New advances in the study on the interaction of [Cr(phen)2(dppz)]3+ complex with biological models; association to transporting proteins

The present study reports a detailed investigation with the interaction of [Cr(phen)₂(dppz)]³⁺ with serum albumins, the key protein for the transport of drugs in the blood plasma, which allows us to understand further the role of [Cr(phen)₂(dppz)]³⁺ as sensitizer in Photodynamic Therapy (PDT).Chromium(III) complex [Cr(phen)₂(dppz)]³⁺, (dppz = dipyridophenazine and phen = 1,10-phenanthroline), where dppz is a planar bidentate ligand with an extended π system, has been found to bind strongly with bovine and human serum albumins (BSA and HSA) with an intrinsic binding constants, K_b, of (1.7 ± 0.3) × 10⁵ M^− 1 and (2.2 ± 0.3) × 10⁵ M^− 1 at 295 K, respectively. The interactions of serum albumins with [Cr(phen)₂(dppz)]³⁺ were assessed employing fluorescence spectroscopy, circular dichroism and UV-vis absorption spectroscopy. The serum albumins-[Cr(phen)₂(dppz)]³⁺ interactions caused conformational changes with the loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the albumin (BSA or HSA) bound to the Cr(III) complex decreased, suggesting that perturbation around the Trp 214 residue took place. The analysis of the thermodynamic parameters ΔG, ΔH, ΔS indicated that the hydrophobic interactions played a major role in both BSA-Cr(III) and HSA-Cr(III) association processes. The binding distances and transfer efficiencies for BSA-Cr(III) and HSA-Cr(III) binding reactions were calculated according to the Föster theory of non-radiation energy transfer. All these experimental results suggests that [Cr(phen)₂(dppz)]³⁺ binds to serum albumins, by which these proteins could act as carriers of this complex for further applications in PDT.     

A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent,pinostrobin

The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, K_a in the range of 1.49 – 6.12 × 10⁴ M⁻¹, with 1:1 binding stoichiometry. Based on the PS–albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics.     

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.     

Interaction of loratadine with serum albumins studied by fluorescence quenching method

The interactions between loratadine and bovine serum albumin (BSA) and human serum albumin (HSA) were studied using tryptophan fluorescence quenching method. The fluorescence intensity of the two serum albumins could be quenched 70% at the molar ratio [loratadine]:[BSA (or HSA)] = 10:1. In the linear range (0–50 μmol L^− 1) quenching constants were calculated using Stern–Volmer equation. Temperature in the range 298 K–310 K had a significant effect (p < 0.05) on the two serum albumins through ANOVA analysis and t-test. Furthermore the conformation changes in the interactions were studied using FTIR spectroscopy.     

Flavonoid–serum albumin complexation: determination of binding constants and binding sites by fluorescence spectroscopy

After a meal rich in plant products, dietary flavonols can be detected in plasma as serum albumin-bound conjugates. Flavonol–albumin binding is expected to modulate the bioavailability of flavonols. In this work, the binding of structurally different flavonoids to human and bovine serum albumins is investigated by fluorescence spectroscopy using three methods: the quenching of the albumin fluorescence, the enhancement of the flavonoid fluorescence, the quenching of the fluorescence of the quercetin–albumin complex by a second flavonoid. The latter method is extended to probes whose high-affinity binding sites are known to be located in one of the two major subdomains (warfarin and dansyl-l-asparagine for subdomain IIA, ibuprofen and diazepam for subdomain IIIA). Overall, flavonoids display moderate affinities for albumins (binding constants in the range 1–15×10⁴ M⁻¹), flavones and flavonols being most tightly bound. Glycosidation and sulfation could lower the affinity to albumin by one order of magnitude depending on the conjugation site. Despite multiple binding of both quercetin and site probes, it can be proposed that the binding of flavonols primarily takes place in subdomain IIA. Significant differences in affinity and binding location are observed for the highly homologous HSA and BSA.     

Effects of thiols, sugars, and proteins on nitric oxide activation of guanylate cyclase.

Purification of soluble guanylate cyclase from rat liver resulted in an apparent loss of enzyme activation by nitric oxide that could be restored by dithiothreitol. methemoglobin, bovine serum albumin, or sucrose. Although hemoglobin also permitted some activation with nitric oxide, the effect of other agents to restore enzyme activation was prevented with hemoglobin. As a result of enzyme purification, there is an alteration of the dose-response relationship for nitric oxide activation. After partial enzyme purification, relatively high concentrations of nitric oxide that were stimulatory in crude enzyme preparations had no effect on enzyme activity. However, partially purified or homogeneous enzyme was activated by lower concentrations of nitric oxide. The bell-shaped dose-response curve for nitric oxide was shifted to the left with guanylate cyclase purification. The addition of dithiothreitol, methemoglobin, bovine serum albumin, or sucrose to enzyme markedly broadens the dose-response curve for nitric oxide. Thus, the apparent loss of responsiveness to nitric oxide with purification is a function of increased sensitivity of guanylate cyclase to nitric oxide. Increased sensitivity to nitric oxide with enzyme purification probably results from the removal of heme, proteins, and small molecules that can serve as scavengers or sinks for nitric oxide and prevent excessive oxidation of the enzyme.     

Photo-induced riboflavin binding to the tryptophan residues of bovine and human serum albumins

Summary The photobinding between riboflavin and the Trp residues from human and bovine serum albumins at two pH-dependent protein conformations was studied. At pH 7.0 both proteins showed photo-adduct formation with hyperbolic kinetics. In the bovine serum albumin this is attributed to the different locations of the two Trp residues. In the case of the human serum albumin, which has only one Trp residue, this behaviour may be related to different molecular conformations of the protein, as is also manifest in the iodide quenching experiments. At pH 3.5, the kinetics of the photo-adduct formation were found to be slower and showed a monophasic behaviour. These results are due to the conformational change of these proteins at acidic pH; the Trp residues of both proteins being now located in a more hydrophobic environment. When bovine serum albumin was anaerobically irradiated at pH 7.0 in the presence of¹⁴C-riboflavin and then cleaved by CNBr, two peptides were obtained, containing the Trp-134 and Trp-212 residues, respectively. The incorporation of¹⁴C-riboflavin in these samples was significantly higher at the level of the peptide containing the Trp-134 residue. Furthermore, it was demonstrated, that the energy transfer from enzymatically generated triplet acetone to riboflavin can also promote the binding of this vitamin to the Trp residues of human and bovine serum albumins.     

The Effect Of Commercial Bovine Serum Albumin And Other Proteins On The Activity Of Bovine Milk Galactosyltransferase

Abstract

The widespread use of bovine serum albumin preparations for the stabilization of purified glycosyltransferases has prompted us to study the effects of different preparations of albumins on the galactosyltransferase activity of bovine milk. For comparison, several other proteins were tested as well. The albumins caused a large stimulation of transferase activity (400–700%) which varied depending on the source of the albumin and the treatment to which it had been subjected. Several other unrelated proteins were tested for their effects on transferase activity. Some proteins stimulated, while others had little effect. Lysozyme stimulated the activity by 178% and poly-L-lysine had Vittle effect. Other proteins stimulated to variable extents. The stimulations obtained with albumin and myelin basic protein were noteworthy. The stimulation was considerably less marked when the enzyme was incorporated into lipid vesicles.     

Role of thiols, pH and cathepsin D in the lysosomal catabolism of serum albumin.

Attempts were made to assess the role of thiols and to determine the cathepsins involved in the degradation of serum albumin in mouse liver and kidney lysosomes. Unlike cysteine or beta-mercaptoethanol, reduced glutathione (GSH) did not stimulate the degradation of formaldehyde-treated albumin in liver lysosomes, suggesting that the tripeptide did not penetrate the membrane. However, GSH was a much more effective stimulant of proteolysis in kidney lysosomes than was cysteine at low concentrations, and the effect was saturable at 1-2 mM concentrations. Thiols did not stimulate proteolysis in lysosomes when the disulphide bonds of albumin were reduced and alkylated, suggesting that the stimulatory effects were solely due to disulphide-bond reduction in protein substrates. Results obtained with thiols and iodoacetamide suggested that albumins denatured by disulphide-bond reduction and alkylation, disulphide-bond reduction without alkylation, or by treatment with 8 M-urea, were all degraded primarily by cathepsin D in lysosomes, but formaldehyde-denatured albumin was attacked by thiol proteinases. These findings correlated well with studies on the degradation of these proteins by rat liver lysosome (tritosome) extracts. Studies with the proteinase inhibitors leupeptin and pepstatin and the stimulatory effects of thiols in these extracts suggested that formaldehyde-denatured albumin was degraded primarily by the thiol proteinases, but that native albumin or albumins denatured by disulphide-bond reduction or by treatment with 8 M-urea were attacked by cathepsin D. Denaturation of serum albumin by any of the methods used caused a shift in the pH optimum of albumin catabolism by tritosome extracts or by purified cathepsin D from approx. 3-4 to 5-6. These results were discussed in terms of a possible mechanism for the catabolic aspect of serum albumin turnover.     

Quenching of the emission of tryptophan, tyrosine, and serum albumins by cupric ion

The effect of cupric ion on the emission of tryptophan, tyrosine, and serum albumins is studied by emission spectroscopy and lifetime measurements. It is found that whenever cupric ion is bound to tryptophan or tyrosine, their emissions are quenched completely. The quenching may be due to an electron transfer mechanism. The fluorescence of complexes of cupric ions with serum albumins is partially quenched; this is because energy is transferred from tryptophan to the complexed cupric ions by a dipolar energy transfer mechanism. It is deduced from the present study that the tryptophan in the human serum albumin molecule is between 11 and 16 Å from the nearest eupric ion binding sites (assumed to be at the surface of the protein) and that one of the tryptophan in the bovine serum albumin molecule is very close to the cupric ion binding sites and the other is near the center of the bovine serum albumin molecule. It is also found that the deuterium solvent effect on serum albumin fluorescence is very small, and that the quenching of bovine serum albumin fluorescence at the N-F transition is the result of quenching of the fluorescence of both tryptophans. The phosphorescence lifetime apparatus, capable of measuring decay times of signals with intensities changing over a few orders of magnitude, and the ratio spectrofluorometer, both of which were constructed in this laboratory, are also described.     

Investigations of interaction mechanism and conformational variation of serum albumin affected by artemisinin and dihydroartemisinin

In this work, binding interactions of artemisinin (ART) and dihydroartemisinin (DHA) with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated thoroughly to illustrate the conformational variation of serum albumin. Experimental results indicated that ART and DHA bound strongly with the site I of serum albumins via hydrogen bond (H-bond) and van der Waals force and subsequently statically quenched the intrinsic fluorescence of serum albumins through concentration-dependent manner. The quenching abilities of two drugs on the intrinsic fluorescence of HSA were much higher than the quenching abilities of two drugs on the intrinsic fluorescence of BSA. Both ART and DHA, especially DHA, caused the conformational variation of serum albumins and reduced the α-helix structure content of serum albumins. DHA with hydrophilic hydroxyl group bound with HSA more strongly, suggesting the important roles of the chemical polarity and the hydrophilicity during the binding interactions of two drugs with serum albumins. These results reveal the molecular understanding of binding interactions between ART derivatives and serum albumins, providing vital information for the future application of ART derivatives in biological and clinical areas.     

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (T_c) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [¹⁴C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle: (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the T_c of the lipid and increases with temperature above the T_c. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.     

Inactivation by detergents of the proline transport system in membrane vesicles from Escherichia coli and its reactivation by bovine serum albumin

The proline transport system of membrane vesicles from Escherichia coli was inactivated by a low concentration of detergents such as deoxycholate, dodecyl sulfate and Triton X-100. The addition of a large amount of bovine serum albumin to membrane vesicles which had been treated with one of these detergents resulted in the restoration of the proline transport activity. The restoration of the transport activity by bovine serum albumin was most remarkable with the deoxycholate-inactivated membrane vesicle. 80% inactivation of the transport system with 0.005% deoxycholate was completely overcome by the addition of albumin. The degree of restoration was dependent on the concentration of albumin. Although albumin stimulated the proline transport activity itself, the stimulatory effect could not account for the restoration transport activity. The binding of deoxy[¹⁴C]cholate to the membrane vesicle was roughly proportional to the amount of detergent added. Deoxycholate once bound to the membrane vesicle was removed almost completely by the incubation with albumin. It is concluded that the removal of detergent from the membrane vesicle by bovine serum albumin results in the restoration of the proline transportactivity.     

The effect of commercial bovine serum albumin and other proteins on the activity of bovine milk galactosyltransferase

The widespread use of bovine serum albumin preparations for the stabilization of purified glycosyltransferases has prompted us to study the effects of different preparations of albumins on the galactosyltransferase activity of bovine milk. For comparison, several other proteins were tested as well. The albumins caused a large stimulation of transferase activity (400-700%) which varied depending on the source of the albumin and the treatment to which it had been subjected. Several other unrelated proteins were tested for their effects on transferase activity. Some proteins stimulated, while others had little effect. Lysozyme stimulated the activity by 178% and poly-L-lysine had little effect. Other proteins stimulated to variable extents. The stimulations obtained with albumin and myelin basic protein were noteworthy. The stimulation was considerably less marked when the enzyme was incorporated into lipid vesicles. These results emphasize the need for caution when adding proteins such as bovine serum albumin to purified enzymes for the purpose of stabilizing the activity of the enzyme.     

Comparison of the antioxidant activity of albumin from various animal species

We measured the antioxidant activity of human, rat, bovine, rabbit, and guinea pig albumins against the superoxide, hydroxyl, and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals. The albumins of different animal species did not differ in antioxidant activity against superoxide. Human and rat albumins exhibited antioxidant activity against hydroxyl radicals, but bovine, rabbit, and guinea pig albumins showed weaker antioxidant activity than human and rat albumins. Human, rat, rabbit, and guinea pig albumins, but not bovine albumin, exhibited strong antioxidant activity against DPPH radicals. Human and rat albumins with strong antioxidant activity against hydroxyl radicals contained methionine-123 in domain 1, but bovine, rabbit, and guinea pig albumins did not. Rat, rabbit, and guinea pig albumins with strong antioxidant activity against DPPH radicals had methionine-264 in domain 2. Human albumin did not have methionine-264, but methionine-298 and methionine-329 in domain 2. Bovine albumin, with the weakest antioxidant activity against DPPH radicals, contained no methionine residues in domain 2. These results suggest that methionine residues in domain 1 or 2 influence the antioxidant activity of albumin.     

Structural studies of bovine,equine, and leporine serum albumin complexes with naproxen

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein‐ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA‐NPS), equine (ESA‐NPS), and leporine (LSA‐NPS) determined to 2.58 Å (C2), 2.42 Å (P6₁), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA‐NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA‐NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein. Proteins 2014; 82:2199–2208. © 2014 Wiley Periodicals, Inc.     

Interaction of loratadine with serum albumins studied by fluorescence quenching method

The interactions between loratadine and bovine serum albumin (BSA) and human serum albumin (HSA) were studied using tryptophan fluorescence quenching method. The fluorescence intensity of the two serum albumins could be quenched 70% at the molar ratio [loratadine]:[BSA (or HSA)]=10:1. In the linear range (0-50 micromol L(-1)) quenching constants were calculated using Stern-Volmer equation. Temperature in the range 298 K-310 K had a significant effect (p<0.05) on the two serum albumins through ANOVA analysis and t-test. Furthermore the conformation changes in the interactions were studied using FTIR spectroscopy.     

Identification of Staphylococcus aureus Colony-Spreading Stimulatory Factors from Mammalian Serum

Staphylococcus aureus forms giant colonies on soft-agar surfaces, which is called colony-spreading. In the present study, we searched for host factors that influence S. aureus colony-spreading activity. The addition of calf serum, porcine serum, or silkworm hemolymph to soft-agar medium stimulated S. aureus colony-spreading activity. Gel filtration column chromatography of calf serum produced a high molecular weight fraction and a low molecular weight fraction, both of which exhibited colony-spreading stimulatory activity. In the low molecular weight fraction, we identified the stimulatory factor as bovine serum albumin. The stimulatory fraction in the high molecular weight fraction was identified as high-density lipoprotein (HDL) particles. Delipidation of HDL abolished the stimulatory activity of HDL. Phosphatidylcholine, which is the major lipid component in HDL particles, stimulated the colony-spreading activity. Other phosphatidylcholine-containing lipoprotein particles, low-density lipoprotein and very low-density lipoprotein, also showed colony-spreading stimulatory activity. These findings suggest that S. aureus colony-spreading activity is stimulated by albumin and lipoprotein particles in mammalian serum.     

Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol.

Methoxypolyethylene glycols of 1900 and 5000 daltons have been attached covalently to bovine serum albumin using cyanuric chloride as the coupling agent. When sufficient polymer is attached, the modified bovine serum albumin appears to lose its immunogenicity in the rabbit and, on intramuscular or intravenous injection, elicits antibodies neither to itself nor to native bovine serum albumin. It does not react with antibodies raised against native bovine serum albumin. Bovine serum albumin to which methoxypolyethylene glycol has been attached exhibits a blood circulating life in the rabbit rather similar to native bovine serum albumin, except that it is not removed from circulation by the eventual development of antibodies. Modified bovine serum albumins which had been iodinated with 125I, or prepared with [14C]cyanuric chloride, were injected intravenously in rabbits. Both labels appeared almost quantitatively in the urine after 30 days. The modified bovine serum albumins showed substantial changes in properties, such as solubility, electrophoretic mobility in acrylamide gel, ion exchange chromatography, and sedimentation, as compared with the unmodified protein.     

Spontaneous and enzymic rearrangement of naringenin chalcone to flavanone

The isomerisation of 2′,4,4′,6′-tetrahydroxychalcone by the enzyme chalcone isomerase is difficult to assay accurately in view of the spontaneous cyclisation of this chalcone at the alkaline pH optimum of the enzyme. We report here that self-cyclisation of naringenin chalcone is dramatically reduced at pH-values ? 6.5 and in the presence of high concentrations of serum albumin (5–10 mg ml ^?1). We have critically evaluated existing assay procedures of chalcone isomerase, utilizing the effects of a monospecific anti-(chalcone isomerase) serum to distinguish between spontaneous and enzymic cyclisation of chalcone. We conclude that the modifications listed above considerably facilitate the measurement of chalcone isomerase kinetics.