Kinetics of the Binding of Salicylazosulfapyridine to Human Serum Albumin

Abstract 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 salicylazosulfa-pyridine (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 · 10⁶, 1.4 · 10⁵ and 3.0 · 10³ M^-1, respectively. The association rate constants for the first and second classes are 4.4 · 10⁶ and 1.5 · 10⁷ 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.     

The Effect of Octanoic Acid on the Binding of the Enantiomers of Ibuprofen and Naproxen to Human Serum Albumin: A Chromatographic Implication

Purpose. The heats of reaction between the enantiomers and racemates of ibuprofen and naproxen and human serum albumin (HSA) are to be measured with and without the addition of octanoic acid. The effects of octanoic acid on the free energies of interaction between the drugs and HSA is to be determined and compared to that estimated from theoretical equations. Methods. The heats of reaction have been measured directly by flow microcalorimetry. Results. The data showed that octanoic acid lowered the 1:1 binding constants for all the drug-HSA interactions investigated. The effect of octanoic acid was greater on the R than on the S forms of the drugs as shown by the differences in free energies of interaction in the presence and absence of octanoic acid. Conclusions. The increased free energy differences for the binding of the enantiomers of both drugs to HSA in the presence of octanoic acid is closer to the value deemed to be necessary for the separation of enantiomers by Davenkov, and shows the importance of the addition of octanoic acid to the mobile phase in the separation of these enantiomers on immobilized albumin columns.     

Binding of Sulfinpyrazone and its Metabolites in Human Serum and in Solutions of Human Serum Albumin

Abstract: The binding of sulfinpyrazone, its sulfone metabolite and its sulfide metabolite to serum protein was studied by equilibrium dialysis. At 20 μg/ml 99.1% of the parent compound was bound in serum, whereas 99.8% of the sulfide and 98.3% of the sulfone were bound at this concentration. The binding of the three compounds were studied in diluted serum and in solutions of human serum albumin (HSA). There was no evidence of binding to proteins other than albumin. The association constants to primary and secondary binding sites and the number of binding sites were calculated. For the sulfide a lower K₁-value in serum (0.76·10⁶ M^?1) than in the HSA solution (1.8·10⁶ M^?1) indicated the possible presence of a competitively bound substance in serum. In undiluted serum no displacing effect of the sulfide on sulfinpyrazone binding was found when both compounds were present in a concentration of 20 μg/ml, but in a HSA solution a pronounced sulfide induced displacement of the sulfinpyrazone from its primary binding site was shown. Acetylation of HSA depressed the binding of sulfinpyrazone but in undiluted serum there was no other effect on sulfinpyrazone binding by the addition of acetylsalicylic acid than could be explained by the displacing effect of salicylic acid. At concentrations at 20 μg/ml of sulfinpyrazone and above 50 μg/ml of the displacing agent significant displacement was demonstrated with phenylbutazone, tolbutamide and salicylic acid.     

Binding Study of the Fluorescence Probe l-Anilino-8-naphthalenesulfonate to Human Plasma and Human and Bovine Serum Albumin Using Potentiometric Titration

The binding of l-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA), human serum albumin (HSA), and human plasma has been studied by potentiometric titration utilizing a laboratory constructed ion selective electrode (ISE) of ANS. Three classes of ANS binding sites were found on BSA, HSA, and plasma at 25 and 37°C. Computer analysis of the data resulted in estimates for the association constants, number of binding sites (HSA, BSA), and binding capacity of each class. The association constants for the first class of binding sites at 25°C were found to be 7.53 (±0.59) × 10⁵, 2.70 (±0.20) × 10⁵, and 2.64 (±0.26) × 10⁵ M ^–l for BSA, HSA, and plasma, respectively. Lower values for the association constants of all binding classes were estimated at the higher temperature (37°C). The binding capacity for ANS decreased in the order BSA, plasma, HSA.     

Binding of Digitoxin to Human Serum Proteins: Influence of pH on the Binding of Digitoxin to Human Albumin

Abstract: The binding of digitoxin to albumin was studied under various conditions with regard to electrolytes and pH. Considerable variations of Na, K, Ca and Mg within the range of clinical relevance did not influence the binding of digitoxin to albumin. Under the influence of excessive concentrations of digitoxin this binding followed the simple law of mass action with an average number of binding sites of about 0.5 independent of pH. The intrinsic association constants were about twice the apparent association constants, calculated in another way and with the assumption of one binding site per molecule of albumin. The binding of digitoxin to albumin was shown to depend on the pH of the medium with a maximum association constant at pH = 4.8 (intrinsic association constant, K = 5 x 10⁵ l/mol). The binding of digitoxin to human serum proteins was shown to be equal to that of the binding to albumin. Under conditions relevant for clinical interpretations the binding of digitoxin to other proteins was insignificant.     

The Binding of Thiopental to Human Serum Albumin at Variable pH and Temperature

Abstract: The binding of thiopental was studied in vitro by equilibrium dialysis over the concentration range 0.8–80 μg/ml in solutions with 45 g albumin (HSA) per litre. The binding at 37° was from 79% to 67.6% at pH 6 while it was 94.5% to 93.0% at pH 9. At intermediate values of pH the observed values for binding were compatible with the assumption of a gradual transition of an N-form of HSA present exclusively at pH 6 to a B-form present exclusively at pH 9. The variation in binding could not be explained by changes in the balance between ionised and unionised drug (pK₂=7.6). An increase in r (number of thiopental molecules bound per HSA molecule) was demonstrated when the temperature was lowered to 2°. Graphs showing the relation between drug concentrations and percentual binding all showed a very modest slope and it was concluded that a traditional binding model with calculation of the number of binding sites and association constants was of little use in the evaluation of our binding studies of thiopental. The binding between thiopental and albumin may be characterized best by the solubility of thiopental in hydrofobic areas in the albumin solutions.     

Mode of Interaction of Loop Diuretics with Human Serum Albumin and Characterization of Binding Site

Purpose. The purpose of this study was to investigate the binding mechanism of loop diuretics with HSA and to characterize the binding site on HSA. Methods. Quantitative analysis of potential interaction between ligands bound to HSA was performed by equilibrium dialysis and data for binding of the two ligands to HSA were analyzed on the basis of a theoretical model of simultaneous binding of two ligands. Results. The binding of loop diuretics is dependent upon the N-B transition, conformational change of albumin. Furthermore, from the results of binding of the drugs to modified HSA, the lysine residue seems to be involved in the binding of loop diuretics to HSA. Conclusions. Analysis using models describing independent, competitive, cooperative and anti-cooperative binding led to the conclusion that loop diuretics bind to site I, particularly to the warfarin region on HSA.     

Study of Competitive Displacement of Curcumin on α-zearalenol Binding to Human Serum Albumin Complex Using Fluorescence Spectroscopy

α-zearalenol (α-ZOL) is a mycotoxin with a strong estrogen effect that affects the synthesis and secretion of sex hormones and is transported to target organs through human serum albumin (HSA). Additionally, it has been reported that curcumin can also bind to HSA with high affinity at the same binding site as α-ZOL. Additionally, several studies reported that reducing the bound fraction of α-ZOL contributes to speeding up the elimination rate of α-ZOL to reduce its hazard to organs. Therefore, to explore the influence of a nutrition intervention with curcumin on α-ZOL effects, the competitive displacement of α-ZOL from HSA by curcumin was investigated using spectroscopic techniques, ultrafiltration techniques and HPLC methods. Results show that curcumin and α-ZOL share the same binding site (subdomain IIA) on HSA, and curcumin binds to HSA with a binding constant of 1.12 × 10⁵ M⁻¹, which is higher than that of α-ZOL (3.98 × 10⁴ M⁻¹). Ultrafiltration studies demonstrated that curcumin could displace α-ZOL from HSA to reduce α-ZOL’s binding fraction. Synchronous fluorescence spectroscopy revealed that curcumin could reduce the hydrophobicity of the microenvironment of an HSA–α-ZOL complex. This study is of great significance for applying curcumin and other highly active foodborne components to interfere with the toxicokinetics of α-ZOL and reduce its risk of its exposure.     

Simultaneous determination of unbound thyroid hormones in human plasma using high performance frontal analysis with electrochemical detection

A direct injection HPLC method in combination with high-performance frontal analysis (HPFA) and electrochemical detection (ECD) was developed for the simultaneous and sensitive determination of unbound thyroid hormones (thyroxine, triiodothyronine, and reverse triiodothyronine) in human plasma. The present on-line HPLC/HPFA system consists of an HPFA column, an extraction column and an analytical HPLC column connected through a column-switching device, and the eluent from the analytical column was monitored by ECD. The calibration lines showed good linearity (rsq.>0.999) within 7.4-148.2 pM for T4 and 1.5-74.1 pM for T3 and rT3. Unbound T4 and T3 concentrations determined by the present system were 16.4+/-2.4 pM (n=15) and 7.14+/-1.04 pM (n=15), which were in agreement with those determined by the EIA method. The unbound rT3 concentration was 2.30+/-0.27 pM (n=15). The CV% values of intra-day and inter-day assays (n=15) were less than 14.9% for T4, 14.5% for T3 and 13.2% for rT3. The present system was also applied to a competitive binding study of these thyroid hormones in human plasma.     

Interaction of Benzothiadiazides with Human Serum Albumin Studied by Dialysis and Spectroscopic Methods

The interaction of a series of benzothiadiazides with human serum albumin (HSA) was investigated by equilibrium dialysis (ED) and spectroscopic methods including circular dichroism (CD). The primary binding site of benzothiadiazides was designated site II, the diazepam site on the HSA molecule, as indicated by displacement experiments using different site-selective probes. Tyrosine and lysine amino acid residues were probably involved in the binding site of these compounds to HSA. Both electrostatic and hydrophobic interactions were found to play a role in the binding of these compounds to HSA. Among the compounds tested, chlorothiazide had the highest affinity (K₁ = 5.5 × 10⁴M^–1, K₂ = 5.8 × 10³ M^–1).The primary binding affinity of the compounds for HSA was of the order: chlorothiazide > cyclopenthiazide > polythiazide > ethiazide > trichlormethiazide = methyclothiazde > hydrochlorothiazide. Binding was insensitive to the N-B transition of HSA. The binding site is proposed to consist of a cationic site on the surface of the HSA molecule with a hydrophobic crevice to accommodate the aromatic ring of the compounds. Positions 3 and 7 of the benzothiadiazide molecule is thought to affect the binding affinity to HSA.     

Covalent Binding Between Bucillamine Derivatives and Human Serum Albumin

Purpose. To clarify the mechanism of covalent binding between human serum albumin (HSA) and drugs containing thiol groups, we studied the interactions between HSA and bucillamine (BA) and its derivatives. Methods. To determine the concentration of HSA-drug conjugate, we used columns of N-methylpyridium polymer cross-linked with ethylene glycol dimethacrylate (4VP-Me), and analyzed the reaction between HSA and B A derivatives kinetically. Following pseudo first-order reaction kinetics, the rate constants of reduction of non-mercaptoalbumin (HNA) to mercaptoalbumin (HMA) (k_a) and formation of HSA-drug conjugate (k_c) were determined. Results. Formation of HSA-drug conjugate was observed only for drugs containing one thiol group. In compound IV, the plots of k_a and k_c against pH were found to be linear. The HSA-drug conjugate was affected by various factors such as pK_a, pH, temparture and the microenviroment of Cys³⁴. The increases in k_a and k_c. against pH were mainly due to the increase in mercaptide ion concentration. Further, fatty acid affected the microenviroment of Cys³⁴, which increased HSA-drug formation. Conclusions. Cys³⁴ located in a crevice on the surface of the protein plays an important role on the formation of HSA-drug conjugate. These results may be useful for elucidating the reaction mechanisms between various proteins and thiol compounds.     

Identification of the Cephalosporin Human Serum Albumin Binding Sites

Abstract: We have demonstrated that the cephalosporins which possess heterozyclic pentagonal group substituents bind to the bilirubin binding site on albumin, while the cephalosporins which possess hexagonal hydrocarbon cycle substituents bind to the benzodizepine binding sites (site II) on albumin. No binding of cephalosporins to the so-called site I and fatty acids binding site on human albumin was demonstrated.     

左氧氟沙星和氧氟沙星与清蛋白的结合位点及荧光淬灭分析

摘要目的研究左氧氟沙星和氧氟沙星与人血清清蛋白的结合作用。方法通过荧光光谱法分析左氧氟沙星和氧氟沙星对人血清清蛋白荧光淬灭光谱,同步荧光光谱,根据热力学方程讨论两者间主要的作用力类型。结果在生理条件（pH＝7.4,37 ℃）下,根据Stem Volmer方程和荧光淬灭双倒数图,左氧氟沙星和氧氟沙星对人血清清蛋白淬灭类型为静态淬灭,左氧氟沙星对人血清清蛋白的结合常数K＝1.46×105 L•mol－1,结合位点n＝1.11,氧氟沙星对人血清清蛋白的结合常数K＝4.31×104 L•mol－1,结合位点n＝1.04,根据热力学方法确定作用力类型均为疏水作用力。结论与左氧氟沙星比较,氧氟沙星对人血清清蛋白的荧光淬灭减弱,结合常数和结合位点均变小,结合位置也有明显区别;这些数据给研究左氧氟沙星和氧氟沙星的药理作用和生物学效应,以及左氧氟沙星和氧氟沙星对蛋白质构象的影响等提供了重要信息。     

Effect of Long-Chain Fatty Acids on the Binding of Triflupromazine to Human Serum Albumin: A Spectrophotometric Study

Human serum albumin (HSA) in the blood binds long-chain fatty acids (LCFAs), and the number of bound LCFAs varies from 1 to 7 depending on the physical condition of the body. In this study, the influence of LCFA-HSA binding on drug-HSA binding was studied using triflupromazine (TFZ), a psychotropic phenothiazine drug, in a buffer (0.1 M NaCl, pH 7.40, 37°C) by a second-derivative spectrophotometric method which can suppress the residual background signal effects of HSA observed in the absorption spectra. The examined LCFAs were caprylic acid (CPA), lauric acid (LRA), oleic acid (OLA), and linoleic acid (LNA), respectively. Using the derivative intensity change of TFZ induced by the addition of HSA containing LCFA, the binding mode of TFZ was predicted to be a partition-like nonspecific binding. The binding constant (K M⁻¹) showed an increase according to the LCFA content in HSA for LRA, OLA, and LNA up to an LCFA/HSA molar ratio of 3–4. However, at higher ratios the K value decreased, i.e. for OLA and LNA, at an LCFA/HSA ratio of 6–7, the K value decreased to 40% of the value for HSA alone. In contrast, CPA, having the shortest chain length (8 carbons) among the studied LCFAs, induced a 20% decrease in the K value regardless of its content in HSA. Since the pharmacological activity of a drug is closely related to the unbound drug concentration in the blood, the results of the present study are pharmaco-kinetically, pharmacologically, and clinically very important.     

Comparison of Binding Characterization of Two Antiviral Drugs to Human Serum Albumin

Ribavirin and lamivudine are representatives of antiviral drugs that are widely used to treat viral infections, especially chronic liver disease. To compare binding mechanism and behavior of antiviral drugs with human serum albumin (HSA), we performed fluorescence spectroscopy and X‐ray crystallography to investigate the interactions of ribavirin and lamivudine with HSA. Fluorescence spectroscopy showed ribavirin and lamivudine inhibit binding affinity each other. Our results further demonstrated that ribavirin and lamivdudine interaction with HSA could be affected by the presence of other compounds, including the non‐steroidal anti‐inflammatory drugs, indometacin. X‐ray structures revealed that ribavirin and lamivudine bind in IIA subdomain of HSA mainly by forming hydrogen bond and hydrophobic interactions forces. The carboxamido of ribavirin forms hydrogen bonds with Arg222; Hydroxyl group (6) of ribavirin forms hydrogen bond with Arg257. Hydroxyl group (15) of lamivudine forms hydrogen bond with Arg222; amino group (4) of lamivudine forms hydrogen bond with carbonyl of Arg257. Our results reveal the key biochemical and structural characteristics of the HSA interaction with ribavirin and lamivudine, providing guidance for future development of ribavirin‐ and lamivudine‐based compounds and a drug‐HSA delivery system.     

Binding of [3H]mianserin to bovine serum albumin,human serum albumin and α1-acid glycoprotein

Scatchard plots which were curvilinear with negative slopes were obtained when the binding of [³H]mianserin to bovine serum albumin (BSA), human serum albumin (HSA), defatted human serum albumin (D-HSA) and α₁-acid glycoprotein (α₁-AGP) was studied with equilibrium dialysis with constant protein concentrations and various ligand concentrations. Binding parameters were estimated graphically and with a non-linear least-squares computer program, assuming two classes of independent binding sites. α₁-AGP had the highest binding affinity (K) and binding capacity (nK). The binding parameters, n and K were not independent of protein concentration when the BSA concentration was varied. Linear atypical Scatchard plots with positive slopes were obtained when the protein concentration was varied for BSA, HSA and D-HSA, at a fixed ligand concentration.     

Spectroscopic Analysis of the Binding of Amylobarbitone,Secbutobarbitone, Pentobarbitone,Phenobarbitone and Quinalbarbitone to Human Serum Albumin

The binding of five barbiturates: amylobarbitone, secbutobarbitone, pentobarbitone, phenobarbitone and quinalbarbitone to human serum albumin (HSA) was studied by difference spectroscopy and spectro-fluorimetric titrations. There were no changes in the HSA spectral properties. Our result suggest that there are two classes of binding sites on HSA for these barbiturates. A detailed investigation of the effect of their binding to HSA by deconvoluted spectra, suggests that the interaction of barbiturate-HSA takes place principally on the subdomain IIIA of HSA.     

Regulation of Amantadine Hydrochloride Binding with IIA Subdomain of Human Serum Albumin by Fatty Acid Chains

Human serum albumin (HSA) is a major protein component of blood plasma that has been exploited to bind and transport a wide variety of endogenous and exogenous organic compounds. Although anionic drugs readily associate with the IIA subdomain of HSA, most cationic drugs poorly associate with HSA at this subdomain. In this study, we propose to improve the association between cationic drugs and HSA by modifying HSA with fatty acid chains. For our experiments, we tested amantadine hydrochloride, a cationic drug with antiviral and antiparkinsonian effects. Our results suggest that extensive myristoylation of HSA can help stabilize the interaction between amantadine and HSA in vitro. Our X‐ray crystallography data further elucidate the structural basis of this regulation. Additionally, our crystallography data suggest that anionic drugs, with a functional carboxylate group, may enhance the association between amantadine and HSA by a mechanism similar to myristoylation. Ultimately, our results provide critical structural insight into this novel association between cationic drugs and the HSA IIA subdomain, raising the tempting possibility to fully exploit the unique binding capacity of HSA's IIA subdomain to achieve simultaneous delivery of anionic and cationic drugs.     

Tocainide analogues binding to human serum albumin: A HPLAC and circular dichroism study

A series of synthesised tocainide analogues were characterized for their human serum albumin (HSA) binding, using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). The synthesis and physico-chemical characterization of compounds 7a–7d is reported here. For the HPLAC investigation HSA was covalently immobilized to the silica matrix of the HPLC column, using an anchoring procedure, which allows the binding properties of the protein to be maintained. The HSA-based column was used for getting information on the high affinity binding sites of the tocainide analogues to HSA. According to the displacement chromatography approach, the retentions of the analytes were determined in the absence and in the presence of increasing concentrations of competitors known to bind to specific binding sites on the protein. The same system, drug/protein, was investigated in solution by CD.     

Irreversible Binding of Tolmetin Glucuronic Acid Esters to Albumin in Vitro

Tolmetin glucuronide (TG), extracted and purified from human urine, was incubated with albumin in vitro. The degradation profile and irreversible binding to protein were investigated and kinetic parameters calculated. Standard conditions were as follows: TG, 30 µg/ml; human serum albumin (HSA), 3%; pH 7.45; 37°C. Lower pH enhanced TG stability and reduced both the extent and the rate of irreversible binding. HSA also increased TG stability, compared to protein-free buffer, but the opposite was observed with bovine serum albumin (BSA). With BSA, irreversible binding was much less, but the rate of adduct formation was the same as with HSA. Essentially fatty acid free HSA behaved similarly to HSA. Preincubation of HSA with warfarin, or diazepam, or an excess of tolmetin, did not influence irreversible binding significantly. In buffer, acyl migration led predominantly to one isomer. This isomer bound irreversibly to HSA, although more slowly and to a lesser extent than the 1-isomer. Incubation of TG with poly-L-lysine also resulted in irreversible binding but to a lesser extent than with HSA. Our results suggest that there is more than one binding mechanism, with the preferential pathway a function of the isomers present and the experimental conditions.