Alginate as an antiglycating agent for human serum albumin

Hyperglycemia and the accumulation of advanced glycation endproducts (AGEs) in tissues and serum have important roles in diabetic complications. Therefore, the identification of anti-glycation compounds is attracting considerable interest. In this study, the interaction of human serum albumin (HSA) with fructose, in the absence and presence of alginate, was studied by circular dichroism, absorbance and fluorescence techniques. The characterization study of AGEs was performed using autofluorescence, fibrillar formation, the increase in absorbance and the quantification of free lysine side chains. The results indicate that alginate inhibits the fructation of HSA as observed by a reduction in the formation of fluorescent AGEs and fibrils. Furthermore, alginate reduces the amount of modified lysine side chains, signified by the lack of increase in absorbance, and increases the helicity of this protein.     

Inhaled anesthetic binding sites in human serum albumin

Previous evidence suggests multiple anesthetic binding sites on human serum albumin, but to date, we have only identified Trp-214 in an interdomain cleft as contributing to a binding site. We used a combination of site-directed mutagenesis, photoaffinity labeling, amide hydrogen exchange, and tryptophan fluorescence spectroscopy to evaluate the importance to binding of a large domain III cavity and compare it to binding character of the 214 interdomain cleft. The data show anesthetic binding in this domain III cavity of similar character to the interdomain cleft, but selectivity for different classes of anesthetics exists. Occupancy of these sites stabilizes the native conformation of human serum albumin. The features necessary for binding in the cleft appear to be fairly degenerate, but in addition to hydrophobicity, there is evidence for the importance of polarity. Finally, myristate isosterically competes with anesthetic binding in the domain III cavity and allosterically enhances anesthetic binding in the interdomain cleft.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

Interaction of anesthetic supplement thiopental with human serum albumin

Thiopental (TPL) is a commonly used barbiturate anesthetic. Its binding with human serum albumin (HSA) was studied to explore the anesthetic-induced protein dysfunction. The basic binding interaction was studied by UV-absorption and fluorescence spectroscopy. An increase in the binding affinity (K) and in the number of binding sites (n) with the increasing albumin concentration was observed. The interaction was conformation-dependent and the highest for the F isomer of HSA, which implicates its slow elimination. The mode of binding was characterized using various thermodynamic parameters. Domain II of HSA was found to possess a high affinity binding site for TPL. The effect of micro-metal ions on the binding affinity was also investigated. The molecular distance, r, between donor (HSA) and acceptor (TPL) was estimated by fluorescence resonance energy transfer (FRET). Correlation between the stability of the TPL-N and TPL-F complexes and drug distribution is discussed. The structural changes in the protein investigated by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy reflect perturbation of the albumin molecule and provide an explanation for the heterogeneity of action of this anesthetic.     

Halothane, an inhalation anesthetic, activates protein kinase C and superoxide generation by neutrophils

The rate of superoxide generation of guinea pig intraperitoneal neutrophils by a chemotactic peptide or 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased by 2-bromo-2-chloro-1,1,1,-trifluoroethane (halothane), an inhalation anesthetic. This increase was inhibited by 1-(5-isoquinolinesulfonyl)methylpiperazine dihydrochloride (H-7), a specific inhibitor of Ca2+- and phospholipid-dependent protein kinase C (PKC). Halothane was found to significantly activate partially purified PKC. The activation required phosphatidylserine (PS) and Ca2+. Dioleoylglycerol- or TPA-activated PKC activity was further increased by halothane. The cytoplasmic proteins of guinea pig neutrophils phosphorylated by halothane-activated PKC were similar to those phosphorylated by PMA-activated PKC. The phosphorylation of a 48 kDa protein, a phosphorylated protein required for NADPH oxidase activation, was also increased by halothane. These data suggest that the increase of superoxide production by halothane is correlated with its activation of PKC.     

Swine serum increases hybridoma stability

Three hybridoma cell lines producing monoclonal antibodies to lambda phage were cultured for 9 months in Dulbecco's modified Eagl's medium supplemented with 10% fetal calf serum or with 5% swine serum. Under these conditions all the clones had the same proliferation rates and maximum population densities. The cultuvation in the medium with swine serum enhanced the stability of hybridomas: all clones retained the ability to produce antibodies throughout the experiment; one of the clones examined enhanced the activity by 10(3)-10(4) times. When the same three clones were cultured in the medium with the fetal calf serum, two of them lost their ability to produce antibodies 3-15 weeks after the beginning of the experiment.     

Influence of fluoro, chloro and alkyl alcohols on the folding pathway of human serum albumin

Urea-induced equilibrium unfolding of human serum albumin (HSA) when studied by mean residue ellipticity at 222 nm (MRE(222)) or intrinsic fluorescence measurements showed a two-step, three-state transition with a stable intermediate around 4.6-5.2 M urea. The presence of 2,2,2-trifluoroethanol (TFE) resulted in a single-step, two-state transition with a significant shift towards higher urea concentration, suggesting the stabilizing effect of TFE. The free energy of stabilization (DeltaDeltaG(D)(H(2)O)) in the presence of 3.0 M TFE was determined to be 2.68 and 2.72 kcal/mol by MRE(222) and fluorescence measurements, respectively. The stabilizing potential of other alcohols on the refolding behavior of HSA at 5.0 M urea (where the intermediate exists) as studied by MRE(222) and intrinsic fluorescence measurements showed the following order: 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) > TFE > 2-chloroethanol > tert-butanol > iso-propanol > ethanol > methanol. Further, the extent of refolding at the highest concentration of alcohol was similar in all cases. The stabilizing effect of TFE on guanidine hydrochloride (GdnHCl)-induced unfolding of HSA was nearly equal to that found for urea denaturation, as reflected in the DeltaDeltaG(D)(H(2)O) value (2.38 kcal/mol). Taken together, these results suggest that the stabilizing effect of TFE and other alcohols on urea/GdnHCl-induced unfolding of HSA is higher for alcohols that contain bulky groups or fluorine atoms.     

Farnesylation of the SNARE protein Ykt6 increases its stability and helical folding

The evolutionarily conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in the fusion of vesicles with their target membranes. While most SNAREs are permanently anchored to membranes by their transmembrane domains, the vesicle-associated SNARE Ykt6 has been found both in soluble and in membrane-bound pools. The R-SNARE Ykt6 is thought to mediate interactions between various Q-SNAREs by a reversible membrane-targeting cycle. Membrane attachment of Ykt6 is achieved by its C-terminal prenylation and palmitoylation motif succeeding the SNARE motif. In this study, we have analyzed full-length farnesylated Ykt6 from yeast and humans by biochemical and structural means. In vitro farnesylation of the C-terminal CAAX box of recombinant full-length Ykt6 resulted in stabilization of the native protein and a more compactly folded structure, as shown by size exclusion chromatography and limited proteolysis. Circular dichroism spectroscopy indicated a specific increase in the helical content of the farnesylated Ykt6 compared to the nonlipidated form or the single-longin domain, which correlated with a marked increase in stability as observed by heat denaturation experiments. Although highly soluble, farnesylated Ykt6 is capable of lipid membrane binding independent of the membrane charge, as shown by surface plasmon resonance. The crystal structure of the N-terminal longin domain of yeast Ykt6 (1-140) was determined at 2.5 Å resolution. As similarly found in a previous NMR structure, the Ykt6 longin domain contains a hydrophobic patch at its surface that may accommodate the lipid moiety. In the crystal structure, this hydrophobic surface is buried in a crystallographic homomeric dimer interface. Together, these observations support a previously suggested closed conformation of cytosolic Ykt6, where the C-terminal farnesyl moiety folds onto a hydrophobic groove in the N-terminal longin domain.     

Identification of anesthetic binding sites on human serum albumin using a novel etomidate photolabel

We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(+) enantiomer being significantly more potent than the S(-) enantiomer. Its effects on alpha1beta2gamma2s GABA(A) receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (beta2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds approximately 75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABA(A) receptor or other putative targets.     

Photocontrol of protein folding: the interaction of photosensitive surfactants with bovine serum albumin

The photoresponsive interaction of light-sensitive azobenzene surfactants with bovine serum albumin (BSA) at neutral pH has been investigated as a means to control protein folding with light irradiation. The cationic azobenzene surfactant undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, with the visible-light (trans) form of the surfactant being more hydrophobic than the UV-light (cis) form. As a consequence, the trans form exhibits enhanced interaction with the protein compared to the cis form of the surfactant, allowing photoreversible control of the protein folding/unfolding phenomena. Small-angle neutron-scattering (SANS) measurements are used to provide detailed information on the protein conformation in solution. A fitting of the protein shape to a low-resolution triaxial ellipsoid model indicates that three discrete forms of the protein exist in solution depending on the surfactant concentration, with lengths of approximately 90, 150, and 250 A, respectively, consistent with additional dynamic light-scattering measurements. In addition, shape-reconstruction methods are applied to the SANS data to obtain relatively high-resolution conformation information. The results confirm that BSA adopts a heart-shaped structure in solution at low surfactant concentration, similar to the well-known X-ray crystallographic structure. At intermediate surfactant concentrations, protein elongation results as a consequence of the C-terminal portion separating from the rest of the molecule. Further increases in the surfactant concentration eventually lead to a highly elongated protein that nonetheless still exhibits some degree of folding that is consistent with the literature observations of a relatively high helical content in denatured BSA. The results clearly demonstrate that the visible-light form of the surfactant causes a greater degree of protein unfolding than the UV-light form, providing a means to control protein folding with light that, within the resolution of SANS, appears to be completely reversible.     

Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.     

Osmotic pressure method to measure salt induced folding/unfolding of bovine serum albumin

A new approach has been developed to monitor protein folding by utilizing osmotic pressure and a range of salt concentrations in a well characterized protein, bovine serum albumin (BSA). It is hypothesized that both the ‘effective’ osmotic molecular weight, A_e, and the solute/solvent interaction parameter, I, in the empirical relation $\text{M}{}_{\mathrm{solvent}}\text{M}{}_{\mathrm{solute}}\text{= (}\text{RTϱ}\text{A}{}_{\mathrm{e}}\text{)}\text{1}\text{gp}\text{+ I}$ [1] can be used as measures of protein folding. I is a measure of solvent perturbed by the solute and is thought to depend directly upon the solvent accessible surface area (ASA). It is reasoned that larger solvent accessible surface area of an unfolded or denatured protein should perturb more water and produce larger I-values. Thus I-values allow calculation of a unfolded protein fraction, f_u^a, due to changes in relative solvent accessible surface area. It has been observed that A_e decreases for filamentous, denatured proteins due to segmental motion of the molecule [2]. This allows calculation of unfolded protein fraction from the effective molecular weight, f_u^m. Colloid osmotic pressure of BSA was measured in a range of salt concentrations at 25°C, and pH = 7 (above the isoelectric point of BSA at pH = 5.4). Both S and I were used to monitor protein folding as the salt concentration was varied. In general, larger and variable I-values and smaller A_e were observed at salt concentrations less than 50 mmolal NaCl (I_max = 8.9), while constant I = 4.1 and A_e = 66,500 were observed above 50 mmolal NaCl. The two expressions for fractional unfolding (f_u^a and f_u^m) are in general agreement. Small differences in the parameters below 50 mmolal salt concentration are explained with well known shifts in the relative amounts of α-helix, β-sheet and random coil in denatured BSA. The relative amounts of these shifts agree with predictions in the literature attributed to continuous BSA expansion rather than an ‘all-or-none’ conversion.     

Structural dynamics, stability and folding of proteins

The present concepts of protein folding in vitro are reviewed. According to these concepts, amino acid sequence of protein, which has appeared a result of evolutionary selection, determines the native structure of protein, the pathway of protein folding, and the existence of free energy barrier between native and denatured states of protein. The latter means that protein macromolecule can exist in either native or denatured state. And all macromolecules in the native state are identical but for structural fluctuations due to Brownian motion of their atoms. Identity of all molecules in native state is of primary importance for their correct functioning. The dependence of protein stability, which is measured as the difference between free energy of protein in native and denatured states, on temperature and denaturant concentration is discussed. The modern approaches characterizing transition state and nucleation are regarded. The role of intermediate and misfolded states in amorphous aggregate and amyloid fibril formation is discussed.     

Structure, stability and folding of the alpha-helix

Pauling first described the alpha-helix nearly 50 years ago, yet new features of its structure continue to be discovered, using peptide model systems, site-directed mutagenesis, advances in theory, the expansion of the Protein Data Bank and new experimental techniques. Helical peptides in solution form a vast number of structures, including fully helical, fully coiled and partly helical. To interpret peptide results quantitatively it is essential to use a helix/coil model that includes the stabilities of all these conformations. Our models now include terms for helix interiors, capping, side-chain interactions, N-termini and 3(10)-helices. The first three amino acids in a helix (N1, N2 and N3) and the preceding N-cap are unique, as their amide NH groups do not participate in backbone hydrogen bonding. We surveyed their structures in proteins and measured their amino acid preferences. The results are predominantly rationalized by hydrogen bonding to the free NH groups. Stabilizing side-chain-side-chain energies, including hydrophobic interactions, hydrogen bonding and polar/non-polar interactions, were measured accurately in helical peptides. Helices in proteins show a preference for having approximately an integral number of turns so that their N- and C-caps lie on the same side. There are also strong periodic trends in the likelihood of terminating a helix with a Schellman or alpha L C-cap motif. The kinetics of alpha-helix folding have been studied with stopped-flow deep ultraviolet circular dichroism using synchrotron radiation as the light source; this gives a far superior signal-to-noise ratio than a conventional instrument. We find that poly(Glu), poly(Lys) and alanine-based peptides fold in milliseconds, with longer peptides showing a transient overshoot in helix content.     

Circular dichroic study of the conformational stability of sulfhydryl-blocked bovine serum albumin

1. The blockage of the single sulfhydryl-group of bovine serum albumin does not alter the secondary structure, although the alpha-helical structure is destabilized since lower concentrations of guanidine and of urea unfold the protein. 2. What happens to the previously helical structure depends upon the reagent used to block the sulfhydryl-group. Bovine serum albumin derivatized with 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate preferentially acquire the beta-structure in high concentrations of guanidine and urea, whereas iodoacetamide-derivatized bovine serum albumin acquires primarily the random coil structure. 3. Part of the helical structure is also lost in 5-6 mM sodium dodecyl sulfate; thionitrobenzoate-bovine serum albumin shows an increase in the random coil, whereas the two alkylated proteins display the increase both in beta-structure and random coil. 4. Carboxymethylation or carboxamidomethylation of fully reduced bovine serum albumin results in a drastic change in the secondary structure of the protein with a substantial decrease in alpha-helix and a corresponding increase in both beta-structure and random coil. These extensively alkylated proteins also display differences in denaturation profiles in solutions of guanidine and urea.     

Effect of genetic variation on the thermal stability of human serum albumin

Reversible thermal denaturation of 33 genetic variants of human serum albumin (HSA) appeared to be a two-state process when studied by circular dichroism (CD). Fourteen single-residue variants have Tm values (midpoint of denaturation) higher than, and nine have Tm values lower than, their endogenous, wild-type counterpart. Nine single-residue variants have DeltaHv values (van't Hoff enthalpy) higher than, and 14 have DeltaHv values lower than, normal albumin. All types of combinations of positive and negative DeltaTm values and Delta(DeltaHv) values were found. Good linear correlations between mutation-induced changes of alpha-helical content and Delta(DeltaHv) values, but not DeltaTm values, were found especially for the variants mutated in domains I and III. The effect of altered chain length and glycosylation on Tm and DeltaHv was also studied. For all variants, no clear relationship was found between the changes in the thermodynamic parameters and the type of substitution, changes in protein charge or hydrophobicity. However, the protein changes taking place in domain I have a rather uniform effect (almost all of the nine variants have positive DeltaTm values and negative Delta(DeltaHv) values, i.e., they denature more easily than normal albumin but they do so at a higher temperature). The present results can be of both protein chemical relevance and of clinical interest, because they could be useful when designing stable, recombinant HSAs for clinical applications.     

Interaction,cytotoxicity and sustained release assessment of a novel anti-tumor agent using bovine serum albumin nanocarrier

Abstract

The interaction ability of bovine serum albumin (BSA) with 2,6-divanillylidenecyclohexanone (DVH) as a stable curcumin derivative was investigated using fluorescence and circular dichroism (CD) spectroscopy techniques under simulative physiological conditions (pH = 7.2). Following the obtained results of binding studies, bovine serum albumin nanoparticles (BSANPs) were synthesized and characterized using Fourier transform infrared spectroscopy (FT-IR), filed emission scanning electron microscopy (FE-SEM), atomic force microscope (AFM) and dynamic light scattering (DLS). The stable BSANPs showed a spherical shape with a diameter of 149.14?±?46.69?nm and the formulation of BSA had no change during the fabrication process. DVH was loaded on BSANPs (DVH@BSANPs) and the release studies showed sustained release of DVH from BSANPs. The validation of DVH@BSANPs system confirmed that the Fickian release mechanism of DVH followed on Korsmeyer–Pepas model. The in vitro studies on HFFF2 and MDA-MB-231 were investigated using MTT assay, DAPI and annexinV/PI staining that showed biocompatible BSANPs reduced the cytotoxicity of DVH in normal cell lines significantly, and antitumor activity of DVH was increased when it was loaded onto BSANPs without necrosis. These results suggest that DVH@BSANPs are a novel biocompatible sustained release system for effective therapeutic approach.

Communicated by Ramaswamy H. Sarma     

热稳定性试验对人血白蛋白质量的影响

目的观察热稳定性试验对人血白蛋白质量的影响。方法依据《中国药典》三部对人血白蛋白待检品进行热稳定性试验,并通过可见异物、多聚体含量和纯度等指标对其进行稳定性评价。结果人血白蛋白经热稳定性试验后可见异物检查无肉眼可见的明显变化。热稳定性试验对多聚体含量有明显影响(t=-2.65,P=0.0160.05),试验品多聚体含量增加。试验品和对照品纯度变化有显著差异(t=4.19,P=0.0010.05;t=12.87,P=0.0000.01)。SDS-PAGE结果显示,试验品有两条杂带含量增多,表明主带人血白蛋白含量降低,纯度下降。结论人血白蛋白应严格按照《中国药典》规定的温度进行储存和运输,确保制品的质量。     

Conformation, stability, and folding of interleukin 1 beta

Recombinant human interleukin 1 beta has been studied in solution with respect to its conformation, stability, and characteristics of unfolding and refolding. It is an all-beta-type, stable globular protein with a high cooperativity under conditions where refolding is reversible. The tryptophan residue is approximately 40% exposed to solvent, and the four tyrosines are 50% exposed. The fluorescence of the single tryptophan residue is quenched at pH 7.5 but dequenched by high salt, by titration to lower pH with a pK of 6.59, and by denaturants, resulting in an unusual biphasic change in fluorescence on unfolding. Both histidine and thiol residues have been excluded as being responsible for the pH dependence of fluorescence by site-directed mutagenesis and by chemical modification, respectively. The likely candidate is an aspartate or glutamate.     

Anion-binding centers of human serum albumin during an N-F conformation transition and in isolated albumin and blood serum

Fluorescent probe N-phenyl-1-amino-8-sulfonaphthalene (ANS) was used for studying pH-dependent structural N-F-transition in human serum albumin of two kinds: in commercial albumin and in natural blood serum. The kinetics of ANS fluorescence decay in albumin solutions was measured. There were found two types of the sites occupied by ANS in albumin under physiological conditions (pH 7.4). In the first binding site ANS fluorescence decay time was 16.6 +/- 0.3 nsec and it was not significantly changed at N-F transition (pH 4.0). In the second binding site the decay time was dependent on pH in commercial albumin and was not significantly changed in serum. In the second binding site there were individual differences of ANS decay time (4.3 +/- 0.6 nsec). The observed ANS fluorescence intensity enhancing (about 40-50%) in N-F transition may be explained by an increase of albumin binding sites capacity for ANS.