Intramolecular structure of mammalian serum albumin

Some intramolecular peculiarities of the structure of the blood albumin serum of some mammals were studied by means of probe luminescence polarization, phase fluorimetry, and solubilization. It has been shown that microviscosity of the "universal" hydrophobic nucleus where N-phenylnaphtylamine is localized is much above that in the surface regions of the protein globule filled with the probe I-anilinonaphtalene-8-sulphonate (ANS). High values of microviscosity of universal nuclei and surface hydrophobic regions were found in the albumins of man and guinea pig. In protein molecules of rabbit and bull lower microviscosity of probe surroundings was recorded both for the nucleus and peripheral surface regions. Rat albumin had higher microviscosity of ANS localization sites, while the density of the hydrophobic nucleus was comparatively low. At the same time an opposite phenomenon was observed in pig albumin preparations.     

Studies on the interaction between Ag(+) and human serum albumin

The interaction between Ag(+) and human serum albumin (HSA) has been intensively studied by means of equilibrium dialysis, ligand-to-metal charge transition (LMCT) bands, circular dichroism (CD) and Raman spectroscopy. Scatchard analysis of the results of equilibrium dialysis indicates the presence of two types of binding sites for Ag(+) on HSA, and the orders of magnitude of binding stability constants are found to be 10(5) and 10(4), respectively. During the binding process, a gradual increase in absorbance values of LMCT bands is observed with time-scanning UV absorption spectra, implying the Ag(I) centers are continually formed in HSA. The time-scanning CD spectra provide evidence that the binding of Ag(+) induces HSA to undergo a slow rearrangement of tertiary structure, and to change from the original conformation in the absence of Ag(+) (B-state) to conformation binding with Ag(+) (A-state). The rate constants and activation free energy of A-B transition are calculated. The Raman spectrum of Ag(I)-HSA system shows distinct vibration bands at 224 and 246 cm(-1) in the low-frequency region, which significantly reveal the formation of Ag-S and Ag-N bonds. In addition, the electrostatic interaction between Ag(+) and negatively charged oxygen is also detected with Raman spectroscopy.     

Dielectric behavior and atomic structure of serum albumin

After 1946, serum albumin was available for studies. Its residue sequence and internal disulfide bonding was developed by 1976. We began to make dielectric dispersion studies and apply Perrin's equations for rotational relaxation times around the two axes of revolution in 1938. These data indicated that albumin should have an elongated shape. In 1992 atomic structure data indicated the molecule was heart-shaped. A similar 1998 study of albumin complexed with fatty acid showed that the molecule was substantially rearranged. We found that the dielectric constant of albumin solutions was sensitive to fatty acid content, making this property an attractive probe in stop–flow kinetic studies. Such studies show that the fatty acid reaction is a two-step process. The fatty acid first binds to exterior sites in a diffusion-limited second order reaction complete in 1 ms. Then a first order rearrangement reaction with 400 ms half-life follows. Thus the highly specialized serum albumin sequence of amino acid residues determines not only the structure of the unligated molecule, but also the distinctive structures of the numerous multiligated molecules.     

Analysis of the structure and dynamics of human serum albumin

A computational approach to designing a peptide-based ligand for the purification of human serum albumin (HSA) was undertaken using molecular docking and molecular dynamics (MD) simulation. A three-step procedure was performed to design a specific ligand for HSA. Based on the candidate pocket structure of HSA (warfarin binding site), a peptide library was built. These peptides were then docked into the pocket of HSA using the GOLD program. The GOLDscore values were used to determine the affinity of peptides for HSA. Consequently, the dipeptide Trp–Trp, which shows a high GOLDscore value, was selected and linked to a spacer arm of Lys[CO(CH₂)₅NH] on the surface of ECH-lysine sepharose 4 gel. For further evaluation, the Autodock Vina program was used to dock the linked compound into the pocket of HSA. The docking simulation was performed to obtain a first guess of the binding structure of the spacer–Trp–Trp–HSA complex and subsequently analyzed by MD simulations to assess the reliability of the docking results. These MD simulations indicated that the ligand–HSA complex remains stable, and water molecules can bridge between the ligand and the protein by hydrogen bonds. Finally, absorption spectroscopic studies were performed to illustrate the appropriateness of the binding affinity of the designed ligand toward HSA. These studies demonstrate that the designed dipeptide can bind preferentially to the warfarin binding site. Graphical Abstract

Three-step computational approach to the design of a dipeptide ligand for human serum albumin purification exploiting structure-based docking and molecular dynamics simulation     

Biosynthesis of serum albumin in rat liver. Isolation and probable structure of ''proalbumin'' from rat liver.

1. Two methods are described for the preparation of 'proalbumin' in good yields from rat liver. 2. One of the methods does not depend on the use of specific antisera. 3. The product from both methods is identical as judged by electrophoresis on polyacrylamide gel, isoelectric focusing on pH gradients, ion-exchange chromatography and quantitative immunoelectrophoresis. The protein also appears to be radiochemically pure by these criteria. 4. The protein is free from serum albumin as judged by isoelectric focusing and co-chromatography on ion-exchange columns. It is judged to be free from other proteins by these same criteria and by specific precipitation with antibody. 5. It is converted into serum albumin by limited tryptic hydrolysis. The albumin so produced has the same N-terminal (glutamic acid) and C-terminal (alanine) amino acids as reported for rat serum albumin. 6. A hexapeptide is liberated from the N-terminal end of 'proalbumin' simultaneously. It contains three arginine, one phenylalanine, one valine and one glycine residues.     

Studies on the interaction between tetraphenylporphyrin compounds and bovine serum albumin.

The interaction of three porphyrin compounds with bovine serum albumin (BSA) was examined by fluorescence emission spectra at the excitation wavelength 280 nm and in UV-Vis absorption spectra. Through fluorescence quenching experiments, it was confirmed that the combination of three porphyrin compounds with BSA was a single static quenching process. The binding constant K(A), the thermodynamic parameters enthalpy change (DeltaH(0)), Gibbs free energy change (DeltaG(0)) and entropy change (DeltaS(0)) were obtained. It was found that hydrophobic interaction played a main role in tetraphenylporphyrin (TPP) or tetraparacholophenylporphyrin (TClPP) binding to BSA, while tetraparamethoxyphenylporphyrin (TMEOPP) mainly based on van der Waals' force. According to F?ster energy transfer, the separate distance r, the energy transfer efficiency E and F?ster radium R(0) were calculated. The results obtained from the above experiments showed that three porphyrin compounds were tightly bound to BSA.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Effect of Chinese medicine alpinetin on the structure of human serum albumin

Alpinetin (7-hydroxy-5-methoxyflavanone), one of the main constituents from the seeds of Alpinia katsumadai Hayata, belongs to flavonoids with its usefulness as antibacterial, anti-inflammatory and other important therapeutic activities of significant potency and low systemic toxicity. In this paper, the interaction of alpinetin to human serum albumin (HSA) has been studied for the first time by spectroscopic method including Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD), and UV-absorption spectroscopy in combination with fluorescence quenching study under physiological conditions with drug concentrations of 3.3 x 10(-6)-2.0 x 10(-5)mol/L. The results of spectroscopic measurements and the thermodynamic parameters obtained (the enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be -10.20 kJ/mol and 53.97 J/molK(-1) according to the Van't Hoff equation) suggest that hydrophobic interaction is the predominant intermolecular forces stabilizing the complex, which is also good agreement with the results of molecule modeling study. The alterations of protein secondary structure in the presence of alpinetin in aqueous solution were quantitatively estimated by the evidences from FT-IR and CD spectroscopy with reductions of alpha-helices about 24%, decreases of beta-sheet structure about 2%, and increases of beta-turn structure about 21%. The quenching mechanism and the number of binding site (n approximately 1) were obtained by fluorescence titration data. Fluorescent displacement measurements confirmed that alpinetin bind HSA on site III. In addition, the effects of common ions on the constants of alpinetin-HSA complex were also discussed.     

Effect of ionic liquids on the solution structure of human serum albumin

The effect of several ionic liquids (ILs) on the solution structure of human serum albumin (HSA) is revealed by continuous wave electron paramagnetic resonance (EPR) spectroscopy and nanoscale distance measurements with double electron-electron resonance (DEER) spectroscopy. HSA, the most abundant protein in human blood, is able to bind and transport multiple fatty acids (FAs). Using spin-labeled FA, the uptake of the FA by the protein and their spatial distribution in the protein can be monitored. The FA distribution provides an indirect yet effective way to characterize the structure of the protein in solution. Addition of imidazolium-based ILs to an aqueous solution of HSA/FA conjugates is accompanied by significant destabilization and unfolding of the protein's tertiary structure. In contrast, HSA maintains its tertiary structure when choline dihydrogenphosphate (dhp) is added. The comparison of FA distance distributions in HSA with and without choline dhp surprisingly revealed that with this IL, the FA anchoring units are in better agreement with the crystallographic data. Furthermore, the FA entry point distribution appears widened and more asymmetric than in pure buffer. These results indicate that choline dhp as a cosolvent may selectively stabilize HSA conformations closer to the crystal structure out of the overall conformational ensemble.     

Studies on the interaction between benzophenone and bovine serum albumin by spectroscopic methods

The interaction between benzophenone (BP) and bovine serum albumin (BSA) was investigated by the methods of fluorescence spectroscopy combined with UV–Vis absorption and circular dichroism (CD) measurements under simulative physiological conditions. The experiment results showed that the fluorescence quenching of BSA by BP was resulted from the formation of a BP–BSA complex and the corresponding association constants (K _a) between BP and BSA at four different temperatures had been determined using the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be –43.73 kJ mol⁻¹ and −53.05 J mol⁻¹ K⁻¹, respectively, which suggested that hydrogen bond and van der Waals force played major roles in stabilizing the BP–BSA complex. Site marker competitive experiments indicated that the binding of BP to BSA primarily took place in site I (sub-domain IIA). The conformational investigation showed that the presence of BP decreased the α-helical content of BSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules.     

Studies on the interaction between benzidine and bovine serum albumin by spectroscopic methods

The interaction between bovine serum albumin (BSA) and benzidine (BD) in aqueous solution was investigated by fluorescence spectroscopy, circular dichroism (CD) spectra and UV–Vis spectroscopy, as well as resonance light scattering spectroscopy (RLS). It was proved from fluorescence spectra that the fluorescence quenching of BSA by BD was a result of the formation of BD–BSA complex, and the binding constants (K _a) were determined according to the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be ?34.11 kJ mol^?1 and ?25.89 J mol^?1 K^?1, respectively, which implied that van der Waals force and hydrogen bond played predominant roles in the binding process. The addition of increasing BD to BSA solution caused the gradual enhancement in RLS intensity, exhibiting the forming of the aggregate. Moreover, the competitive experiments of site markers suggested that the binding site of BD to BSA was located in the region of subdomain IIA (sudlow site I). The distance (r) between the donor (BSA) and the acceptor (BD) was 4.44 nm based on the Förster theory of non–radioactive energy transfer. The results of synchronous fluorescence and CD spectra demonstrated the microenvironment and the secondary conformation of BSA were changed.     

Studies on the interaction between Oxaprozin-E and bovine serum albumin by spectroscopic methods

The interaction between Oxaprozin-E and bovine serum albumin (BSA) was studied by spectroscopic methods including fluorescence and UV–vis absorption spectroscopy. The quenching mechanism of fluorescence of BSA by Oxaprozin-E was discussed to be a dynamic quenching procedure. The number of binding sites n and apparent binding constant K was measured by fluorescence quenching method. The thermodynamics parameter ΔH, ΔG, ΔS were calculated. The results indicate the binding reaction was mainly entropy-driven and hydrophobic forces played major role in the binding reaction. The distance r between donor (BSA) and acceptor (Oxaprozin-E) was obtained according to Förster theory of non-radioactive energy transfer.