Bovine Serum Albumin System Research Articles

Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application

In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.

In vitro study on the binding of anti-coagulant vitamin to bovine serum albumin and the influence of toxic ions and common ions on binding

The mechanism of binding of vitamin K 3 (VK 3) with bovine serum albumin (BSA) was investigated by fluorescence, absorption and circular dichroism (CD) techniques under physiological conditions. The analysis of fluorescence data indicated the presence of static quenching mechanism in the binding. Various binding parameters have been evaluated. Thermodynamic parameters, the standard enthalpy change, Δ H 0 and the standard entropy change, Δ S 0 were observed to be −164.09 kJ mol −1 and −465.08 J mol −1 K, respectively. The quantitative analysis of CD spectra confirmed the change in secondary structure of the protein upon interaction with VK 3. The binding average distance, r between the donor (BSA) and acceptor (VK 3) was determined based on the Förster's theory and it was found to be 3.3 nm. The effects of toxic ions and common ions on VK 3–BSA system were also investigated.

Interaction between Proteins and Cationic Gemini Surfactant

Surface tension, fluorescence, and circular dichroism (CD) methods have been used to investigate the interaction between cationic gemini surfactant 1,2-ethane bis(dimethyldodecylammonium bromide) (C12C2C12) and proteins including bovine serum albumin (BSA) and gelatin. Surface tension measurements show that the complexes of gelatin--C12C2C12 form more easily than that of BSA--C12C2C12. Addition of C12C2C12 has a different effect not only on the polarity of the microenvironment in BSA and gelatin systems but also on their fluorescence spectra. It can be seen from far-UV CD spectra that the alpha-helical network of BSA is disrupted and its content decreases from 41.7% to 27.6% while the random coil content of gelatin increases from 53.0% to 55.9% with increasing C12C2C12 concentration. The results from near-UV CD spectra show that the binding of C12C2C12 induces changes of the microenvironment around the aromatic amino acid residues and disulfide bonds of BSA at high C12C2C12 concentrations.

Studies on the Interaction of Cinnamic Acid with Bovine Serum Albumin

The interaction between cinnamic acid and bovine serum albumin (BSA) have been studied at three temperatures, 296, 303 and 310 K. Fluorescence quenching spectra in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to investigate the drug-binding mode, the binding constant and the protein structure changes in the presence of cinnamic acid in aqueous solution at pH 7.40. The fluorescence quenching constant K(q), K(sv) and the binding constant K were calculated according to Stern-Volmer equation based on the quenching of the fluorescence of BSA in the presence of cinnamic acid. The thermodynamic parameters, the enthalpy (DeltaH) and the entropy change (DeltaS) were estimated to be -16.457 kJ mol(-1) and 38.028 J mol(-1) K(-1) according to the van't Hoff equation. The displacement experiment shows that cinnamic acid can bind to the subdomain IIA (corresponding to Sudlow's drug binding site I). The distance between the tryptophan residues in BSA and cinnamic acid bound to site I was estimated to be 1.63 nm using Föster's equation on the basis of fluorescence energy transfer. The decreased binding constant in the presence of common ions indicates that common ions have effect on drug-BSA system.

The effect of sodium dodecyl sulfate on the conformation of bovine serum albumin

The effect of sodium dodecyl sulfate (SDS) on the conformation of bovine serum albumin (BSA) was first determined by cyclic voltammetry, and verified by isothermal titration microcalorimetry, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and freeze-fracture transmission electron microscopy (FF-TEM). In a very low SDS concentration range, SDS makes the globular protein loose through a specific binding in the structure, and then surface adsorption of SDS around the protein occurs. This makes the cathodic peak current of BSA arising from disulfide bonds increase first and then decrease steeply. When the SDS concentration is larger than 1.0 × 10 −3 mol L −1, the electrostatic repulsion between the head groups of SDS could initiate the partial unfolding, and the gradual exposure of disulfide bonds facilitate the redox of BSA, leading to a slow decrease in the peak current. When the SDS concentration reaches the critical aggregation concentration (4.4 × 10 −3 mol L −1), the SDS binding and resulting protein denaturation are largely enhanced. As only a small amount of disulfide bonds are surrounded by the micelle-like clusters, the decrease of the cathodic peak current of BSA becomes even slow. Above the critical micellar concentration, the repulsion between the charged micelle-like clusters and the increased hydrophobic interactions of hydrophobic SDS chains with the hydrophobic backbone of BSA results in the complete unfolding of the protein. The cathodic peak current is hardly affected with SDS addition due to the complete surrounding of disulfide bonds by micelle-like clusters. The corrected mixing enthalpy of SDS with BSA was exothermic at all SDS concentration (0–1.1 × 10 −2 mol L −1), and could be interpreted in terms of various molecular events: specific binding, cooperative binding, protein unfolding, micelle cluster, and free micelle formation, which confirms the electrochemical behavior. The above results can be further verified by fluorescence spectral feature of BSA and FF-TEM images of SDS–BSA system. CD spectra studies indicate that the interactions of SDS with BSA only cause partial loss of the secondary structure of BSA.

Resonance light scattering technique for the determination of proteins with polymethacrylic acid (PMAA)

As a resonance light scattering (RLS) probe, the polyelectrolyte polymethacrylic acid (PMAA) was applied in this assay. The bovine serum albumin (BSA) and human serum albumin (HSA) were determined by the electrostatic interaction of PMAA and proteins. At pH 3.8 Na 2HPO 4–citric acid buffer solution, the RLS intensities of PMAA–BSA (HSA) system were greatly enhanced. The characteristic peaks were appeared at the wavelength 320, 546 and 594 nm. The optimization conditions of the reaction were also examined and selected. Under the selected conditions, the RLS intensities were proportional to the protein concentrations in the range of (0.0200–2.00) × 10 −6 mol/L for BSA and (0.0200–2.40) × 10 −6 mol/L for HSA. The influences of some foreign substances were also examined. The synthetic samples containing proteins and some real samples were analyzed and the results obtained were satisfactory.

Influence of metal ions on phosphatidylcholine–bovine serum albumin model membrane, an FTIR study

FTIR spectroscopy was used to study the interaction of K +, Ca 2+ and Eu 3+ ions and the Phosphatidylcholine (PC)–bovine serum albumin (BSA) complex. First, a PC–BSA interaction system was constructed. The analytical results of transmission electron microscope (TEM), quasi-elastic light scattering (QELS) techniques and FTIR-ATR spectroscopy indicated that PC molecules interacted with BSA in aqueous solutions. However, IR inspection was limited for aqueous solutions. Solid experimental condition was then employed, and FTIR spectra showed that the PC and BSA molecules incorporated with each other, which could represent their interactions in solutions. Then, the influence of metal ions on PC–BSA system was studied in solid experimental conditions, and FTIR spectroscopy was used in this study. The spectral results showed that: (1) K +, Ca 2+ and Eu 3+ ions all decreased the rigidities of acyl chains of PC in PC–BSA systems. (2) The interactions between Ca 2+, Eu 3+ ions and the hydrophilic phosphate ester and carbonyl ester groups of PC were stronger than that of K + ions, while the influent modes of Ca 2+ and Eu 3+ ions on these regions were different. (3) When the relative molar content of Eu 3+ ions to PC ( R i/p) reached 2, the coordination effect between Eu 3+ ions and PO 2 − groups of PC was saturated. (4) The addition of these ions increased the content of α-helix structures of BSA, and decreased the content of β-turn structures. By comparing these results with the interactions of K +, Ca 2+, Eu 3+ ions with phospholipid system in the absence of protein, some special characters were discovered in the acyl regions of PC, while their interactions results with the hydrophilic regions of PC were alike. It might be interpreted that these metal ions influenced the acyl chains of PC mediated from BSA molecules, and coordinated directly with the hydrophilic regions of PC. As for biological membrane was a system included both phospholipid and proteins, these characters suggested that phospholipid–protein mixture system could be a better model in the studies of interaction between metal ions and biological membranes using FTIR spectroscopy.

Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems

The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems.

Studies on bovine serum albumin-sodium dodecyl sulfate complexes using pyrene fluorescence probe and 5-doxylstearic acid spin probe.

Interactions and characteristics of 0.1% bovine serum albumin (BSA)-sodium dodecyl sulfate (SDS) in 20 mM phosphate buffer solution were investigated by means of fluorescence spectroscopy and electron spin resonance (ESR) spectroscopy. In BSA-SDS system, the intensity ratio, Im3/Im1, of the third vibronic band of the pyrene monomer to the first vibronic band showed a small peak at about 0.1 mM SDS in the phosphate buffer below cmc. In accordance with this Im3/Im1 ratio, the intensity ratio, Ie/Im1, of fluorescence from the pyrene excimer to that from the monomer showed a pseudo-plateau (0.08-0.8 mM) and suggested the existence of micelle-like aggregates below the cmc. Temperature dependence of ln(Ie/Im1) in pyrene fluorescence in the SDS-BSA system was examined as a function of SDS concentration. The activation energy of pyrene diffusion for excimer formation in a micelle was estimated to be 19.2 kJ mol(-1) for the BSA-SDS system. ESR spectra of 5-doxylstearic acid (5-DSA) showed that the probe location is restricted at SDS concentrations above the cmc, and that the probe also is highly restricted in motion for BSA-bound SDS micelles.

Mechanisms of Interaction Between Vanillin and Milk Proteins in Model Systems

ABSTRACT: The effects of milk proteins on vanillin intensity reduction were studied in a phosphate buffer (pH 6.5) using urea and sodium dodecyl sulfate (SDS) as bond disrupting agents. The reduction of vanillin occurred immediately as the protein was introduced to the system. Bovine serum albumin (BSA) interacted more with vanillin than sodium caseinate (CAS) did. Heat treatment had no effect on vanillin reduction of the CAS system; however, free vanillin content was higher in the heated than nonheated BSA system. Hydrogen bonding appeared to be a major force for the interaction of vanillin and CAS. However, hydrophobic interaction seemed to be more important than hydrogen bonding in the vanillin and BSA system.

The subsequent effect of interaction between Co(2+) and human serum albumin or bovine serum albumin.

A notable hysteretic effect has been observed in the interaction of Co(II) with human serum albumin (HSA) or bovine serum albumin (BSA) using UV-Visible spectrometry at physiological pH (7.43), which shows that the binding between Co(II) and HSA or BSA may induce a slow transition of HSA or BSA from the conformation of weaker affinity for Co(II) to one of stronger affinity (A-B transition). The rate constants and activation parameters of this transition were measured and are discussed. It is inferred that such a conformation transition may occur due to the binding of the first Co(II) ion with the peptide segment of N-terminal residues 1-3, which results in a 'hinged movement' of the relatively hydrophobic 'valley' in the IA subdomain. This process leads to a slow conformational transition in the albumins, makes the other binding sites of Co(II) exposed, and shows a positive cooperativity effect. The LMCT (ligand-to-metal charge transition) bands of the Co(II)-HSA and Co(II)-BSA systems also show a kind of hypochromic effect featuring a dipole-dipole interaction mechanism. This phenomenon is rarely reported.

Binding equilibrium study between Mn(II) and HSA or BSA

AbstractThe binding of Mn(II) to human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by equilibrium dialysis at physiological pH (7.43). The Scatchard analysis indicates that there are 1.8 and 1.9 strong binding sites of Mn(II) in HSA and BSA, respectively. The successive stability constants which are reported for the first time are obtained by non‐linear least‐squares methods fitting Bjerrum formula. For both Mn(II)‐HSA and Mn(II)‐BSA systems, the order of magnitude of K1 was found to be 104. The analyses of Hill plots and free energy coupling show that the positive cooperative effect was found in bom Mh(II)‐HSA and Mn(II)‐BSA systems. The results of Mn (II) competing with Cu (II) Zn(II), Cd(II) or Ca(II) to bind to HSA or BSA further support the conjecture that there are two strong binding sites of Mn(II) in both HSA and BSA. One is most probably located at the tripeptide segment of N‐terminal sequence of HSA and BSA molecules involving four groups composed of nitrogen atoms, and the fifth coordination atom is the carboxyl oxygen of Asp1. The coordinated atoms of the other are most probably almost all oxygen atoms.

Step-and-Repeat Photopatterning of Protein Features Using Caged-Biotin−BSA: Characterization and Resolution

This paper characterizes the caged-biotin−BSA system developed for selectively patterning biotinylated proteins into patterns on glass slides. Methyl α-nitropiperonyloxycarbonyl biotin, caged biotin, is coupled to a bovine serum albumin (BSA) carrier molecule using succinimide chemistry and then employed in a four-step patterning process: (1) caged-biotin−BSA compound is adsorbed onto a glass slide; (2) the slide is irradiated with 353-nm light through a chrome-on-glass photomask; (3) streptavidin is selectively bound to the irradiated regions; and (4) biotinylated protein is bound to the selectively immobilized streptavidin. A step-and-repeat scheme was used to demonstrate that streptavidin and then biotinylated BSA can be sequentially immobilized with reproducible feature density and little interfeature binding. Eight-minute irradiation of a mixed monolayer of 25% native BSA and 75% caged-biotin−BSA yielded the highest feature contrast, required the minimum use of reagent, and produced the least nonspe...

Equilibrium dialysis study on the interaction between Cu(II) and HSA or BSA

The interaction of Cu(II) and human serum albumin (HSA) or bovine serum albumin (BSA) at physiological pH is studied by equilibrium dialysis. The successive stability constants are obtained by non-linear least square methods fitting Bjerrum formula. For both the Cu(II)-HSA and Cu(II)-BSA systems, the order of magnitude of K1 and K2 was found to be ≈104 mol−1· dm3. There are about twenty stoichiometry binding sites found in one HSA or BSA molecule. They can be divided into two or three sets. Results of equilibrium dialysis experiments suggest that there exists one strong metal binding site in both Cu(II)-HSA and Cu (II)-BSA. It is the imidazol group nitrogen atoms of His3 that are primarily concerned with copper binding site. After reaching dialysis equilibrium, there is the interaction among the different binding sites, the values ofK all deviate from the simple statistical effect except forK 1 and K2 in both Cu(II)-HSA and Cu(II)-BSA systems, and the positive cooperative effect is found.

Equilibrium dialysis of metal-serum albumin (II)——Allosteric effect in Ni(II)-serum albumin systems

Detailed studies were carried out on equilibrium dialysis of the binding of Ni2+ ion to human serum albumin (HSA) and bovine serum albumin (BSA). The successive stability constants were obtained by the leastsquares fitting. The eight binding sites found for both Ni(II)-HSA and Ni(II)-BSA systems can be divided into two different sets; and for both systems, there exist two identical prior binding sites where the bound Ni2+ ions can be considered as allosteric effectors, which induce the allosteric effect in accordance with the model proposed by Monodet al. As indicated by allosteric parameters, the ability of R-state to bind Ni2+ ionions is ca. 100 times as much as that of T-state, and the conformation of HSA is markedly tenser than that of BSA.

Equilibrium dialysis of metal-serum albumin. I. Successive stability constants of Zn(II)-serum albumin and the Zn 2+-induced cross-linking self-association

The binding of Zn(II) to human serum albumin (HSA) and bovine serum albumin (BSA) was intensively studied by equilibrium dialysis. The successive stability constants were obtained by least-squares fitting. For both the Zn(II)-HSA and Zn(II)-BSA systems, the successive stability constants are basically similar, though such constants for the latter generally slightly larger than those for the former; the order of magnitude of K 1 and K 2 was found to be ≈ 10 5 M −1. The twelve binding sites found for Zn(II)-HSA and fourteen binding sites for Zn(II)-BSA can be divided into three and two different sets, respectively, but in both systems, there exist approximately three identical strongest binding sites. The binding equilibrium of Zn(II)-HSA markedly depends on the concentration of HSA. The type of Scatchard plots indicates the existence of the Zn 2+-induced cross-linking self-association of HSA, which was found from Wyman plots to be the strongest at a concentration of Zn 2+ in the range (4-26) × 10 −6 M. The maximum of the apparent self-association constants k 2 so obtained was approximately 2.6 × 10 4 M −1. Such a self-association mechanism and the order of magnitude of K 1, and K 2 both to some extent support the inference that the zinc group ions are bound to the cysteinyl sulphur atoms.

Characterization of covalent protein conjugates using solid-state carbon-13 NMR spectroscopy

Cross-polarization magic-angle spinning (CPMAS) 13C NMR spectroscopy has been used to characterize covalent conjugates of alachlor, an alpha-chloroacetamide hapten, with glutathione (GSH) and bovine serum albumin (BSA). The solid-state NMR method demonstrates definitively the covalent nature of these conjugates and can also be used to characterize the sites of hapten attachment to proteins. Three different sites of alachlor binding are observed in the BSA system. Accurate quantitation of the amount of hapten covalently bound to GSH and BSA is reported. The solid-state 13C NMR technique can easily be generalized to study other small molecule/protein conjugates and can be used to assist the development and refinement of synthetic methods needed for the successful formation of such protein alkylation products.

Distribution behavior of some drug enantiomers in an aqueous two-phase system using counter-current extraction with protein

The partial resolution of some drug enantiomers by counter-current extraction in an aqueous two-phase system using bovine serum albumin (BSA) or ovomucoid (OVM) as chiral discriminator is described. The phase system was prepared with dextran 40, polyethylene glycol and a protein. Ofloxacin (OFLX) enantiomer was well recognized by the BSA system, whereas the enantiomers of carvedilol (β-blocker) and DG-5128 (a new oral hypoglycaemic drug) were hardly recognized by the OVM system. The distribution coefficients of (S)-, (R)- and rac-OFLX varied with pH. Good enantioselectivity for OFLX was obtained at relatively high pH (≥ pH 9). The crude optically enriched OFLX enantiomers were extracted from the aqueous fractions and finally purified by thin-layer chromatography.

Volatile retention during freeze drying of protein solutions

Retention of 14C-labeled 2-propanol was studied in freeze-dried systems containing either bovine serum albumin (BSA) or pepsin. Retention during freeze drying varied with: initial concentration of solids and 2-propanol, rate of freezing and sample thickness. Heat denaturation of the proteins did not significantly affect the volatile retention. When freeze-dried BSA was rehumidified, volatile retention dropped to a new level which depended on the final water content. Heating the freeze-dried BSA systems in vacuum had no effect on the 2-propanol retention. The results are indicative of structurally dependent retention and can be mainly interpreted by the “microregions” theory of volatile retention.