Reversible fast-dimerization of bovine serum albumin detected by fluorescence resonance energy transfer

Self-association of bovine serum albumin (BSA) was explored using fluorescence resonance energy transfer (FRET) between two populations of the protein labeled separately with either fluorescein-5'-isothiocyanate (FITC) or eosin-5'-isothiocyanate (EITC). The energy transfer reached the steady state after 5 s at 25 degrees C, indicating a fast exchange between oligomer subunits. The dependence of the energy transfer efficiency on the protein concentration and its reversion by unlabeled BSA demonstrate that association between BSA monomers occurs through a reversible path that involves specific interactions between the protein molecules. Because energy transfer took place even after blocking Cys 34 with iodoacetamide, this residue might not be involved in the reversible self-association process. The number of subunits forming the oligomer and its dissociation constant were determined from measurements of energy transfer as a function of the donor-acceptor ratio and of the total protein concentration. Analysis of these data indicated that BSA is in a monomer-dimer equilibrium with a dissociation constant of 10 +/- 2 microM at 25 degrees C in 10 mM MOPS-K (pH 5.8).     

Increased resonance energy transfer between fluorophores bound to DNA in proximity to metallic silver particles

We examined the effects of metallic silver particles on resonance energy transfer (RET) between fluorophores covalently bound to DNA. A coumarin donor and a Cy3 acceptor were positioned at opposite ends of a 23-bp double helical DNA oligomer. In the absence of silver particles the extent of RET is near 9%, consistent with a Forster distance R(0) near 50 A and a donor to acceptor distance near 75 A. The transfer efficiency increased when the solution of AMCA-DNA-Cy3 was placed between two quartz plates coated with silver island films to near 64%, as determined by both steady-state and time-resolved measurements. The apparent R(0) in the presence of silver island films increases to about 110 A. These values of the transfer efficiency and R(0) represent weighted averages for donor-acceptor pairs near and distant from the metallic surfaces, so that the values at an optimal distance are likely to be larger. The increased energy transfer is observed only between two sandwiched silvered slides. When we replaced one silvered slide with a quartz plate the effect vanished. Also, the increased energy transfer was not observed for silvered slides separated more than a few micrometers. These results suggest the use of metal-enhanced RET in PCR, hybridization, and other DNA assays, and the possibility of controlling energy transfer by the distance between silver surfaces.     

Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin

A simple adaptation of a commercial spectrofluorometer allows selective excitation of fluorescent biomolecules adsorbed to a solid surface while they are in equilibrium with a bulk solution. As a demonstration of this technique, we have detected a change in the effective singlet-singlet energy transfer in fluorescence-labeled bovine serum albumin (BSA) upon adsorption to a fused silica surface. The technique combines total internal reflection fluorescence excitation of surface-adsorbed BSA with a fluorescence spectroscopic examination of energy transfer between two different fluorophores that are covalently bound to amino groups in each BSA molecule. Two donor--acceptor pairs were used, 4-chloro-7-nitro-2,1,3-benzoxadiazole-rhodamine and dansyl-eosin. For studies of surface-adsorbed BSA, we constructed a device in which the excitation light of a standard fluorescence spectrometer totally internally reflects from a surface at which adsorbed BSA is in equilibrium with the bulk solution. A shallow evanescent wave is created, which excites fluorescence from only those BSA molecules in close proximity to the surface. Spectral examination shows significantly less effective singlet-singlet energy transfer from the donor to the acceptor in surface-adsorbed BSA relative to that in native bulk-dissolved BSA. Under appropriate and reasonable assumptions, the energy transfer change between native and adsorbed states of fluorescent BSA can be interpreted as a conformational change of BSA upon adsorption.     

Fluorescence resonance energy transfer in ferritin labeled with multiple fluorescent dyes

We simultaneously labeled ferritin with two Alexa Fluor fluorophores (AF350 and AF430). When both fluorophores label the same ferritin subunit, fluorescence resonance energy transfer (FRET) takes place from the excited AF350 to the acceptor AF430. By varying the number and the ratio of labeling fluorophores, we can modulate FRET such that the ferritin particles can exhibit multiple colors under UV illumination. Labeling of the ferritin shell does not affect the properties of the metallic core. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of intermediates on aggregation of native bovine serum albumin

Protein aggregation has been recognized to be a pathological indicator for several fatal diseases, such as Alzheimer's disease, transmissible spongiform encephalopathies, Creutzfeldt-Jacob disease, etc. Aggregation usually involves conformational changes of proteins that have acquired an intermediate beta-structure-rich conformation and can occur even at low protein concentration. Recent work in our laboratory has shown that bovine serum albumin (BSA), even at low-concentration, exhibits self-association properties related to conformational changes, so providing a very convenient model system to study this class of problems. Here we report data (obtained by different experimental techniques) on a mixture of BSA in native and intermediate (beta-structure-rich) form. Results show that the interaction between the two species is responsible for a decrease in the thermodynamic stability of the solution. This occurs without requiring noticeable conformational changes of the native protein. Results presented here can provide new insight on the "protein only" hypothesis proposed for the formation of plaques involved in several neurodegenerative diseases.     

Effects of oxidation on changes of compressibility of bovine serum albumin

The methods of ultrasound velocity and density measurements were used to study the adiabatic compressibility of bovine serum albumin (BSA) during its oxidation by the prooxidants Cu2+ and 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). We did not find changes of compressibility of BSA in the presence of copper ions at rather high molar ratio Cu2+/BSA = 0.66 mol/mol. This can be explained by binding of the Cu2+ to the binding site of BSA and thus protecting the prooxidant action of the copper. However, AAPH-mediated oxidation of BSA resulted in an increase of its apparent specific compressibility (psik/beta0). These changes could be caused by the fragmentation of the protein.     

Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin

The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.     

Biodistribution and long-term fate of silver nanoparticles functionalized with bovine serum albumin in rats

Silver nanocrystals (Ag NCs) hold promising antibiotic and antiviral properties in biological systems. The biodistribution of silver nanostructures injected into animals in vivo is currently unknown, remaining as a fundamental issue for potential therapeutic applications. Here, we injected Ag NCs capped with bovine serum albumin (BSA) in live rats to elucidate their fate in several organs including liver, heart and brain. Very significant accumulations of nanoparticles were confirmed by inductively coupled plasma mass spectroscopy (ICPMS) and transmission electron microscopy (TEM) techniques on the liver and heart. In contrast, the brain tissue did not reveal evidence of particles content. Our results suggest that Ag+ permeated across the blood-brain barrier (BBB), and followed swift clearance from the organ.     

Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes

Bovine serum albumin (BSA) and fetal calf serum (FCS) were evaluated as protein supplements for in vitro maturation and fertilization of oocytes from cows and hamsters. BSA and low doses of FCS (0.1 or 1.0%) did not support viability or maturation of the cumulus-oocyte complex as well as higher doses of FCS (5, 10, or 20%) for either species. BSA failed to support cumulus expansion for bovine or hamster cumulus-oocyte complexes. All doses of FCS examined supported cumulus expansion in bovine cumulus-oocyte complexes, whereas the hamster complexes required at least 1.0% FCS to induce cumulus expansion. The addition of a serum filtrate, Solcoseryl, with BSA improved viability of the cumulus in the bovine but did not support cumulus expansion or completion of Meiosis I in bovine complexes. In vitro fertilization could be accomplished in media containing FCS by increasing the heparin concentration in the bovine system or reducing FCS for the hamster system. Polyspermy was increased when FCS was the protein supplement. It is not known whether this is an interaction of FCS with the sperm or oocyte. In conclusion, FCS was found necessary for follicle-stimulating-hormone (FSH)-induced cumulus expansion. It also improved cumulus cell viability and completion of the first meiotic division in complexes of both species compared with BSA.     

Calcium-promoted resonance energy transfer between fluorescently labeled proteins during aggregation of chromaffin granule membranes

Proteins of the chromaffin granule membrane were covalently labeled in situ with sulfhydryl-specific fluorophores. Using MIANS (maleimide iodoaminonaphthyl sulfonate) as the donor and fluorescein mercury acetate or fluorescein-5-maleimide as the acceptor, Förster fluorescence resonance energy transfer (FRET) could be employed to measure the degree of inter-membrane and intra-membrane protein-protein contact upon Ca²⁺-induced aggregation of the membranes. The four major findings were: (1) Raising the Ca²⁺ concentration to approx. 500 μM causes the proteins to aggregate in the plane of the membrane. This is demonstrated by Ca²⁺-induced increases in the fluorescence resonance energy transfer in double labeled membranes. This effect is not protein-concentration dependent and occurs at calcium concentrations too low for granule aggregation, implying intra-membrane protein clustering or patching. To our knowledge this is the first direct demonstration of the fluid mosaic nature of subcellular organelles. (2) If two sets of granules are labeled separately, Ca²⁺-induced aggregation brings at least 74% of the labeled proteins into close transmembrane proximity. This effect is also observed at 10–100-fold slower rates in the absence of calcium and can be greatly reduced by depleting the granule membrane of labeled peripheral proteins. It is enhanced if the granules are aggregated by Ca²⁺ or K⁺. We conclude that (some) peripheral proteins can transfer from one membrane surface to another. (3) Aggregation of separately labeled sets of membranes by Ca²⁺ also produces transmembrane energy transfer since: (a) the K_m for Ca²⁺-induced quantum transfer is in the same range as the K_m for aggregation; (b) the reaction is protein-concentration dependent; (c) reversal of aggregation also (partially) reverses donor quenching. (4) A kinetic analysis of the transmembrane effect shows it to be 5–10-fold slower than aggregation itself, supporting earlier suggestions (Haynes, D.H., Kolber, M. and Morris, S.J., (1979) J. Theor. Biol. 81, 713–743) that lipid and protein rearrangements are secondary to granule membrane aggregation.     

Toxic effects of ethanol on bovine serum albumin

The toxic effects of ethanol on bovine serum albumin (BSA) were measured by resonance light scattering (RLS), fluorescence spectroscopy, ultraviolet spectrophotometry (UV), circular dichroism (CD), and transmission electron microscopy (TEM). The results indicated that ethanol had toxic effects on BSA, which led to protein denaturation and the effects increased with the ethanol dose. By means of RLS, BSA was found to aggregate in the presence of ethanol and particles smaller than 100 nm were observed from TEM. The fluorescence spectra showed that the intensity of the characteristic peak of BSA decreased and blue shifted, because of changes in the BSA skeleton structure, as well as alteration of the microenvironment of tryptophan (Trp) residues. The conformation changes of BSA were also shown by UV and CD spectrometry. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:66–71, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20314     

Urea-induced structural transformations in bovine serum albumin

Urea-induced unfolding of bovine serum albumin and one of its fragments containing domain II + III has been studied by difference spectral and fluorescence emission measurements. The unfolding-refolding curves of both the proteins showed the presence of at least one stable intermediate when the transition was monitored at 288 nm. The presence of the intermediate was not detectable at 293 nm where only tryptophan contributed towards the protein absorption. However, both the proteins did show the presence of intermediate when the denaturation was monitored fluorometrically. Since domain III of the albumin is devoid of tryptophan, it is concluded that the formation of intermediate in the unfolding-refolding transition of serum albumin involves (i) unfolding of domain III, (ii) minor structural transformations in domain II, and/or (iii) the separation of the sub-domains of domain III from each other.