Comparative investigations of the binding of perazine dimalonate (PDM) to human and bovine serum albumin (author's transl)]

Comparative Investigations of the Binding of Perazine Dimalonate (PDM) to Human and Bovine Serum Albumin The unbound portion of perazine dimalonate (PDM) in buffered solutions of human and bovine serum albumin (pH 7.4) was determined by means of gel filtration and ultrafiltration. Both procedures were optimized by applying techniques which are normaly used in high-pressure liquid chromatography. The binding of the drug to serum albumin was characterized by determination of the overall binding constant (K₁), the apparent binding constant (k⁺), the number of binding sites per molecule of albumin (n) and the free reaction energy (ΔF°). It was found that less PDM is bound by bovine serum albumin than by human serum albumin.     

Azapropazone binding to human serum albumin

Summary Azapropazone, a new non-steroidal antiinflammatory drug, is strongly bound to human serum albumin. As revealed by Scatchard analysis, one high-affinity binding site with an association constant of about 1.2×10⁶ M^–1 and two low-affinity binding sites with association constants of about 0.05×10⁶ M^–1 were found. While the high-affinity binding site of azapropazone is clearly not identical with the diazepam or digitoxin binding sites of human serum albumin, contradictory evidence was found by optical measurements and displacement studies for the similarity of the azapropazone and the warfarin binding site of human serum albumin. At present, it is suggested that both drugs bind to different areas of the same binding site. Therefore, the pronounced effects of various disease states on the plasma protein binding of azapropazone can not be explained by a binding to an unusual binding site, but seem to be due to an extreme sensitivity of the azapropazone binding area to the putative endogenous binding inhibitors, present in the blood during those disease states.     

Palmitate and stearate binding to human serum albumin

Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 °C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions. The experimental data were analysed by a computerised curve fitting procedure using equilibrium equations for multiple binding of ligands, containing relative binding constants, valid whether the ligands are truly insoluble or are slightly soluble and irrespective of aggregation in aqueous solution. A best-fit set of relative binding constants was found, and subsequently 30 sets of acceptable constants for each set of data in order to evaluate the variation. The data were first fitted by the relative Scatchard's equation, then by the relative, stoichiometric equation. Scatchard's equation is deduced on the presumption that cooperativity is absent while the stoichiometric equation is valid even when cooperativity is present. It was found with palmitate as well as with stearate that the two equations fitted the data equally well, and it was concluded that the observations were compatible with absence of cooperativity. The relative Scatchard binding constants were converted to relative, stoichiometric constants and it was found that the variations of the latter were slight. © Munksgaard 1997.     

The binding of physostigmine to human serum albumin

The binding of [3H]physostigmine to crystallized human serum albumin (HSA) has been investigated using equilibrium dialysis. The percentage bound to 1% (w/v) HSA decreased from 18 to 4% as the total concentration of physostigmine increased from 3.3 nM to 2.7 microM (0.9 to 750 ng mL-1). A single class of specific binding sites with a large affinity constant, K = 8 x 10(7) L mol-1, was identified. The concentration of binding sites was approximately 3 nM. The Michaelis constants for human serum cholinesterase and albumin were the same; an explanation for these results is that the drug is binding to a trace cholinesterase, in the albumin.     

Diclofenac binding to albumin and lipoproteins in human serum

The binding of diclofenac to human serum albumin (HSA) and to lipoproteins was studied in vitro by equilibrium dialysis. Binding to HSA is characterized by two classes of sites with one site each (K1 = 5 X 10(5) M-1 and K2 = 0.6 X 10(5) M-1). The binding to lipoproteins was shown to be saturable with a larger number of binding sites and low association constants. The evidence of two specific binding sites on HSA was confirmed by circular dichroism data. In addition, an identification of those sites was performed by displacement of fluorescent probes. The data show that the high affinity site (K1 = 5 X 10(5) M-1) is likely to be shared by benzodiazepines while the second one (K2 = 0.6 X 10(5) M-1) is common with the warfarin site.     

Warfarin-sulfinpyrazone interaction on binding to human serum albumin

Sulfinpyrazone displacement of warfarin from human serum albumin was studied in-vitro. At low sulfinpyrazone concentrations one molecule of warfarin is displaced on binding by one molecule of sulfinpyrazone. Clinical plasma concentrations of sulfinpyrazone are, however, too low to cause significant displacement.     

Characterization of the binding of benzodiazepines to human serum albumin

Summary The binding of eleven benzodiazepine derivatives to human serum albumin (HSA) was determined by means of sephadex gel filtration.The albumin binding of the substances was characterized by the percentage of bound drug, the binding constants k ⁺, K ₁ and m, the number of binding sites per albumin molecule, and the free binding energy. Under the conditions chosen in these experiments there seems to exist only one binding site of the same type for all investigated benzodiazepines at the HSA molecule. The affinities of the benzodiazepines to this binding site are very different. It is discussed which part of the benzodiazepine molecule represents the main binding group.     

On the binding of cinmetacin and indomethacin to human serum albumin

The binding of two non-steroidal anti-inflammatory drugs, indomethacin and cinmetacin, to human serum albumin was studied by dynamic dialysis at 37 degrees C and pH 7.4. Cinmetacin is bound more than indomethacin. The affinity constant for the primary binding site is 4.28 X 10(6) M-1 for cinmetacin and 1.4 X 10(6) M-1 for indomethacin. The protein binding of indomethacin is decreased in the presence of cinmetacin.     

The binding of oxicam derivatives to human serum albumin

The binding characteristics of several oxicam derivatives (tenoxicam, 4-hydroxy-2-methyl-N-phenyl-2H-1,2-benzothiazixine-3- carboxamide-1,1-dioxide (CP 14,304), 4-hydroxy-2-methyl-N-2-(3-methyl)-pyridyl-2H-1,2-benzothiazixine-3 -carboxamide- 1,1-dioxide (CP 16,460), piroxicam, meloxicam [corrected], isoxicam, 5-hydroxy-piroxicam) to 2% and 4% human serum albumin (HSA) were determined using a modified ultrafiltration process. The binding properties to HSA were characterized by determining the overall binding constant, the apparent binding constant, the slope, the free reaction energy, and the unbound portion of the drug. The following results were obtained: 1. These oxicam derivatives show a high affinity to HSA. The unbound fraction amounts to 1-3%. 2. The affinity of the compounds to HSA decreases in the order mentioned above. 3. Doubling of the HSA concentration reduces the unbound fraction, to the half, with piroxicam being the only exception.     

Allosteric modulation of drug binding to human serum albumin

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its extraordinary ligand binding capacity. The three homologous domains of HSA (labeled I, II, and III), each in turn composed of two subdomains (named A and B), give rise to the three-dimensional structure of HSA. This flexible structural organization allows the protein structure to adapt to a variety of ligands. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA similar to a multimeric protein. Although kinetic and thermodynamic parameters for ligand binding to HSA calculated by quantitative structure-activity relationship models are in excellent agreement with those obtained in vitro, cooperative and allosteric equilibria between different binding sites and competition between drugs or between drugs and endogenous ligands make difficult the interpretation of HSA binding properties in vivo. Binding of exogenous and endogenous ligands to HSA appears to be relevant in drug therapy and management. Here, the allosteric modulation of drug binding to HSA is briefly reviewed.     

The binding of thioureylene compounds to human serum albumin

The binding interactions of some thioureylene compounds to human serum albumin were studied in vitro by ultraviolet spectroscopy and equilibrium dialysis. Binding of 6-n-propyl-2-thiouracil, 6-n-benzyl-2-thiouracil and 2-thiouracil to human serum albumin results in a red shift of the ultraviolet absorption maximum, suggesting that the binding site is a hydrophobic area of the protein. Bindings of 6-n-propyl-2-thiouracil and 6-n-benzyl-2-thiouracil to human serum albumin are characterized by two classes of sites while 6-n-propyl-uracil and 2-thiouracil bind to one low-affinity binding site. In addition, an identification of those sites was performed by measuring the displacement of these drugs. The data show that the moderate-affinity site is common with the warfarin site while the low-affinity site is likely to be shared by benzodiazepines. It is concluded that the binding is enhanced by the hydrophobicity of the substituent in the thioureylene compounds, and it is further shown that thiol-group substitutions in the thioureylene ring will weaken the binding.     

Kinetics of the binding of salicylazosulfapyridine to human serum albumin.

In order to elucidate the possibility of the dissociation rate of drugs from plasma proteins presenting a rate limiting step in the elimination of drugs by secretion (renal or biliary) and metabolism, the kinetics of salicylazosulfapyridine (SASP) binding to human serum albumin (HSA) has been investigated by stopped-flow photometry. Equilibrium dialysis showed that HSA has three classes of binding sites for SASP with 0.93, 2.3 and 8.4 sites, respectively, and association constants of 2.1.10(6), 1.4.10(5) and 3.0.10(3) M-1, respectively. The association rate constants for the first and second classes are 4.4.10(6) and 1.5.10(7) M-1 sec.-1, and the dissociation rate constants are 2.1 and 109 sec.-1. At SASP concentrations resulting from the usual therapeutic doses about 83% will bind to the first class binding sites. The dissociation "half time" for this class being 0.34 sec., leads to the conclusion that dissociation rates of this order of magnitude are unlikely to reduce the rate of metabolism or biliary secretion whereas it may reduce renal tubular secretion. Whether this is the case depends on the intrinsic rate constant of secretion.     

齐墩果酸在人血浆蛋白和血清白蛋白中结合率的测定

齐墩果酸(oleanolic acid,OA)又名庆四素(图1),系五环三萜类化合物,以游离或结合成苷的形式广泛存在于白花蛇舌草、山楂、丁香、大枣、女贞子、枇杷叶、橡木和夏枯草等植物中。临床上主要用于急     

Influence of pH on the binding of suramin to human serum albumin

The pH dependence of the binding of suramin to albumin has been studied by means of equilibrium dialysis and circular dichroism. Dialysis experiments have revealed that the association constants of the high and low affinity binding sites are strongly influenced by the pH. At pH 6.0 K1 = 1.4 x 10(6) M-1/n1 = 2.0 and K2 = 1.3 x 10(5) M-1/n2 = 1.0; at pH 9.2 K1 = 2.0 x 10(5) M-1/n1 = 2.0. At the high pH no low affinity sites could be demonstrated any more. The pH dependence of the induced ellipticity of the suramin-albumin complex at low molar drug-to-protein ratio r = 0.1 can be superimposed upon the neutral-to-base (N-B) transition of albumin alone. By means of the Linderstr?m-Lang equation for electrostatic interaction and a two-state model for the N-B transition of albumin, evidence is obtained of a link of the pH dependent binding behaviour of suramin to albumin and the neutral-to-base transition of albumin. The possible correlation of this link with transport processes of suramin in the body and with selective uptake of suramin in cells and parasites is discussed.     

Reviewing the binding of a series of parabens to human serum albumin

To better understand the factors that contribute to the accumulation of unmetabolized parabens (p‐hydroxybenzoic acid esters) in breast cancer tissue, the binding of a series of parabens (methyl‐, ethyl‐, butyl‐, benzyl‐paraben) to human serum albumin (HSA) was investigated by fluorescence spectroscopy and also their ability to modify the binding parameters of albumin site markers. Emission spectra of HSA upon fluorescence excitation of Trp 214 residue at 295 nm were recorded at different molar ratios of PB/HSA and data were corrected for the inner‐filter effect. A significant inner‐filter effect was obtained for molar ratios of 2.0 and above. For lower molar ratios, a slight increase in fluorescence of HSA was detected. p‐Hydroxybenzoic acid, the main metabolite of parabens, did not modify the fluorescence of HSA whatever the molar ratio used. Binding parameters for compounds that are markers of site I, bilirubin and warfarin, were determined in the absence and presence of methyl, butyl and benzyl paraben at molar ratios of PB/HSA of 0, 1 and 2. No variation of the binding constants of these markers was observed. The results indicate that parabens weakly interact with HSA thus suggesting that they are in a free form in blood and therefore more available to reach tissues. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of the binding epitope of ciprofloxacin bound to human serum albumin

Aqueous solutions of ciprofloxacin in phosphate buffer were measured by NMR under physiological conditions. The chemical shifts differ substantially compared to earlier investigations at low pH or in DMSO. Protein binding experiments using saturation transfer were optimized to measure proton resonances of ciprofloxacin that are in close proximity to human serum albumin. The relative intensities were mapped on the molecule to define the binding epitope. According to this methodology the cyclopropane ring and the chinolon ring constitute the binding epitope. Competition experiments with increasing amounts of salicylic acid did not change the saturation transfer to the ciprofloxacin protons indicating at least two different binding sites.     

Intravascular factors affecting diazepam binding to human serum albumin.

The binding of a benzodiazepine derivative (diazepam) to human serum albumin was studied by equilibrium dialysis. The influence on this interaction of various endogenous substances (fatty acids, uric acid and bilirubin) and the metabolites of diazepam (desmethyldiazepam and oxazepam) were investigated. Binding studies were analyzed by fitting a model which utilizes the two independently measured quantities, the free drug concentration and the total amount of drug, to the experimental data points with a least squares method. Oleic acid decreases the binding of diazepam at all molar ratios of fatty acid:albumin. In contrast, palmitic acid at ratios of < 1:1 enhanced diazepam binding. Both uric acid and bilirubin had negligible effects on diazepam binding. The metabolites were bound to a less extent than diazepam (diazepam > desmethyldiazepam > oxazepam), and both metabolites reduced diazepam binding to albumin. In vivo, the absolute ratio of specific fatty acids to each other is probably as important a source of variations as the quantitative changes in total fatty acids or metabolites.