding sites were identified through competitive displacement of molecules that bind specifically to each site. Thus, binding of molecules to these sites can be followed by their displacement of oleic acids. Furthermore, the amount of bound ligand at each site can be determined from changes in resonance intensities. For molecules containing fluorine, binding results were further validated by direct observations of the bound ligands using (19)F NMR. Identifying the binding sites for drug molecules on HSA can aid in determining the structure-activity relationship of albumin binding and assist in the design of molecules with altered albumin binding.     

A spectroscopic study of the interaction of isoflavones with human serum albumin

Genistein and daidzein, the major isoflavones present in soybeans, possess a wide spectrum of physiological and pharmacological functions. The binding of genistein to human serum albumin (HSA) has been investigated by equilibrium dialysis, fluorescence measurements, CD and molecular visualization. One mole of genistein is bound per mole of HSA with a binding constant of 1.5 +/- 0.2 x 10(5) m(-1). Binding of genistein to HSA precludes the attachment of daidzein. The ability of HSA to bind genistein is found to be lost when the tryptophan residue of albumin is modified with N-bromosuccinimide. At 27 degrees C (pH 7.4), van't Hoff's enthalpy, entropy and free energy changes that accompany the binding are found to be -13.16 kcal x mol(-1), -21 cal x mol(-1) K(-1) and -6.86 kcal x mol(-1), respectively. Temperature and ionic strength dependence and competitive binding measurements of genistein with HSA in the presence of fatty acids and 8-anilino-1-naphthalene sulfonic acid have suggested the involvement of both hydrophobic and ionic interactions in the genistein-HSA binding. Binding measurements of genistein with BSA and HSA, and those in the presence of warfarin and 2,3,5-tri-iodobenzoic acid and F?rster energy transfer measurements have been used for deducing the binding pocket on HSA. Fluorescence anisotropy measurements of daidzein bound and then displaced with warfarin, 2,3,5-tri-iodobenzoic acid or diazepam confirm the binding of daidzein and genistein to subdomain IIA of HSA. The ability of HSA to form ternery complexes with other neutral molecules such as warfarin, which also binds within the subdomain IIA pocket, increases our understanding of the binding dynamics of exogenous drugs to HSA.     

NMR study of Ni2+ binding to the H-N-H endonuclease domain of colicin E9.

Ni2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2- at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)N]2-. Dialysis against > or =0.2 M NaCl was required to remove the [Ni(EDTA)(H2O)n]2-. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni(EDTA)(H2O)n]2- should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+.     

Proton nuclear magnetic resonance study on the multimode interactions of human serum albumin with drug molecules

The interactions of human serum albumin (HSA) with a number of ligands (mostly drugs) were examined by proton nuclear magnetic resonance spectroscopy. Ligand presence-absence difference spectra of HSA solutions were measured. Nonspecifically bound drugs such as tiaramide showed difference spectrum patterns which were similar to the spectra of the drugs themselves but were broadened as to the line-widths of signals. Thus, the difference spectra of these drugs reflect only the changes in the surroundings of the drug molecules, that is, between the bound and free states. In contrast, specifically bound drugs like ibuprofen and warfarin showed difference spectra in which signals from the HSA molecule only were observed. Furthermore, according to the characteristic peaks in these difference spectrum patterns, specifically bound drugs may be classified into several groups; the drugs in the first group bind to the ibuprofen binding site, those in the second group to the warfarin binding site, and those in the third group to sites other than the warfarin and ibuprofen sites. These findings suggest that the specific binding of drugs to HSA brings about a conformational change of this protein which is specifically correlated to the binding site.     

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.     

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.     

Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis.

Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.     

Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations

Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer’s disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn²⁺, Cu²⁺, Mg²⁺ and Ca²⁺ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn²⁺ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1–2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn²⁺-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca²⁺/Mg²⁺-binding, Zn²⁺ or Cu²⁺ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca²⁺-binding to HSA synergistically support each other. Zn²⁺ and Cu²⁺ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.     

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 1. Evidence from deuterium NMR measurements.

Deuterium NMR has been used to investigate the structure and dynamic state of cytochrome c complexed with bilayers of cardiolipin. Reductive methylation was employed to prepare [N epsilon, N epsilon-C2H3]lysyl cytochrome c, and deuterium exchange provided labeling of backbone sites to give [amide-2H]cytochrome c or more selective labeling of just histidine residues in [epsilon-2H]histidine cytochrome c. Deuterium NMR measurements on [N epsilon, N epsilon-C2H3]lysyl cytochrome c in the solid state showed restricted motions, fairly typical of the behavior of aliphatic side-chain sites in proteins. The [amide-2H]cytochrome c provided "immobile" amide spectra showing that only the most stable backbone sites remained labeled in this derivative. Relaxation measurements on the aqueous solution of [amide-2H]cytochrome c yielded a rotational correlation time of 7.9 ns for the protein, equivalent to a hydrodynamic diameter of 4.0 nm, just 0.6 nm greater than its largest crystallographic dimension. Similar measurements on [epsilon-2H]histidine cytochrome c in solution showed that all labeled histidine residues were also "immobile" compared with the overall reorientational motion of the protein. The interaction with cardiolipin bilayers appeared to create a high degree of mobility for the side-chain sites of [N epsilon, N epsilon-C2H3]lysyl cytochrome c and perturbed backbone structure to instantaneously release all deuterons in [amide-2H]cytochrome c. The [epsilon-2H]histidine cytochrome c derivative, when complexed with cardiolipin, failed to produce any detectable wide-line 2H NMR spectrum, demonstrating that the overall reorientational motion of bound protein was not isotropic on the NMR time scale, i.e., tau c greater than 10(-7)s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin. I. The filling of individual fatty acid binding sites

13C NMR chemical shift and intensity results for a series of carboxyl 13C-enriched saturated fatty acids (8-18 carbons) bound to bovine serum albumin (BSA) are presented as a function of increasing fatty acid (FA)/BSA mole ratio. Spectra for long-chain (greater than or equal to 12 carbons) FA X BSA complexes exhibited up to five FA carboxyl resonances, designated a, b, b', c, and d. Only three resonances (peaks b, b', and d) were observed below 3:1 FA X BSA mole ratio, and at greater than or equal to 3:1 mole ratio, two additional resonances were observed (peaks c and a). In a spectrum of 5:1 stearic acid X BSA complexes, peaks b, b', and d each represented approximately one-fifth, and peak c approximately two-fifths, of the total FA carboxyl intensity. Plots of total carboxyl/carbonyl intensity ratio as a function of FA X BSA mole ratio were linear up to 7-9 mole ratio. Deviation from linearity at mole ratios greater than or equal to 7 was accompanied by the detection of crystalline unbound FA (as 1:1 acid/soap) by X-ray diffraction. In contrast to long-chain FA X BSA complexes, 13C NMR spectra of octanoic acid X BSA complexes yielded only one FA carboxyl resonance (peak c) at FA X BSA mole ratios between 1 and 20. We conclude: peaks b, b', and d represent FA bound to three individual high affinity (primary) long-chain FA binding sites on BSA; peak c represents FA bound to several secondary long-chain (or primary short-chain) FA binding sites on BSA; peak a represents long-chain FA bound to an additional lower affinity binding site. We present a model that correlates the observed 13C NMR resonances with individual binding site locations predicted by a recent three-dimensional model of BSA.     

Histidine146 of human serum albumin plays a prominent role at the interface of subdomains IA and IIA in allosteric ligand binding

Fatty acids are endogenous ligands of human serum albumin (HSA) that induce conformational changes and participate in allosteric ligand binding to HSA. In a previous study, we showed that, when myristate (MYR) is present, the binding of [(14) C]ketoprofen (KP) to subdomain IA of HSA was increased, indicating that, when MYR binds to HSA, a new binding site in formed in that region. Meanwhile, an N-B transition has been reported to increase the binding of ligands at alkaline pH when the status of albumin is the B-conformer. Six histidine single mutants of HSA, H9A, H39A, H67A, H105A, H128A and H146A were produced and photolabeled with [(14) C]KP at pH 6.5, 7.4 and 8.2 and the role of each histidine in causing the N-B transition induced allosteric ligand binding was examined. Cyanogen bromide cleavage of the photolabeled native HSA showed that subdomain IA was the site of the allosteric binding of KP at pH 8.2. From the photolabeling results, H146 was found to play a prominent role whilst H128 played little or no role in the allosteric binding. However, the remaining 4 mutants did not show a clear photolabeling pattern that was similar to either native HSA or H146A and, as a result, no firm conclusions can be made. An additional histidine mutant, H146I, was produced to confirm the results for H146A. A similar experiment using H146I showed that a benzene ring-like structure at position 146 is required for the allosteric ligand binding to occur.     

The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography

Human serum albumin (HSA) has been shown to bind 2–3 mol of Zn²⁺, Ni²⁺, or Cd²⁺ per mole of protein with apparent dissociation constants (K_d) in the range of 10 μm. Rabbit histidine-rich glycoprotein (HRG) binds 13, 9, and 6 mol of Zn²⁺, Ni²⁺, and Cd²⁺ per mole of protein, respectively, with apparent K_ds also near 10 μm. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent K_ds may underestimate HRGs true affinity for metal ions. The relative affinities of HSA and HRG for metal ions were found to be Zn²⁺ > Ni²⁺ > Cd²⁺. In addition, histidine (a serum metal chelator) affected the binding of Ni²⁺ by both proteins but not that of Zn²⁺ or Cd²⁺. At physiological concentrations of HSA (250 μm), HRG (2.5 μm), and histidine (100 μm), HRG bound 36% of the Zn²⁺, 9% of the Ni²⁺, and 13% of the Cd²⁺ at a total metal concentration of 25 μm. Under the same conditions HSA held 37% of the Zn²⁺, 14% of the Ni²⁺, and 56% of the Cd²⁺. Thus, HSA appears to have a lower intrinsic affinity for the three metals than HRG but would be expected to bind a higher proportion of these metals in serum. A specific immunoadsorbent column was prepared and used to study the metal binding by HRG in serum directly. Both ⁶⁵Zn²⁺ and ⁶³Ni²⁺ were associated with HRG in aliquots of rabbit serum after incubation with the corresponding metal ion. This evidence indicates that HRG must be considered as a metal binding component of serum.     

Characterization of the interaction between prostacyclin and human serum albumin using a fluorescent analogue, 2,6-dichloro-4-aminophenol iloprost

We synthesized a fluorescent probe, 2,6-dichloro-4-aminophenol iloprost or dichlorohydroxyphenylamide of iloprost (DCHPA-iloprost) by reacting the stable prostacyclin analog, iloprost (ZK 35 374), with 2,6-dichloro-4-aminophenol with a yield of 60%. This probe exhibited an optical spectrum which overlapped with the emission spectrum of the sole tryptophan of human serum albumin (HSA). Energy transfer from the tryptophan residue to the phenol moiety of DCHPA-iloprost was observed. We utilized this donor-quenching phenomenon to quantitate the binding stoichiometry and affinity as well as the association rate of DCHPA-iloprost binding to HSA. As DCHPA-iloprost showed similar binding characteristics similar to those of iloprost and prostacyclin and competed with iloprost for HSA binding sites, we used DCHPA-iloprost as a probe to locate the binding domain of prostacyclin (PGI2) in HSA. The distance between the tryptophan indole and the phenol group of DCHPA-iloprost was estimated to be 15-18 A. Because iloprost binding to HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA molecules. A possible molecular mechanism by which HSA reduces the chemical degradation of PGI2 and stabilizes its activity could be derived from this model.     

Photochemical reactions of fentichlor with soluble proteins

The photochemical reactions of the photoallergen fentichlor with soluble proteins have been studied. [35S]Fentichlor was shown to bind covalently to human serum albumin (HSA) when irradiated with UV light (313 nm). HSA had the ability to bind at least eight molecules of fentichlor per molecule of protein. Fractionation of fentichlor-HSA photoadducts after (a) treatment with cyanogen bromide and (b) reduction, carboxymethylation and digestion with trypsin showed that the bound fentichlor was distributed fairly evenly throughout the sequence of the HSA molecule. Fentichlor was also shown to form photoadducts with human gamma-globulin and with bovine insulin. Its binding to insulin was restricted to the B chain of the molecule. Fundamental differences between the photochemical reactions of the photo-allergens fentichlor and tetrachlorosalicylanilide (T4CS) with soluble proteins are discussed. The reactions of fentichlor with soluble proteins are not restricted to specific binding sites (unlike T4CS). Fentichlor has the potential to react photochemically with a wide range of proteins in the epidermis and dermis, to form antigens.     

Characterisation of oxidised 7Fe dicluster ferredoxins with NMR spectroscopy

Dicluster ferredoxins (Fds) from Sulfolobus acidocaldarius and Desulfovibrio africanus (FdIII) have been studied using 1H NMR. Both wild-type proteins contain a [3Fe-4S]+/0 and a [4Fe-4S]2+/+ cluster as isolated. The [4Fe-4S]2+/+ cluster (cluster II) is bound by cysteine residues arranged in a classic ferredoxin motif: CysI-(Xaa)2-CysII-(Xaa)2-CysIII-(Xaa)n-CysIV-Pro , whilst the binding motif of the [3Fe-4S]+/0 cluster (cluster I) has a non-ligating aspartic acid (Asp14) at position II, i.e. CysI-(Xaa)2-Asp-(Xaa)2-CysIII. D. africanus FdIII undergoes facile cluster transformation from the 7Fe form to the 8Fe form, but S. acidocaldarius Fd does not. Many factors determine the propensity of a cluster to undergo interconversion, including the presence, and correct orientation, of a suitable ligand. We have investigated this using 1H NMR by introducing a potential fourth ligand into the binding motif of cluster I of D. africanus FdIII. Asp14 has been mutated to cysteine (D14C), glutamic acid (D14E) and histidine (D14H). Cluster incorporation was performed in vitro. The cluster types present were identified from the chemical shift patterns and temperature-dependent behaviour of the hyperfine-shifted resonances. Factors influencing cluster ligation and cluster interconversion, in vitro, are discussed. Furthermore, the data have established that the residue at position II in the cluster binding motif of cluster I is influential in determining the chemical shift pattern observed for a [3Fe-4S]+ cluster when a short/symmetric binding motif is present. Based on this, a series of rules for characterising the 1H NMR chemical shifts of mono- and di-cluster [3Fe-4S]+ cluster-containing ferredoxins is given.     

In vitro binding of leukotriene B4 (LTB4) to human serum albumin: evidence from spectroscopic, molecular modeling, and competitive displacement studies

Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B4 (LTB4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB4 was enhanced fivefold in the presence of HSA. The red-shifted, hypochromic, and reduced vibrational fine structure of the ligand/protein UV absorption spectrum indicated complexation of the two molecules in solution. Results obtained from CD titration experiments were subjected to non-linear regression analysis to estimate the binding parameters (Ka = 6.7 x 10(4) M(-1), n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB4/HSA complex, suggesting the role of a high-affinity fatty acid HSA binding site in the leukotriene complexation. Molecular modeling calculations based on the crystal structure of HSA predicted that the long-chain fatty acid site that overlaps with drug binding site II in subdomain IIIA was the most likely binding location for LTB4. Using the drug site II-specific marker ligand rac-ibuprofen, this prediction was confirmed with induced-CD displacement measurements. To the authors' knowledge, the current study represents the first demonstration of binding of LTB4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.     

Design, synthesis and spectroscopic studies of resveratrol aliphatic acid ligands of human serum albumin

As one of the natural polyphenols, resveratrol possesses hydroxyl substituted trans-stilbene structure and exerts impact on health by inhibiting multiple human enzymes, such as cyclooxygenase, F1 ATPase, and tyrosinase. Resveratrol has to be bound by human serum albumin (HSA) to keep a high concentration in serum, since its solubility is low in water. To improve water solubility and bioavailability, two resveratrol aliphatic acids and their esters have been designed and synthesized. The solubilities of the resveratrol and its derivatives have been measured using a standard procedure. The two aliphatic acids showed better solubilities in pure water and phosphate buffer (pH 7). The binding affinities of resveratrol derivatives for HSA were also measured, and the drug-protein interaction mechanism was investigated using fluorescence, UV-vis, and NMR spectroscopies. Interestingly, resveratrol hexanoic acid (5) was found to be a much better ligand (K(a)=(6.70+/-0.10)x10(6) M(-1)) for HSA than resveratrol (K(a)=(1.64+/-0.07)x10(5) M(-1)), and there was 41-fold improvement for the binding affinity. It was the first time that the increase of fluorescence of resveratrol moiety was observed during the binding to HSA, suggesting that 5 should be bound tightly by HSA. The UV-vis absorption spectroscopy revealed a maximum absorption shift from 318 to 311 nm with decreasing intensity by 20% upon complexation, suggesting that the pi-pi conjugation of the stilbene structure was impaired during the binding. Although HSA was reported to have only one binding site for resveratrol, the Job's and molar ratio plots suggested that HSA should bind two molecules of 5. NMR study suggested that phenyl group (B ring) in the center of the molecule of 5 should be involved in the pi-pi stacking interactions with HSA aromatic amino acid residues. Molecular geometry calculation of 5 with Spartan software showed that the stilbene structure had two conformers, orthogonal and planar ones. The former (E=-1.432 KJ/mol) was more stable than the latter (E=-0.128 KJ/mol), suggesting that the former should be the conformer of 5 in the complexation with HSA.     

Reversible binding of ethacrynic acid to human serum albumin: Difference circular dichroism study

The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anticooperative) and to site III (cooperative). Chirality 11:33–38, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis,characterization, and binding assessment with human serum albumin of three bipyridine lanthanide(III) complexes

In this work, the terbium(III), dysprosium(III), and ytterbium(III) complexes containing 2, 2′-bipyridine (bpy) ligand have been synthesized and characterized using CHN elemental analysis, FT-IR, UV–Vis and ¹H-NMR techniques and their binding behavior with human serum albumin (HSA) was studied by UV–Vis, fluorescence and molecular docking examinations. The experimental data indicated that all three lanthanide complexes have high binding affinity to HSA with effective quenching of HSA fluorescence via static mechanism. The binding parameters, the type of interaction, the value of resonance energy transfer, and the binding distance between complexes and HSA were estimated from the analysis of fluorescence measurements and Förster theory. The thermodynamic parameters suggested that van der Waals interactions and hydrogen bonds play an important role in the binding mechanism. While, the energy transfer from HSA molecules to all these complexes occurs with high probability, the order of binding constants (BpyTb > BpyDy > BpyYb) represents the importance of radius of Ln³⁺ ion in the complex-HSA interaction. The results of molecular docking calculation and competitive experiments assessed site 3 of HSA, located in subdomain IB, as the most probable binding site for these ligands and also indicated the microenvironment residues around the bound mentioned complexes. The computational results kept in good agreement with experimental data.     

Identification of Lys190 as the primary binding site for pyridoxal 5'-phosphate in human serum albumin.

The covalent binding of pyridoxal 5'-phosphate (PLP) to human serum albumin (HSA) is important in the regulation of PLP metabolism. In plasma, PLP is bound to HSA at a single high-affinity and at two or more nonspecific sites. To characterize the primary PLP binding site, HSA was incubated with [3H] PLP, and the Schiff base linkage was reduced with potassium borohydride. Tryptic peptides were purified, and the major labeled peptide was sequenced. Amino acid analysis confirmed a homogeneous peptide Leu-Asp-Glu-Leu-Arg-Asp-Glu-Gly-Xaa-Ala-Ser-Ser-Ala-Lys which corresponds to residues 182-195 of HSA. The data indicate that Lys190 is the primary PLP binding site. This Lys residue is distinct from other sites of covalent adduct formation; namely, the primary sites for nonenzymatic glycosylation (Lys525) and acetylation by aspirin (Lys199).