Secondary Structure Of Albumin Research Articles

X-ray Microscopy Studies of Protein Adsorption on a Phase Segregated Polystyrene/Polymethylmethacrylate Surface. 2. Effect of pH on Site Preference

X-ray photoemission electron microscopy (XPEEM) using synchrotron radiation illumination has been used to study the adsorption of human serum albumin (HSA) onto a phase segregated polystyrene/polymethylmethacrylate (PS/PMMA) blend surface from solutions of five different pH values. The absolute coverage of albumin on each of three chemically distinct components of the surface, PS domains, PMMA domains, and the interface between the domains, was determined from a quantitative analysis of C 1s image sequences. At all pH values, the preferred adsorption site is the interface. At neutral pH (7.0), albumin showed a slight preference for PS regions relative to PMMA. At strongly acidic pH (2.0) and strongly basic pH (10.0), similar amounts of albumin adsorb on the PS and PMMA regions. However, at pH 4.0, the amount of albumin adsorbed on PMMA domains is approximately 1.6 times greater than that on PS domains, while at pH 8.6 the amount of albumin adsorbed on PMMA is one-half that adsorbed on PS domains. The pH dependence of the site preference is rationalized in terms of the known changes of albumin conformation with pH [Peters, T., Jr. All About Albumin: Biochemistry, Genetics, and Medical Applications; Academic Press: New York, 1995]. We infer from our results that the site preference of albumin adsorption on PS/PMMA blends is related mainly to changes in hydrophobic interactions, which are driven by pH-dependent electrostatic effects, that is, changes to the protein surface structure as the charge on the protein changes. The results provide insight into changes in the secondary structure of albumin in acid and basic media.

Effects of sodium tetraborate and boric acid on nonisothermal mannitol crystallization in frozen solutions and freeze-dried solids

The purpose of the present study was to elucidate the effects of sodium tetraborate (borax) and boric acid on the crystallization of mannitol in frozen aqueous solutions and freeze-dried solids. Thermal analysis of frozen solutions showed that sodium tetraborate inhibits mannitol crystallization at sodium tetraborate/mannitol molar concentration ratios of approximately 0.05, which is much lower than the other co-solutes studied (boric acid, sucrose, sodium phosphate buffer). Inhibition of the mannitol crystallization in frozen solutions resulted in highly amorphous mannitol in the freeze-dried solids. Mannitol remained in an amorphous state in some of the combination freeze-dried solids, even at elevated temperatures. Changes in the thermal transition temperatures (glass transition temperature of maximally freeze-concentrated solute ( T g′) and glass transition temperature of freeze-dried solid ( T g)) suggested reduced mannitol molecular mobility with increases in the sodium tetraborate ratio. Fourier-transform infrared spectroscopy (FT-IR) analysis of the bovine serum albumin secondary structure showed apparent protein structure-stabilizing effects of the amorphous mannitol and sodium tetraborate combination during the freeze-drying process. The mannitol and sodium tetraborate combination also protected lactate dehydrogenase (LDH) from inactivation during freeze-drying. We conclude that the complex formation and the accompanying reduction in molecular mobility make sodium tetraborate an effective mannitol crystallization inhibitor in frozen solutions and freeze-dried solids.

Structure and interactions of albumin–lipid systems as studied by infrared spectroscopy

Infrared spectroscopy has been applied here to study the structure and interactions of albumin–lipid systems in heavy water dispersions. The lipids used were dimyristoylphosphatidylcholine (DMPC) and cholesteryl palmitate (CP). The following results were obtained: (a) lipid vesicles consisting of DMPC and CP modify significantly the albumin secondary structure, particularly at high proportions of CP, as reflected by the amide I profiles and the kinetics of H/D isotopic exchange; (b) as to phospholipid vesicle stability, the transition temperature of DMPC is lowered upon addition of CP, with DMPC/CP ratio higher than 1.0. By contrast, no change has been observed when the above ratio is lower than this value. The presence of albumin, in any case, stabilizes the phospholipid vesicles in such a way that the transition temperature is the same whatever be the lipid composition of the vesicles.

Determination of the secondary structure of isomeric forms of human serum albumin by a particular frequency deconvolution procedure applied to Fourier transform IR analysis.

A new deconvolution procedure was applied to the analysis of Fourier transform ir spectra of human serum albumin secondary structure in the native state and in states denatured by heat and acid treatment. The deconvolution method is based on the use of the Conjugate Gradient Minimization Algorithm, with the addition of suitable constraints directly obtained by the application to the measured spectrum of the second derivative operator. This method computes central band frequency, bandwidth, and amplitude of the different spectral components of conformation-sensitive amide bands. In the specific case, it was applied to analysis of the amide I band, and the quantitative determination of the different secondary structures (alpha-helix, beta-sheet, beta-turns, and random) was attempted for all the samples examined. The precision of the quantitative determination depends on the amounts of these structures present in the protein. The coefficient of variation is < 10% for values of amide I component > 15%. The accuracy was tested by comparing, by means of linear regression, the results obtained for human serum albumin, hemoglobin, alpha-chymotrypsin, and cytochrome c, using our method, with those obtained by x-ray crystallography and CD; the results obtained by other vibrational spectroscopic approaches were also compared. The fit standard error between x-ray and ir secondary structure values estimated by our method is 2.5% for alpha-helix, 7.16% for beta structures, and 5.1% for other structures (turns and random coils). Quantitative results are given for the secondary structures (alpha-helix, turns, and beta-strands) present in the native state (turns and beta-strands up to now unknown in aqueous solution), together with the percentages of these structures and additional ones (random coils and beta-sheets) formed during denaturization.

Contribution of a peroxide adduct of copper(II)-peptide complex to modify the secondary structure of albumin.

We have found that copper(II) compounds containing a peptide group in the chelate exhibit high activity for modification or degradation of albumin in the presence of hydrogen peroxide, whereas no activity was detected for the copper(II) compounds without an amide-group. It is suggested that presence of the amide-group in the ligand may play an important role in the formation of a peroxide adduct and in activation of the peroxide ion, leading to cleavage of the peptide bond of a neighboring protein. It is implied that conversion of normal cellular prion protein PrPC into a disease-causing isoform, PrPSc is attributed to the activated peroxide ion coordinated to a copper(II) captured in the NH2-terminal domain of the PrPC.

Far-infrared radiation effect on the structure and properties of proteins

This paper describes the effect of far-infrared radiation on the activity and secondary structure of albumin, alcohol dehydrogenase, peroxidase and trypsin. For the first time the data obtained allowed a conclusion that enzymatic activity changes under the influence of radiation having the wavelength of 90 μm. No disruptures of polypeptide chains or oligomerization of proteins were obtained. It has been shown that albumin secondary structure calculated from circular dichroism spectra changes lineary according to dose of laser emission.

Influence of aspirin and iron(III) tetrasulfonated phthalocyanine on bilirubin binding by human serum albumin

The interaction of bilirubin with aspirin-modified human serum albumin (HSA) and the influence of iron tetrasulfonated phthalocyanine on bilirubin binding by the native protein has been studied by difference spectroscopy and circular dichroism measurements. Spectroscopic studies of the systems containing bilirubin and aspirin-modified HSA compared to the analogous systems with the native protein have shown that selective acetylation of albumin at lysine 199 inhibits bilirubin binding by this protein. In both cases, interaction between bilirubin and albumin leads to complex formation at a molar ratio of ligand to protein of 2:1. The studies of the reaction of bilirubin with fragments of albumin produced by reaction with CNBr have demonstrated that one of the strong bilirubin binding sites is located in the M fragment and is close to the high-affinity binding site of aspirin. The other one was found in fragment C. Acetylation of albumin brings about marked conformational change in the protein, which probably accounts for the decrease in its ability to react with anti-HSA antibody. Bilirubin does not change the secondary structure of albumin but, like aspirin, lowers its antigenicity. It has been suggested that the decrease in antigenic properties in this case results from cooperation of the closely neighboring antigenic and bilirubin-binding sites. The studies of the influence of iron(III) tetrasulfonated phthalocyanine on bilirubin binding by HSA suggest that there is no competition between strong sites for iron(III) tetrasulfonated phthalocyanine and bilirubin, but these compounds compete for some of the weaker sites.

Association of hemin with protein 4.1 as compared to spectrin and actin

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 · 10 7 M −1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 was unaffected by hemin binding to the primary site, but, at higher hemin concentrations, some reduction in the ellipticity of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.

Electric and magnetic properties of TiCo and related compositions

In the present work, the interaction between HSA with three potential anti-trypanosomal drugs – (E)-2-benzylidene-hydrazine-carbothioamide (UTS); (E)-2-(3,4-difluoro-benzylidene)-hydrazine-carbothioamide (DFTS) and (E)-2-(2,3,4-trifluoro-benzylidene)-hydrazine-carbothioamide (TFTS) – was studied under physiological conditions by spectroscopic techniques (UV–vis, circular dichroism, steady state, time resolved and synchronous fluorescence) at 296 K, 303 K and 310 K, and theoretical calculations (molecular docking). The bimolecular quenching constant values (kq) were higher than 5.00 × 109 M−1 s−1, suggesting a ground state association between albumin and the ligands (static quenching mechanism). Time-resolved fluorescence measurements confirmed the static quenching mechanism, despite the high probability of Förster resonance energy transfer (FRET) (r ≈ 4.00 nm for TFTS and r ≈ 3.00 nm for UTS and DFTS). Modified Stern–Volmer binding constant values (Ka = 104 M−1), CD results and synchronous fluorescence (Δλ = 15 and 60 nm) indicated moderate association between HSA:UTS, HSA:DFTS and HSA:TFTS, with weak perturbation on the secondary structure of albumin, without perturbation on the microenvironment of the Trp and Tyr residues. The calculated thermodynamic parameters (ΔH°, ΔS° and ΔG°) are in agreement with a spontaneous binding process, both entropically and enthalpically driven with hydrogen bonding and hydrophobic interactions as the main binding forces. Competitive binding experiments indicated site I as the main binding site of the protein for all studied compounds and molecular docking results suggested the same intermolecular interactions indicated by the thermodynamic parameters.