Characterization of adducts formed between human serum albumin and the butadiene metabolite epoxybutanediol

1,3-Butadiene (BD) has been classified as a potential human carcinogen. It occurs in the environment as well as in industrial settings. In humans, BD is readily metabolized to reactive epoxides (e.g. 1,2-epoxy-3,4-butanediol). In this study, conjugates between human serum albumin (HSA) and EBD were synthesized (molar ratios of 1:600, 1:1 and 1:0.1; HSA/EBD) under physiological conditions. The 1:600 conjugate and a blank HSA sample were digested with trypsin to obtain specific peptides that were fractionated by preparative liquid chromatography (LC). The fractions were analyzed using nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoES-QqTOFMS). Adducted HSA tryptic peptides were identified and the adducted amino acid residues were identified by sequence analysis based on tandem mass spectrometry (MS/MS). A total of 26 2,3,4-trihydroxybutyl (THB) adducts were identified on 23 tryptic peptides in the HSA/EBD conjugate. The adducted amino acids were the N-terminal aspartic acid residue, six glutamic acid residues, five histidine residues and 14 lysine residues. Results from the nanoES-QqTOFMS experiments were used to set up a more sensitive liquid chromatographic/mass spectrometric (LC/MS) analysis using selected reaction monitoring. Eight of the adducted peptides could be detected in tryptic digests of the 1:0.1 HSA/EBD conjugate.     

Effect of pH and small inorganic ions on binding of sulfadimethoxine and sulfaphenazole to human serum albumin measured by circular dichroism

The binding of sulfadimethoxine and sulfaphenazole to human serum albumin (HSA) has been shown by circular dichroism measurements to be dependent on the N-B transition. The secondary drug binding sites were found to be optically active in the B conformation form in HSA but optically inactive in the N form. Moreover, the drug-HSA interaction in Tris-HCl buffer seems to be more sensitive to the conformational change in HSA, compared with that in the phosphate buffer.     

Glycyrrhizin binding site on human serum albumin.

The binding site of glycyrrhizin (GLZ) on human serum albumin was detected through competitive displacement experiments with GLZ and ibuprofen (IBU) (diazepam site), warfarin (WAR), salicylate (SAL) (digitoxin site), or deoxycholic acid (DCA) by means of an ultrafiltration technique. The specific binding of GLZ was subject to competitive inhibition by IBU, WAR, SAL, or DCA (1 or 4 mM). The extent of displacement was in the order of: DCA greater than IBU greater than WAR greater than SAL. Conversely, the specific bindings of WAR and DCA and the low-affinity bindings of IBU and SAL were subject to competitive inhibition by GLZ (1 or 4 mM). The extent of inhibition by GLZ was in the order of DCA greater than IBU greater than WAR not equal to SAL. In addition, the low-affinity IBU binding and the specific DCA binding showed mutual competitive inhibition at 4 mM, with almost identical displacements. It was concluded that the specific GLZ binding site on human serum albumin may be located mostly within the low-affinity IBU binding site area (probably the same as the specific DCA binding site area) and partially within the specific WAR binding site area and the low-affinity SAL binding site area.     

A novel lophine-based fluorescence probe and its binding to human serum albumin

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510 nm. The binding constant and binding number determined by Scatchard plot was 3.65 × 10⁶ M⁻¹ and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.     

Binding of naproxen and 1-anilino-8-naphtalenesulphonic acid to albumin: Characterization of their binding sites in bovine serum albumin

The binding of naproxen (NP) and 1-anilino-8-naphtalenesulphonic acid (ANS) to bovine serum albumin (BSA) has been studied by equilibrium dialysis and spectrophotofluorometry, respectively. The drug protein (D/P) ratio is found to determine the nature of binding sites for NP but not for ANS. At low D/P ratio, NP possesses a specific site in BSA and one of the tryptophan residues is part of it. Fluorometric investigation reveals non-displacement of ANS from its sites by the binding of NP to its specific site. At high D/P ratio, NP instead causes localized conformational changes in BSA. Furthermore, it is possible to infer the location of NP's specific site as loop 4 of BSA. NP and ANS binding sites have also been examined by employing Bromocresol Green (BCG) as a spectrophotometric probe. Competition studies are also able to establish distinct binding pattern for the two ligands in BSA. The displacement pattern points out the presence of independent sites for NP (specific site) and ANS (initially occupied site) in BSA in spite of their amphipathically similar nature.     

Zinc-human serum albumin association: testimony of two binding sites

The zinc cation (Zn²⁺) binding to human serum albumin (HSA) was studied using a non-equilibrium approach in order to prove two HSA binding sites. The effect of the bulk solvent pH and column temperature T on this binding and the corresponding thermodynamic data were also investigated. It appeared that the association process can be divided into two pH value ranges due to a predominant Zn²⁺ interaction with either HSA site I or site II. It was also demonstrated that the Zn²⁺ affinity for the site II was weakly affected by modifying the mobile phase pH whereas for the site I, the affinity constant increased strongly with increasing the pH of the bulk solvent.     

Characterization of subdomain IIA binding site of human serum albumin in its native, unfolded, and refolded states using small molecular probes

Subdomain IIA binding site of human serum albumin (HSA) was characterized by examining the change in HSA fluorescence in the native, unfolded, and refolded states. The study was carried out in the absence and presence of small molecular probes using steady-state and time-resolved fluorescence measurements. 2-Pyridone, 3-pyridone, and 4-pyridone bear similar molecular structures to those found in many drugs and are used here as probes. They are found to specifically bind in subdomain IIA and cause a reduction in the fluorescence intensity and lifetime of the Trp-214 residue in native HSA which is located in the same subdomain. The efficiency of energy transfer from Trp-214 fluorescence to the probes was found to depend on the degree of the spectral overlap between the donor's fluorescence and the acceptor's absorption. After probe binding in subdomain IIA, the distance between the donor and acceptor was calculated using Forster theory. The calculated quenching rate constants and binding constants were also shown to depend on the degree of spectral overlap. The results point to a static quenching mechanism operating in the complexes. Denaturation of HSA in the presence of guanidine hydrochloride (GdnHCl) starts at [GdnHCl] > 1.0 M and is complete at [GdnHCl] > or = 6.0 M. Upon unfolding, two fluorescence peaks were observed. One peak was assigned to the fluorescence of Trp-214 in a polar environment, and the other peak was assigned to tyrosine fluorescence. A reduction of the fluorescence intensity of the two peaks upon binding of the probes to the denatured HSA indicates that Tyr-263 in subdomain IIA is one of the tyrosine residues responsible for the second fluorescence peak. The results were confirmed by measuring the fluorescence spectra and lifetimes of denatured HSA at different excitation wavelengths, and of L-tryptophan and L-tyrosine free in buffer. The measured lifetimes of denatured HSA are typical of tryptophan in a polar environment and are slightly reduced upon probe binding. Dilution of the denatured HSA by buffer results in a partial refolding of subdomain IIA. This partial refolding is attributed to some swelling of the binding site caused by water. The swelling prevents a full recovery from the denatured state.     

7-Alkylaminocoumarin-4-acetic acids as fluorescent probe for studies of drug-binding sites on human serum albumin

7-Alkylaminocoumarin-4-acetic acids I-IX having alkylamino groups different in alkylchain lengths were synthesized as fluorescence probes for characterization of drug-binding sites on human serum albumin (HSA). The fluorescences of I-IX were quenched or enhanced in the presence of HSA with shifts of the emission maxima to shorter wavelength. The binding constants and the number of binding sites were determined by the spectral changes of the probes I-IX bound to HSA through analysis of Scatchard's and Job's plots. The primary binding sites of the tested probes were found to be site 2 (diazepam site) on HSA from the results of competitive displacement studies. The polarity of site 2 was estimated from the relationship between the emission maximum of the probe of IV and Z-values, and was found to be comparable to that of acetonitrile. Simple attempts to estimate the site 2 region from the molecular size of the probe of VIII obtained using the Corey-Pauling-Koltun molecular model suggest that the hydrophobic cleft at site 2 is about 21-25 A in depth. The distance between the lone tryptophan residue in HSA and probes bound to site 2 was estimated to be 15-17 A using F?rster's equation on the basis of fluorescence energy transfer. The present data suggest that I-IX are useful as fluorescence probes for the characterization of site 2 on HSA.     

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C₄₄), 83% carbamylated (C₈₃) and 85% acetylated (A₈₅) were made by treating the HSA solution with a different molar excess of potassium cyanate and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl S-200 HR column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C₈₃ and A₈₅ HSAs while C₄₄ HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 ​nm to a different extent. Whereas the values of the Stern-Volmer constant (K_SV) and bimolecular quenching rate constant (k_q) suggested, NA-HSA complex formation, binding constant (K_a) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the K_a value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.     

Spectroscopic studies on binding of Puerarin to human serum albumin

The interaction between Puerarin with human serum albumin has been studied for the first time by spectroscopic methods including fluorescence quenching technology, circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy under simulative physiological conditions. The results of fluorescence titration revealed that Puerarin can strongly quench the intrinsic fluorescence of HSA by static quenching and there is a single class of binding site on HSA. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the binding of Puerarin to HSA changed slightly molecular conformation of HSA. Furthermore, the thermodynamic functions ΔH⁰ and ΔS⁰ for the reaction were calculated to be −9.067 kJ mol⁻¹ and 54.315 J mol⁻¹ K⁻¹ according to van’t Hoff equation. These data suggested that both hydrogen bond and hydrophobic interaction play a major role in the binding of Puerarin to HSA, which is in good agreement with the result of molecular modeling study.     

Characterization of hydroxycinnamic acid derivatives binding to bovine serum albumin

Hydroxycinnamic acid derivatives (HCAs) are a group of naturally occurring polyphenolic compounds which possess various pharmacological activities. In this work, the interactions of bovine serum albumin (BSA) with six HCA derivatives, including chlorogenic acid (CHA), caffeic acid (CFA), m-coumaric acid (m-CA), p-coumaric acid (p-CA), ferulic acid (FA) and sinapic acid (SA) have been investigated by NMR spectroscopic techniques in combination with fluorescence and molecular modeling methods. Competitive STD NMR experiments using warfarin sodium and L-tryptophan as site-selective probes indicated that HCAs bind to site I in the subdomain IIA of BSA. From the analysis of the STD NMR-derived binding epitopes and molecular docking models, it was deduced that CHA, CFA, m-CA and p-CA show similar binding modes and orientation, in which the phenyl ring is in close contact with protein surface, whereas carboxyl group points out of the protein. However, FA and SA showed slightly different binding modes, due to the steric hindrance of methoxy-substituents on the phenyl ring. Relaxation experiments provided detailed information about the relationship between the affinity and structure of HCAs. The binding affinity was the strongest for CHA and ranked in the order CHA > CFA > m-CA ≥ p-CA > FA > SA, which agreed well with the results from fluorescence experiments. Based on our experimental results, we also conclude that HCAs bind to BSA mainly by hydrophobic interaction and hydrogen bonding. This study therefore provides valuable information for elucidating the mechanisms of BSA-HCAs interaction.     

Multiple and irreversible binding of cis-diamminedichloroplatinum(II) to human serum albumin and its effect on warfarin binding.

Irreversible bindings of cis-diamminedichloroplatinum(II) (cis-DDP) to human serum albumin (HSA) were investigated in a pH 7.4 buffer containing 0.1 M NaCl at various molar ratios (cis-DDP/HSA) up to 60 over a 14 d period (37 degrees C). The metal binding seemed to reach a plateau when incubated at less than 10 times excess of cis-DDP. As the molar ratio increased, the reaction rate was relatively fast within the first day, followed by a moderate increase in the metal binding. When incubated at 60 times excess of cis-DDP, the metal bound as much as 20 mol per mol of HSA in 14 d. Fluorescence quenching of the metal-bound protein suggested that the tryptophan residue was gradually exposed to a hydrophilic environment as the metal binding increased. Furthermore, cis-DDP cleaved disulfide bonds at the ratio of 1 mol of disulfide bond per 5.3 mol of the metal binding. It was therefore suggested that the metal binding also occurred at several sites other than the disulfide bond. Warfarin binding to the metal-bound protein, examined by fluorescence changes, also decreased with increasing metal binding or cleavage of the disulfide bonds. Thus, cis-DDP bound to multiple sites in addition to the lone sulfhydryl group (Cys-34), suggesting that massive conformational changes of the protein took place.