Association of gold nanorods in water solutions: influence of globular proteins

By absorption spectroscopy method optical properties of gold nanorods (10x38 nm) and their interaction with globular protein bovine hemoglobin and bovine serum albumin were investigated. Nanorods behavior was studied in water solution and in solution of 97 mM NaCl under ultrasound action during 90 min and results were then compared. In water solutions nanorods coagulation (aggregation) was observed with reduced optical density of the longitudinal plasmon band widening at lamda>800 nm. In NaCI solution absorption spectra evolution had complex character and was in some degree analogous to the result that was obtained for two-dimensional grids of gold nanoparticles when changing the distance between them. By interacting with serum albumin stabilization of colloid solution and dissociation of nanorods aggregates were observed.     

Association of gold nanorods in water solutions: Influence of globular proteins

By the absorption spectroscopy method, optical properties of gold nanorods (10 × 38 nm) and their interaction with globular protein bovine hemoglobin and bovine serum albumin were investigated. Nanorod behavior was studied in water solution and in solution of 97 mM NaCl under ultrasound action during 90 min and results were then compared. In water solutions, nanorod coagulation (aggregation) was observed with reduced optical density of the longitudinal plasmon band widening at λ > 800 nm. In NaCl solution, absorption spectra evolution had a complex character and was in some degree analogous to the result that was obtained for two-dimensional grids of gold nanoparticles when changing the distance between them. By interacting with serum albumin, stabilization of colloid solution and dissociation of nanorod aggregates were observed.     

Photooxidation of human serum albumin and its complex with bilirubin.

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole O2, and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen. Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non-oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole O2. Parallel photooxidation of the protein carrier could not be detected. It is considered possible that the fast, anaerobic process is operative in phototherapy of hyperbilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.     

Dye-binding protein assay using a long-wave-absorbing cyanine probe

A simple and fast protein assay that involves the binding of water-soluble sulfonate heptamethylene cyanine to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 778 to 904 nm, and the increase in absorption at 904 nm is monitored. This assay is very reproducible, of good color stability for at least 80 min, and sensitive at the 100 ng/mL level of human serum albumin (HSA) when a spectrophotometer with near-infrared wavelength is used to measure absorbance. Few chemicals except ionic surfactants such as cetyltrimethylammonium bromide and sodium dodecyl sulfonate interfere with the assay. Purified proteins have different capacities to interact with the dye; under the experimental conditions, the linear ranges of bovine serum albumin (BSA), HSA and gamma-IgG were 200-2000, 100-2400, and 200-3000 ng/mL, respectively. The relative standard deviation for the five replicate determinations of 1200 ng/mL BSA is 2.1%.     

Preparation and partial characterization of iron-sulfur, iron-selenium, and iron-tellurium complexes of bovine serum albumin.

An artificial Fe-S* protein was prepared by the reaction of bovine serum albumin with FeSO4 and Na2S or with a synthetic Fe-S*-1,4-butanenedithiol complex. These improved methods enabled us to characterize the derivatives from serum albumin. The Fe-S* albumin complex has about 20 iron ions and 14 labile sulfur atoms per molecule of the protein, whose absorption spectrum closely resembled that of 2Fe-2S* proteins. Its electron paramagnetic resonance spectrum exhibited signals different from those of ferredoxins. The addition of p-chloromercuriphenylsulfonate quenched the optical absorption in the visible region as well as the electron paramagnetic resonance signals. These properties of the albumin-iron complex are similar to those of iron-sulfur dithiothreitol and mercaptoethanol complexes, suggesting that the albumin-iron complex has one or more protein ligands besides sulfur lignads. Presumably, the oxygen atom of the tyrosine residue, or other hydroxyamino acids participates in the complex formation. In this context, the albumin polypeptide appears to be incapable of forming an iron-sulfur cluster identical to those of ferredoxins. Yet, from the albumin-iron derivative, the extrusion of the iron-sulfur core with benzenethiol provided products similar to those from ferredoxins. The iron-selenium and iron-tellurium derivatives of the bovine serum albumin were prepared and partially characterized by optical absorption and electron paramagnetic resonsnace spectroscopies. These results imply that both selenium and tellurium can be incorporated into the protein molecule as the respective labile components.     

Metal-catalyzed oxidation of human serum albumin: conformational and functional changes. Implications in protein aging

Mild oxidative stress, as elicited by ascorbate, oxygen, and trace metals, affects the binding properties of human serum albumin via purely conformational changes. In fact, no gross alteration can be observed in the electrophoretic and chromatographic patterns of albumin, whereas localized modifications are indicated by the changes in absorption and fluorescence spectra and in polarization degree. The oxidized protein presents a small increase of bityrosine production and a time-dependent increase in the content of carbonyl groups, whereas proteolytic susceptibility is unchanged. A higher affinity for cis-parinaric acid and a slight loss of solubility in high salt indicate a greater surface hydrophobicity. Pinpoint denaturation of the albumin molecule is also suggested by a decreased "esterase" activity in the presence of p-nitrophenyl acetate. Conformational stability evaluated through thermal shock and addition of moderate amounts of guanidine indicate that the oxidized protein is more heat-resistant, less flexible, and more rigid than the native one. Although limited, structural damages afforded by the oxidative stress cause alterations of albumin binding properties as documented by experiments with probes and physiological ligands. The loss of biological activity of human serum albumin induced by ascorbate system appears of medical relevance, because it can affect drug metabolism and particularly drug tolerance in the elderly.     

Changes in the antioxidant properties of substituted phenol polydisulfides during interaction with human serum albumin

Gallic acid polydisulfide and poly(2-aminodisulfide-4-nitrophenol) in aqueous solutions were shown to form polycomplexes with human serum albumin. This process was accompanied by considerable changes in the spectrum of protein circular dichroism recorded in distilled water in the far UV range at 20 degrees C. Complex formation between human serum albumin and polydisulfides was followed by a marked decrease in the content of alpha-helices and increase in the count of antiparallel beta-structures in the protein. Stable complexes containing 1.5, 2.8, and 7.7 poly(2-aminodisulfide-4-nitrophenol) molecules per human serum albumin molecule were formed in bicarbonate buffer (pH 9.0). In these complexes, the secondary protein structure underwent changes similar to those in polycomplexes of human serum albumin and polydisulfides. Gallic acid polydisulfide and poly(2-aminodisulfide-4-nitrophenol) inhibited the catalase-induced degradation of 50 mM H2O2. Complexes of human serum albumin and poly(2-aminodisulfide-4-nitrophenol) increased the catalytic activity and operational stability of catalase 1.5 and 4-7-fold, respectively. This was characterized by the effective reaction rate constant (kin, s-1). Our results indicate that complexes of human serum albumin and substituted phenol polydisulfides act as potent protectors and activators of catalase during enzymatic degradation of H2O2 at high concentrations.     

Interaction of antiviral and antitumor photoactive drug hypocrellin A with human serum albumin.

Absorption, resonance Raman, surface-enhanced Raman spectroscopy and differential scanning microcalorimetry were employed to study the interaction of hypocrellin A with human serum albumin. The identification of the binding place for hypocrellin A as well as the model for the albumin-hypocrellin A complex are proposed. In this model hypocrellin A interacts with albumin through more than one binding site placed on the protein surface. This model of non-specific interaction could explain why the absorption spectrum of hypocrellin A does not change in the presence of albumin and why the presence of the drug does not change significantly the thermodynamic parameters of the protein, while the Raman spectra show evident changes concerning both the protein and the drug structure. Even if hypocrellin A does not interact with an interior binding site, it can affect deeply the general albumin structure.     

Effect of the bidistilled modified water on bovine serum albumin conformation. Fluorescent spectroscopy data

It was shown that bidistilled modified water induces a marked decrease in the intensity of intrinsic fluorescence of bovine serum albumin and increases the binding of this protein to the fluorescent probe 1.8 ANS. These effects can be interpreted as a denaturing action of bidistilled modified water on the protein and a change in its conformational state, which is probably caused by changes in the microenvironment of the protein molecule. In addition, a substantial increase in the intrinsic fluorescence of bidistilled modified water, as compared with that of distilled water, was found.     

Enhancement of the viscosity of mucin by serum albumin.

The interaction of serum albumin with a model epithelial mucin from pig stomach was explored by rotary viscometry. During 30 min of incubation of human serum albumin(20mg/ml) and pig gastric mucin (8mg/ml) in iso-osmotic buffers at 37 degrees C, the solution became markedly viscous. Viscosity enhancement was proportional to albumin concentration (2-40mg/ml), was most pronounced under conditions of low shear rate (less than 45S-1), and was considerably greater than the additive or multiplicative viscosity values calculated from albumin or mucin solutions measured separately. The viscous mucin-albumin complex was destroyed by high shear rates (greater than 90S-1), but slowly re-formed under zero shear conditions. Elevation of pH (7 to 9), ionic strength (0.1 to 1.0), and addition of disodium EDTA (5mM) did not cause marked or specific alterations in the viscosity of the mixture, suggesting that electrostatic interactions probably do not stabilize mucin-albumin complexes. Urea (7M) and heating (35 to 55 degrees C) caused a major increase in the viscosity of mucin and mucin-albumin mixtures, suggesting that rupture of hydrogen bonds, unfolding and partial denaturation of mucin promotes greater intertangling (possibly hydrophobic interactions) between mucin and albumin molecules. The implications of mucin-albumin interaction in diseases associated with mucus obstruction are briefly discussed.     

Thickness and density of protein films by optical mixing spectroscopy.

The adsorption of protein films on polystyrene latex spheres was studied by optical mixing spectroscopy. With this technique, we show that both the hydrodynamic thickness of protein films and their optical density can be measured. Thus, we found that films of the glycoproteins isolated from the human erythrocyte membrane were four times as thick as films of either human serum albumin or bovine serum albumin for about the same surface coverage. This result suggests an end-on orientation for the adsorbed glycoprotein molecules, which is consistent with the model proposed by others for the orientation of these molecules at the surface of the red blood cell itself.     

Use of amphotericin B as optical probe to study conformational changes and thermodynamic stability in human serum albumin

The binding of polyene antibiotic amphotericin B to serum albumin was studied using absorption, fluorescence, and circular dichroism techniques. A hypochromic effect was observed in the absorption spectrum of amphotericin B in the presence of albumin with maxima at 366 nm, 385 nm, and 408 nm, which correspond to the absorption of the monomeric form of amphotericin B. A modification on the circular dichroism spectrum of amphotericin B in the presence of albumin was observed at bands 329 nm and 351 nm (excitronic interaction), which suggests that only amphotericin B monomer is bound to the protein. Amphotericin B perturbs the specific markers for sites I, II, and fatty acid binding site bound to these sites, suggesting that amphotericin B interacts with a great binding area in albumin. Lysines 199 and 525 in albumin participate in the molecular interaction between amphotericin B and the protein. The absorption spectrum of amphotericin B bound to albumin was sensitive to the chemical and thermal treatment of the protein, to neutral-basic transition of albumin and to conformational changes induced by the binding of other ligands to this protein.     

Relationship between microtubules and Golgi apparatus in hepatocytes: a quantitative study during experimental nephrosis

In many cell types, microtubules are preferentially associated with the Golgi apparatus. However, the existence of a functional link between these two organelles is still hypothetical. To gain insight into this question, the relationships between microtubules and the Golgi apparatus were studied in rat hepatocytes during experimental nephrosis induced by the aminonucleoside of puromycin. This condition is known to cause prolonged stimulation of plasma protein production by the hepatocytes. Rats were studied 2, 4, 5, 10 and 20 days after aminonucleoside injection. The amount of albumin was measured in serum and hepatic microsomes by laser immunonephelometry. The volume densities of microtubules around the Golgi apparatus and in the remaining cytoplasm were measured by ultrastructural morphometry. Changes of the Golgi apparatus were analysed by measuring the volume density of the whole organelle and the respective proportion of saccules and vesicles. Proteinuria began 5 days after aminonucleoside injection and was accompanied by a decrease in serum albumin and a rise in microsomal albumin. These changes were still more striking after 10 days, but protein and albumin levels were almost back to normal after 20 days. Concomitantly, the volume density of the microtubules increased significantly around the Golgi apparatus (32% after 10 days), and not in the remaining cytoplasm. The Golgi apparatus was enlarged (80% after 10 days) with a higher ratio of secretory vesicle to saccule volume densities. These results show that additional microtubules are present around the Golgi apparatus during the enhanced production of plasma proteins which occurs in nephrosis. They suggest that in hepatocytes, microtubules play a part in the Golgi apparatus function of plasma protein processing.     

Relationship between microtubules and Golgi apparatus in hepatocytes: a quantitative study during experimental nephrosis

In many cell types, microtubules are preferentially associated with the Golgi apparatus. However, the existence of a functional link between these two organelles is still hypothetical. To gain insight into this question, the relationships between microtubules and the Golgi apparatus were studied in rat hepatocytes during experimental nephrosis induced by the aminonucleoside of puromycin. This condition is known to cause prolonged stimulation of plasma protein production by the hepatocytes. Rats were studied 2, 4, 5, 10 and 20 days after aminonucleoside injection. The amount of albumin was measured in serum and hepatic microsomes by laser immunonephelometry. The volume densities of microtubules around the Golgi apparatus and in the remaining cytoplasm were measured by ultrastructural morphometry. Changes of the Golgi apparatus were analysed by measuring the volume density of the whole organelle and the respective proportion of saccules and vesicles. Proteinuria began 5 days after aminonucleoside injection and was accompanied by a decrease in serum albumin and a rise in microsomal albumin. These changes were still more striking after 10 days, but protein and albumin levels were almost back to normal after 20 days. Concomitantly, the volume density of the microtubules increased significantly around the Golgi apparatus (32% after 10 days), and not in the remaining cytoplasm. The Golgi apparatus was enlarged (80% after 10 days) with a higher ratio of secretory vesicle to saccule volume densities. These results show that additional microtubules are present around the Golgi apparatus during the enhanced production of plasma proteins which occurs in nephrosis. They suggest that in hepatocytes, microtubules play a part in the Golgi apparatus function of plasma protein processing.     

Production physiology and properties of a novel fungal fibrinolytic enzyme.

A potent extracellular fibrinolytic enzyme was obtained from cultures of the imperfect fungus Fusarium semitectum under certain growth conditions. Nitrate addition to cultures increased enzyme production. The enzyme showed a versatile proteolytic activity against several protein substrates including casein, gelatin, haemoglobin, bovine serum albumin, and fibrin from both buffalo and human sources. Optimal fibrinolysis occurred at pH values around 7.0. The fibrinolytic activity exhibited marked heat stability in enzyme samples heated at 60 degrees C, and retained more than 40% of its activity in samples heated to 100 degrees C for 10 min. Fibrinolysis proceeded optimally in the temperature range between 50--60 degrees C. Copper ions significantly activated the enzyme. Other biochemical properties are also reported.     

Early stage aggregation of human serum albumin in the presence of metal ions

The heat induced aggregation of human serum albumin (HSA) with and without an equimolar amount of Cu(II) and Zn(II) was investigated by using optical absorption, fluorescence, AFM and EPR spectroscopy. Turbidity experiments as a function of temperature indicate that the protein aggregation occurs after the melting of the protein. The kinetic of HSA aggregation, investigated between 60 and 70 °C by monitoring the optical density changes at 400 nm on a 180 min time window, shows an exponential growth with a rate that increases with the temperature. Fluorescence of the thioflavin T evidences a significant increase of the intensity at 480 nm at increasing incubation time. These results combined with AFM experiments show that the protein aggregates are elongated oligomers with fibrillar-like features. The absence of a lag-phase suggests that the early stage aggregation of HSA follows a downhill pathway that does not require the formation of an organized nucleus. The presence of Cu(II) and Zn(II) ions does not affect the thermally induced aggregation process and the morphology of HSA aggregates. The result is compatible with the binding of the metal ions to the protein in the native state and with the high conformational stability of HSA.     

Absorption and fluorescence spectra of hypericin sodium salt in complexes with albumins

The spectra of absorption and fluorescence of hypericin sodium salt (Na-Hy) in organic solvents and in complexes with human serum albumin, bovine serum albumin, and lipoproteins of low and high density have been studied. It was shown that, as the proton donor properties of the solvent enhance, the absorption and fluorescence maxima shift toward the blue region, and as the proton-accepting properties increase, the maxima shift toward the red region. The absorption spectra of complexes of Na-Hy with bovine serum albumin significantly differ from those of complexes of this ligand with human serum albumin, which is evidenced by a lesser width of absorption bands and a lower value of the Stokes shift. The positions of the absorption and fluorescence maxima and the value of the Stokes shift for the complex of Na-Hy with human serum albumin increases when D₂O instead of common water is used as a solvent. It was concluded that H-bonds of hypericin play a significant role in the interaction with human serum albumin.     

Interaction of bromophenol blue with serum albumin: criteria for using dyes for assessing the state and quantity of protein

The optical properties of the complexes of the pH-dependent dye bromophenol blue (BPB) with human serum albumin were investigated by the spectrophotometric method. The solvatochromic longwave displacement of bound BPB-2 absorption and BPB-1/BPB-2 redistribution were shown to form the optical signal of complexes. Because of the distortion of the bound BPB-2 signal its quantity was determined as delta A630 = A630 - A660 and the use of lambda max as structural parameter was limited to low pH less than or equal to 3. The conclusion was made that BPB is inapplicable as a structural probe on account of low structural dependence of delta A630 and pH-limitation of lambda max used. The maximal absorption delta Amax = Amax - A660 and its structural independence were obtained in the region of 70-100% occupation of the dye-binding centers of the protein. It is the optimal conditions for the quantitative determination of protein. After maximal dye binding (15-16 molecules of BPB per 1 molecule of albumin) the aggregation and precipitation of the complexes occurred.     

The action of corticosteroids on proteolysis

1. The corticosteroids cortisol, cortisone and corticosterone were tested for their ability to affect the hydrolysis of serum albumin, insulin and oxyhaemoglobin incubated with trypsin, chymotrypsin, papain and pepsin. 2. Corticosteroids stimulated the hydrolysis of albumin and oxyhaemoglobin with trypsin between 10% and 200% and inhibited the hydrolysis of insulin by 15% (steroid/substrate molar ratio, 5:1). 3. The degree of stimulation of proteolysis for a given substrate depended on both the nature of the steroid and the protease. Corticosterone did not increase the activity of papain and pepsin with any of the substrates tested. 4. Corticosterone stimulated (fivefold) the denaturation of oxyhaemoglobin measured spectroscopically in 2.4% (w/v) sodium hydroxide. Small changes in the absorption spectrum of haemoglobin solutions were also noted at pH7.8 without a marked change in the basic properties of haemoglobin. 5. With regard to the action of corticosterone on the activity of trypsin, the lack of stimulation when benzoylarginine amide was used as a substrate, the lowering of the stimulation on prior heat denaturation of haemoglobin and the high temperature coefficient for stimulation suggest that the steroid resulted in improved access of the protease to susceptible bonds of the substrate.