Structural changes of ultrasonicated bovine serum albumin revealed by hydrogen-deuterium exchange and mass spectrometry.

The structural changes of bovine serum albumin (BSA) under high-intensity ultrasonication were investigated by fluorescence spectroscopy and mass spectrometry. Evidence for the ultrasonication-induced conformational changes of BSA was provided by the intensity changes and maximum-wavelength shift in fluorescence spectrometry. Matrix-assisted laser desorption-ionization time-of-flight mass spectroscopy (MALDI-TOF MS) revealed the increased intensity of the peak at the charge state +5 and a newly emerged peak at charge state +6, indicating that the protein became unfolded after ultrasonication. Prevalent unfolding of BSA after ultrasonication was revealed by hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS). Increased intensity and duration of ultrasonication further promoted the unfolding of the protein. The unfolding induced by ultrasonication goes through an intermediate state similar to that induced by a low concentration of denaturant.

Development of an albuminous reactive oxygen species assay for photosafety evaluation under experimental biomimetic conditions.

The generation of reactive oxygen species (ROS) from an ultraviolet (UV)-exposed chemical can be an experimental indicator of phototoxic potential. The aim of the present study was to develop a new ROS assay using serum albumin to provide photosafety assessment under experimental biomimetic conditions. To assess assay robustness, a validation study on an albuminous ROS (aROS) assay was conducted with a focus on intra- and inter-day precisions and Z'-factor reflecting both the assay signal-to-noise ratio and variation associated with signal measurements. In the aROS assay on quinine HCl (200 µM), a typical phototoxic drug, both intra- and inter-day precisions (coefficient of variation; CV) were found to be below 4%, and the Z'-factors for singlet oxygen and superoxide suggested a large separation band between samples and blank signals. To evaluate the prediction capacity, the aROS and ROS assays were applied to 21 phototoxins and 10 non-phototoxic chemicals. Upon aROS assay on these model chemicals, the individual specificity was 100%, and the positive and negative predictivities were found to be 100% and 81.8%, respectively. The aROS assay can be employed for poorly soluble chemicals for which the ROS assay is unavailable. Comparing the ROS assay data, there seemed to be a photochemical transition of some chemicals in albuminous solution. A molecular interaction between albumin and chemical was also assessed by UV and fluorescent spectroscopic analyses, and the results suggested the limited relationship between the albumin-chemical interaction and the photochemical change. The aROS assay may allow photosafety assessment of new drug entities with a wide range of applicability partly under experimental biomimetic conditions.

Detection of parathion pesticide by quartz crystal microbalance functionalized with UV-activated antibodies.

Photonic immobilization technique (PIT) has been used to develop an immunosensor for the detection of parathion. An antibody solution has been activated by breaking the disulfide bridge in the triad Trp/Cys-Cys through absorption of ultrashort UV laser pulses. The free thiol groups so produced interact with gold lamina making the antibody oriented upside, that is, with its variable parts exposed to the environment, thereby greatly increasing the detection efficiency. PIT has been applied to anchor polyclonal antiparathion antibodies to the gold electrode of a Quartz Crystal Microbalance (QCM) giving rise to very high detection sensitivity once the parathion is made heavier by complexion with BSA (bovine serum albumin), this latter step only required by the mass based transducer used in this case. The comparison of the sensor response with irradiated antibodies against different analytes shows that the high degree of antibody specificity is not affected by PIT nor is it by the complexion of parathion with BSA. These results pave the way to important applications in biosensing, since the widespread occurrence of the Trp/Cys-Cys residues triads in proteins make our procedure very general and effective to detect light analytes.

Effect of diode-laser and AC magnetic field of bovine serum albumin nanospheres loaded with phthalocyanine and magnetic particles.

This study reports on the development and characterization of bovine serum albumin (BSA) nanospheres containing Silicon(IV) phthalocyanine (NzPc) and/or maghemite nanoparticles (MNP), the latter introduced via ionic magnetic fluid (MF). The nanosized BSA-loaded samples were designed for synergic application while combining Photodynamic Therapy and Hyperthermia. Incorporation of MNP in the albumin-based template, allowing full control of the magnetic content, was accomplished by adding a highly-stable ionic magnetic fluid sample to the albumin suspension, following heat denaturing. The material's evaluation was performed using Zeta potential measurements and scanning electron microscopy. The samples were characterized by steady-state techniques and time-resolved fluorescence. The in vitro assay, using human fibroblasts, revealed no cytotoxic effect in all samples investigated, demonstrating the potential of the tested system as a synergistic drug delivery system.

Femtosecond laser programmed artificial musculoskeletal systems.

Natural musculoskeletal systems have been widely recognized as an advanced robotic model for designing robust yet flexible microbots. However, the development of artificial musculoskeletal systems at micro-nanoscale currently remains a big challenge, since it requires precise assembly of two or more materials of distinct properties into complex 3D micro/nanostructures. In this study, we report femtosecond laser programmed artificial musculoskeletal systems for prototyping 3D microbots, using relatively stiff SU-8 as the skeleton and pH-responsive protein (bovine serum albumin, BSA) as the smart muscle. To realize the programmable integration of the two materials into a 3D configuration, a successive on-chip two-photon polymerization (TPP) strategy that enables structuring two photosensitive materials sequentially within a predesigned configuration was proposed. As a proof-of-concept, we demonstrate a pH-responsive spider microbot and a 3D smart micro-gripper that enables controllable grabbing and releasing. Our strategy provides a universal protocol for directly printing 3D microbots composed of multiple materials.

In vitro radioprotection studies of organoselenium compounds: differences between mono- and diselenides.

Organoselenium compounds belonging to the class of monoselenides, such as selenomethionine (SeM) and methylselenocysteine (MSeCys) and diselenides including selenocystine (SeCys) and selenopropionic acid (SePA), were examined for their comparative radioprotective effects using in vitro models. Effects of these compounds on the inhibition of gamma-radiation induced lipid peroxidation in liposomes, protein carbonylation in bovine serum albumin (BSA) and strand breaks in pBR322 plasmid DNA, assessed, respectively, by the formation of thiobarbituric acid reactive substances, formation of 2,2'-dinitrophenyl hydrazine (DNPH) carbonyl complex and horizontal gel electrophoresis, were used to compare their radioprotective ability. The IC 50 values for SeCys, SePA, SeM and MSeCys for lipid peroxidation were 27 +/- 1, 33 +/- 2, 200 +/- 8 and 163 +/- 4 microM, respectively, and the values for inhibition of protein carbonylation were >200, 300 +/- 6, 464 +/- 8 and 436 +/- 3 microM, respectively. Inhibition of DNA strand break formation was tested at 200 microM for all the compounds and SePA and SeCys exhibited a protective effect on DNA, while SeM and MSeCys did not lead to any protection. The in vitro cytotoxicity studies in normal and tumor cells revealed that MSeCys and SeM were not cytotoxic to lymphocytes and EL4 tumor cells at the concentrations employed. In contrast, SeCys was toxic, with a higher effect on tumor cells than lymphocytes. Our studies suggest that the non-toxic diselenides like SePA should be explored as protective agents against gamma-irradiation induced damage.

Nano titanium dioxide photocatalytic protein tyrosine nitration: a potential hazard of TiO2 on skin.

Protein tyrosine nitration is a prevalent post-translational modification which occurs as a result of oxidative and nitrative stress, it may be directly involved in the onset and/or progression of diseases. Considering the existence of nano titanium dioxide (TiO(2)) in environment and sunscreen products along with the high content of nitrite in sweat, the UV-exposed skin may be a significant target for the photosensitized damage. In this paper, tyrosine nitration of bovine serum albumin (BSA) was initiated in the UV-irradiated reaction mixture containing 0.2-3.0mg/ml of three commercially nano TiO(2) products and 0.25-1.0mM NO2-. It was found that anatase TiO(2) and Degussa P25 TiO(2) showed prominent photocatalytic activity on promoting the formation of protein tyrosine nitration, and the optimum condition for the reaction was around physiological pH. Meanwhile, the photocatalytic effect of rutile on protein tyrosine nitration was subtle. The potential physiological significance of nano TiO(2)-photocatalytic protein nitration was also demonstrated in mouse skin homogenate. Although the relationship between photocatalytic protein tyrosine nitration and chronic cutaneous diseases needs further study, the toxicity of nano TiO(2) to the skin disease should be paid more attention in the production and utilization process.

Structural and functional changes in ultrasonicated bovine serum albumin solutions.

Effects of high-intensity ultrasonication on functional and structural properties of aqueous bovine serum albumin (BSA) solutions were investigated. The functional properties of BSA were altered by ultrasonication. Surface activity of BSA increased. Minimal changes were observed in the global structure of BSA but surface charge increased particularly at basic pH values (e.g. pH>9). While dynamic light scattering measurements indicated that the particle size increased up to 3.4 times after 90 min of sonication, no significant increase in the oligomeric state of BSA using blue native PAGE was observed. The amount of free sulfhydryl groups in BSA after 90 min of sonication decreased. The increased particle size and decreased number of free sylfhydryl groups may be attributed to formation of protein aggregates. Surface hydrophobicity increased and circular dichroism spectroscopy and FTIR analysis indicated changes in the secondary structure of BSA. We hypothesize that mechanical, thermal and chemical effects of ultrasonication resulted in structural changes in BSA that altered the functional properties of the macromolecule which may be attributed to the formation of an ultrasonically induced state that differs from a thermally, mechanically or solvent induced state.

Formation of long-lived reactive species of blood serum proteins induced by low-intensity irradiation of helium-neon laser and their involvement in the generation of reactive oxygen species.

It was demonstrated that low-intensity radiation of helium-neon (He-Ne) laser at 632.8nm, which leads to the transition of oxygen to a singlet state, causes the formation of reactive oxygen species (ROS) - hydrogen peroxide, hydroxyl and superoxide (hydroperoxide) radicals - in aqueous solutions. The oxygen effect - dependence of hydrogen peroxide formation on the concentration of molecular oxygen - was shown, and the participation of singlet oxygen, hydroxyl radicals and superoxide (hydroperoxide) radicals in this process was testified. Laser radiation-induced ROS in solutions of blood serum proteins, bovine serum albumin and bovine gamma-globulin, cause the formation of long-lived reactive protein species (LRPS) with a half-life of about 4h. The generation of LRPS caused by laser irradiation results in prolonged several-hour generation of ROS - hydrogen peroxide, hydroxyl and superoxide radicals. As affected by LRPS, coupled radical reactions lead to conversion of dissolved molecular oxygen to hydrogen peroxide. Irradiation with light sources away from the oxygen absorption band is not attended by formation of ROS and LRPS. A consideration is provided for the possible molecular mechanisms of ROS formation under the influence of He-Ne laser irradiation, the role of proteins in their generation and the biological significance of these processes.

Protein secondary structure controlled with light and photoresponsive surfactants.

The interaction of a light-responsive azobenzene surfactant with bovine serum albumin (BSA) has been investigated as a means to examine photoreversible changes in protein secondary structure. The cationic azobenzene surfactant undergoes a reversible photoisomeriztion upon exposure to the appropriate wavelength of light, with the visible-light (trans) form being more hydrophobic and, thus, inducing a greater degree of protein unfolding than the UV-light (cis) form. Fourier transform infrared (FT-IR) spectroscopy is used to provide quantitative information on the secondary structure elements in the protein (alpha-helices, beta-strands, beta-turns, and unordered domains). Comparing the secondary structure changes induced by light illumination in the presence of the photoresponsive surfactant with previous measurements of the tertiary structure of BSA obtained from small-angle neutron scattering (SANS) allows the three discrete conformation changes in BSA to be fully characterized. At low surfactant concentrations, an alpha-helix --> beta-structure rearrangement is observed as the tertiary structure of BSA changes from a heart-shaped to a distorted heart-shaped conformation. Intermediate surfactant concentrations lead to a dramatic decrease in the alpha-helix fraction in favor of unordered structures, which is accompanied by an unfolding of the C-terminal portion of the protein as evidenced from SANS. Further increases in photosurfactant concentration lead to a beta --> unordered transition with the protein adopting a highly elongated conformation in solution. Each of these protein conformational changes can be precisely and reversibly controlled with light illumination, as revealed through FT-IR spectra collected during repeated visible-light <--> UV-light cycles.

Mutual interaction between glycation and oxidation during non-enzymatic protein modification.

Aging pathogenesis involves non-enzymatic modifications of proteins; protein oxidation, glycation and their interactions have aroused a particular interest. Possible interrelations between oxidation and glycation have been evaluated in vitro: bovine serum albumin was oxidized by gamma-irradiation and then exposed to in vitro glycation. Fluorescence modifications induced by radiolytic oxidation and glycation were similar and tended to be additive. Both non-enzymatic processes provoked a loss of free sulfhydryl groups and a strong increment of protein carbonyl content: this supports that glycation can act through oxidative mechanisms. The observed rearrangement of amino groups after irradiation could predispose proteins to glycation attacks. Protein peroxides generated during irradiation appear able to give birth to further protein modifications leading to the generation of carbonyl groups and to interact with monosaccharides, probably stimulating their autoxidation and in turn glycative protein damage. Glycation increases the oxidation-mediated structural damage revealed by SDS-PAGE. Therefore our data support the hypothesis of mutual enhancement between oxidation and glycation of proteins and suggest possible molecular mechanisms of interactions.

Novel water-soluble bacteriochlorophyll derivatives for vascular-targeted photodynamic therapy: synthesis, solubility, phototoxicity and the effect of serum proteins.

New negatively charged water-soluble bacteriochlorophyll (Bchl) derivatives were developed in our laboratory for vascular-targeted photodynamic therapy (VTP). Here we focused on the synthesis, characterization and interaction of the new candidates with serum proteins and particularly on the effect of serum albumin on the photocytotoxicity of WST11, a representative compound of the new derivatives. Using several approaches, we found that aminolysis of the isocyclic ring with negatively charged residues markedly increases the hydrophilicity of the Bchl sensitizers, decreases their self-association constant and selectively increases their affinity to serum albumin, compared with other serum proteins. The photocytotoxicity of the new candidates in endothelial cell culture largely depends on the concentration of the serum albumin. Importantly, after incubation with physiological concentrations of serum albumin (500-600 microM), WST11 was found to be poorly photocytotoxic (>80% endothelial cell survival in cell cultures). However, in a recent publication (Mazor, O. et al. [2005] Photochem. Photobiol. 81, 342-351) we showed that VTP of M2R melanoma xenografts with a similar WST11 concentration resulted in approximately 100% tumor flattening and >70% cure rate. We therefore propose that the two studies collectively suggest that the antitumor activity of WST11 and probably of other similar candidates does not depend on direct photointoxication of individual endothelial cells but on the vascular tissue response to the VTP insult.

Photophysics in motionally constrained bioenvironment: interaction of norharmane with bovine serum albumin.

Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (DeltaG) for the probe-protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water-dioxane mixture with varying composition.

Effect of gamma-irradiation on the molecular properties of bovine serum albumin.

To investigate the effect of oxygen radicals on the molecular properties of bovine serum albumin (BSA), the secondary and tertiary structures, molecular weight and optical anisotropy of BSA were examined after the irradiation of the protein at various doses. gamma-Irradiation of the protein solution caused the disruption of the ordered structure of protein molecules as well as degradation, cross-linking and aggregation of polypeptide chains. Fluorescence spectroscopy indicated that irradiation quenched the emission intensity excited at 280 nm. Fourier transform infrared spectroscopy (FTIR) indicated that irradiation caused transformation from beta-turns into beta-sheets. A light scattering study showed that increasing the radiation dose decreased the molecular weight of the protein. Optical anisotropy data showed that radiation changed the ordered structure of the protein. Ultraviolet absorption spectroscopy indicated that fragmentation and aggregation might occur in response to radiation exposure.

Modulation of protein behavior through light responses of TiO2 nanodots films.

In this work, the behavior of protein molecules adsorbed on TiO2 nanodots films are modulated through the light responses of the nanodots. TiO2 nanodots films are first prepared through phase separation induced self assembly. Then, bovine serum albumin (BSA) is adsorbed on TiO2 nanodots films and exposed to ultraviolet (365 nm) illumination. It is found the conformation of surface-bound BSA molecules changes with ultraviolet illumination. Moreover, the BSA molecules conjugate to the surface-bound molecules, which are in the overlayer, are released. The reason is ascribed to that TiO2 nanodots absorb ultraviolet and result in the increase of surface hydroxyl groups on nanodots. Such increase further leads to intensified attraction of -NH3 groups in the surface-bound BSA molecules. That not only changes the conformation of the surface-bound BSA molecules, but also weaken the conjugation between surface-bound molecules and other BSA molecules in the overlayer. Eventually, the overlayer of BSA molecules is released. It is believed that such protein conformation variation and release behavior induced through light responses of TiO2 nanodots are crucial in understanding the biomedical performance of TiO2 nanostructures. Also, it could be widely utilized in tailoring of the materials-protein interactions.

Bilirubin attenuates radical-mediated damage to serum albumin.

Oxidative damage to biological macromolecules has been implicated in a number of diseases. Much interest has focused on how non-proteinaceous, low-molecular weight antioxidants prevent oxidative damage to lipids, while comparatively little is known about protein antioxidation. Here we show that bilirubin (BR), the end-product of heme catabolism, when bound to bovine serum albumin (BSA), is oxidised by hydroxyl (.OH), hydroperoxyl (HO2.), and superoxide anion (O2-.) radicals to so far mostly uncharacterised products. The initial oxidation rates of BSA-bound BR decreased in the order OH > HO2. > O2-.. BR protected its carrier protein from oxidative damage inflicted by .OH radicals. This protective action included a reduction in the .OH-mediated cleavage of BSA, conversion of Trp into kynurenine and formation of 'bityrosine-specific' fluorescence. BR also strongly inhibited .OH-mediated formation of protein carbonyls, whereas ascorbate and Trolox (a water-soluble analogue of vitamin E) were much less effective. These results support an antioxidant-protective function of BR and point towards significant differences in the efficacies of various antioxidants in the prevention of oxidative damage to lipids and proteins.

Microwave radiation can alter protein conformation without bulk heating.

Exposure to microwave radiation enhances the aggregation of bovine serum albumin in vitro in a time- and temperature-dependent manner. Microwave radiation also promotes amyloid fibril formation by bovine insulin at 60 degrees C. These alterations in protein conformation are not accompanied by measurable temperature changes, consistent with estimates from field modelling of the specific absorbed radiation (15-20 mW kg(-1)). Limited denaturation of cellular proteins could explain our previous observation that modest heat-shock responses are induced by microwave exposure in Caenorhabditis elegans. We also show that heat-shock responses both to heat and microwaves are suppressed after RNA interference ablating heat-shock factor function.

Increased oxidative modification of albumin when illuminated in vitro in the presence of a common sunscreen ingredient: protection by nitroxide radicals.

We previously reported on the ability of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a ultraviolet A (UVA)-absorbing sunscreen, to generate free radicals upon illumination, and as a consequence, to inflict strand breaks in plasmid DNA in vitro. This study has now been extended to determine the effects of Parsol 1789 and DBM on proteins, under UVA illumination, with the sole purpose of gaining more knowledge on the photobiological effects of sunscreen chemicals. Parsol 1789 (100 microM) caused a 2-fold increase in protein carbonyl formation (an index of oxidative damage) in bovine serum albumin (BSA) when exposed to illumination, and this damage was both concentration- and time-dependent. The degree of protein damage was markedly reduced by the presence of free radical scavengers, namely piperidinic and indolinonic nitroxide radicals, in accordance with our previous study. Vitamin E had no effect under the conditions used. The results obtained corroborate the fact that Parsol 1789 generates free radicals upon illumination and that these are, most probably, responsible for the protein damage observed under the conditions used in our system. However, at present, we cannot extrapolate from these results the relevance to human use of sunscreens; therefore, further studies should be necessary to determine the efficacy at the molecular and cellular level of this UVA-absorber in order to ascertain protection against photocarcinogenic risk.

The effects of derivatives of the nitroxide tempol on UVA-mediated in vitro lipid and protein oxidation.

Derivatives of tetramethylpiperidines are extensively employed in polymers to prevent photooxidation, and their stabilizing effect is attributed to the activity of the nitroxide radical derived from the parent amine. In this study, we examined the photoprotective effect of a commercial polymer photostabilizer, HALS-1, its corresponding nitroxide, bis(2,2,6,6-tetramethyl-piperidine-1-oxyl-4-yl)sebacate (TINO), and two derivatives of the piperidine nitroxide TEMPOL, 2,2,6,6-tetramethyl-piperidin-4-acetyloxy-1-oxyl (TEMP2) and 2,2,6,6-tetramethyl-piperidin-4-octanoyloxy-1-oxyl (TEMP8) synthesized by us, in liposomes exposed to ultraviolet A (UVA) radiation. For comparison, the UVA-absorber, 4-tert-butyl-4'-methoxydibenzoylmethane (Parsol 1789) used in many suncream formulations, was also included. The nitroxide TINO resulted extremely efficient at inhibiting aldehydic breakdown products deriving from 30 min exposure of liposomes to UVA and the protection was dose-dependent (10-100 microM). The corresponding amine HALS-1 was the least efficient while protection increased in the order: TEMP2 < Parsol 1789 < TEMP 8. HALS-1, TINO, and the two TEMPOL derivatives were also tested in a simple protein system consisting of bovine serum albumin (BSA) exposed to UVA. In this case, these compounds did not inhibit nor enhance UVA-mediated protein carbonyl formation in BSA. The differences in protection between the compounds are discussed in relation to their chemical reactivity, UVA-absorbing capacities, and their molecular structure. Overall, the results obtained envisage the potential use of nitroxide compounds as topical antioxidants.

Differential oxidative damage to mitochondrial proteins during aging.

The level of mitochondrial protein oxidative damage, indicated by the protein carbonyl content, has been previously shown to increase with age. The objective of this study was to determine whether such age-associated damage is random or whether some proteins are relatively more susceptible. Mitochondrial proteins were analyzed by gel filtration. High molecular weight proteins were found to be relatively more oxidatively damaged during aging. Results are interpreted to indicate that protein oxidative damage during aging is not random. The question of the validity of the HPLC method for the measurement of protein carbonyls is discussed in light of a recent report questioning its reliability.