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.     

Evidence for the fatty acid-induced heterogeneity of the N and B conformations of human serum albumin

The influence of oleic acid on the interaction between albumin and warfarin, oxyphenbutazone or diazepam has been studied by circular dichroism and equilibrium dialysis. The pH dependences of the molar ellipticity of the drug-albumin complexes and of the free fraction of drug are completely changed by the presence of oleic acid. This phenomenon is attributed to an oleic acid-induced conformational change in both the neutral (N) and the basic (B) conformation of albumin, a change to which the warfarin-oxyphenbutazone binding area and the diazepam binding site is sensitive. The oleic acid-induced conformational states of albumin, the so-called N* and B* conformations, show binding properties that are different from the binding properties of the N and B conformations.     

Differences in the serum protein binding of prazosin in man and rat

The serum protein binding of prazosin in man and rat has been studied in vitro by equilibrium dialysis. Prazosin was more extensively bound in human serum than in rat serum with binding ratios (B/F) of 14.3 +/- 3.4 and 4.4 +/- 0.2 (corresponding to 93.4 and 81.4% bound), respectively. This difference in binding between the species was partly due to qualitative differences between human and rat serum albumin, but also to the lower concentration of albumin in rat serum. Rat serum albumin (RSA) apparently showed two different classes of binding sites for prazosin, one with high (KD = 5.78 X 10(-6) M) and one with low (KD = 1.1 X 10(-4) M) affinity; the former is suggested as representing alpha 1-acid glycoprotein (alpha 1-AGP) with one binding site for prazosin per molecule, the latter as representing RSA with 0.28 binding sites per molecule. Human serum albumin (HSA) and human alpha 1-AGP both showed one class of binding sites with KD values of 2.7 X 10(-5) and 1.95 X 10(-6) M, respectively. HSA possessed 0.5 and human alpha 1-AGP 1 binding site for prazosin per molecule. The binding parameters obtained for the isolated serum proteins overestimated to some degree the total serum protein binding of prazosin in man. This was explained by a specific deviation from the law of mass action. HSA was the major binding protein in human serum at therapeutic concentrations, with ca. 60% of the total binding, the remaining 40% being bound to alpha 1-AGP. Anticipating that the high affinity binding site on the RSA preparation represents the binding of prazosin to alpha 1-AGP, then this protein accounts for 70% of the binding in rat serum, while rat serum albumin accounts for approximately 23%. The binding of prazosin to lipoproteins was insignificant in both species. The observed differences between man and rat in the serum protein binding of prazosin implicate differences in the two species with respect to prazosin pharmacokinetics and the pharmacological effect.     

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.     

Species Differences of Serum Albumins: I. Drug Binding Sites

Purpose. The purpose of this study was the classification and identification of drug binding sites on albumins from several species in order to understand species differences of both drug binding properties and drug interaction on protein binding. Methods. Binding properties and types of drug-drug interaction on the different albumins were examined using typical site I binding drugs, warfarin (WF) and phenylbutazone (PBZ), and site II binding drugs, ibuprofen (IP) and diazepam (DZ) on human albumin. Equilibrium dialysis was carried out for two drugs and the free concentrations of drugs were then treated using the methods of Kragh-Hansen (Mol. Pharmacol. 34. 160–171, (1988)). Results. Binding affinities of site I drugs to bovine, rabbit and rat albumins were reasonably similar to human albumin. However, interestingly, those to dog albumin were considerably smaller than human albumin. On the other hand, binding parameters of DZ to bovine, rabbit and rat albumins were apparently different from those of human albumin. These differences are best explained by microenvironmental changes in the binding sites resulting from change of size and/or hydrophobicity of the binding pocket, rather than a variation in amino acid residues. Conclusions. We will propose herein that mammalian serum albumins used in this study contain specific drug binding sites: Rabbit and rat albumins contain a drug binding site, corresponding to site I on human albumin, and dog albumin contains a specific drug binding site corresponding to site II on the human albumin molecule.     

The Interaction of Fatty Acid Amide Hydrolase (FAAH) Inhibitors with an Anandamide Carrier Protein Using 19 F-NMR

It has been reported that the endocannabinoid anandamide (AEA) binds to a class of fatty acid-binding proteins and serum albumin which can serve as carrier proteins and potentiate the cellular uptake of AEA and its intracellular translocation. Here, we employed ¹⁹F nuclear magnetic resonance spectroscopy to study the interactions of serum albumin with two inhibitors of fatty acid amide hydrolase (FAAH), the enzyme involved in the deactivation of anandamide. We found that, for both inhibitors AM5206 and AM5207, the primary binding site on serum albumin is drug site 1 located at subdomain IIA. Neither inhibitor binds to drug site 2. While AM5207 binds exclusively to drug site 1, AM5206 also interacts with other fatty acid-binding sites on serum albumin. Additionally, AM5206 has an affinity for serum albumin approximately one order of magnitude higher than that of AM5207. The data suggest that interactions of FAAH inhibitors with albumin may provide added advantages for their ability to modulate endocannabinoid levels for a range of applications including analgesia, antiemesis, and neuroprotection.     

Drug-Biomacromolecule Interaction XII: Comparative binding study of sulfaethidole to bovine serum albumin by equilibrium dialysis,fluorescence probe technique,uv difference spectrophotometry and circular dichroism

Binding of sulfaethidole to bovine serum albumin was studied by equilibrium dialysis, fluorescence probe technique, uv difference spectrophotometry and circular dichroism. Equilibrium dialysis method enabled us to estimate the total number of drug binding sites of albumin molecule. For sulfaethidole, albumin had 6 primary and 40 secondary binding sites. The primary and secondary binding constants were 0.9×10⁵ M⁻¹ and 0.2×10⁶ M⁻¹, respectively. 1-Anilino-8-naphthalenesulfonate (ANS) and 2-(4′-hydroxylbenzeneazo)-benzoic acid (HBAB) were used as the fluorescence probe and the uv spectrophotometric probe, respectively. In fluorescence probe technique, results indicated that the number of higher affinity drug binding site of albumin was 1 and the number of lower affinity drug binding sites of albumin was 3, and the primary and secondary drug binding constants for bovine serum albumin were 2.15×10⁵ M⁻¹ and 1.04×10⁵ M⁻¹, respectively. In uv difference spectrophotometry, binding sites were 3 and binding constant was 1.88×10⁵ M⁻¹. The above results suggest that several different methods should be used in ompensation for insufficient information about drug binding to albumin molecule given by only one method.     

Concentration-dependent effects of fatty acids on warfarin binding to albumin

The interrelationships between fatty acid and warfarin binding to albumin were investigated. Various molar ratios of palmitic acid and oleic acid were added to defatted human albumin in the presence of warfarin, and the warfarin binding association constants, K_a, were calculated. Warfarin association constants increased from 0.84 × 10⁵ M ^?1 to 3.66 × 10⁵ M ^?1 as the oleic acid concentration increased from zero to three moles per mole of albumin and from 1.19 × 10⁵ M ^?1 to 3.13 × 10⁵ M ^?1 as the palmitic acid concentration increased from zero to two moles per mole of albumin. Larger amounts of either fatty acid progressively decreased the amount of warfarin bound in a noncompetitive fashion. In addition, two proteolytic fragments were utilized to define the location of the warfarin binding site on albumin. The warfarin site was located between loops 5 and 6 on the albumin molecule in close proximity to the secondary and tertiary binding sites of palmitic acid.     

Kinetics of drug binding to human serum albumin: allosteric and competitive inhibition at the benzodiazepine binding site by free fatty acids of various chain lengths

Summary The inhibition of dansylsarcosine (DS) binding at the benzodiazepine binding site of human serum albumin has been studied in the presence of saturated and unsaturated free fatty acids (FFA) of various chain lengths (C6–C20, C18:1, C18:2). In order to determine the mechanism of displacement, velocity constants for association (k ₂) and dissociation (k _–2) and binding constants (K _A and K _A) have been measured using the stopped-flow method.The inhibitory effect of FFA on DS binding kinetics at site II is dependent of their structure. With increasing amounts of FFA the association velocity constant of DS binding decreases from 520 s^–1 (fatty acid free albumin) by a factor of 3–10 and affinity decreases according to FFA chain length. Inhibition is strongest in the presence of caprylic, capric and lauric acid (C8–C12) i.e. with more than one mole FFA per mole albumin, DS association could no longer be measured. Short chain caproic and the long chain FFA C14–C20 showed only a less inhibitory effect since in the presence of a twofold excess k ₂ ranged between 100 and 200 s^–1. Dissociation velocity of DS from the benzodiazepine binding site could be measured in relationship to FFA chain length using ibuprofene, another drug binding at site II. Dissociation velocity constants k _–2 remained constant up to 2 moles FFA per mole albumin (k _–2 = 16–18 s^–1). A rise in k _–2 to 70 s^–1 was seen, however, when 2–4 moles capric, lauric, myristic and palmitic (C10–C16) acid were bound, whereas no change was observed when increasing concentrations of caproic, caprylic, stearic and arachic acid. The more compact unsaturated FFA oleic and linoleic acids did not inhibit DS binding to the same extent as their saturated homologue stearic acid. k ₂ and k _–2 values resembled those when comparable amounts of myristic acid were bound indicating that chain length is the relevant parameter.It can be concluded that two different types of FFA inhibition of binding at the benzodiazepine binding site exist. Short-chain FFA (C6–C8) may be specifically bound at binding site II thereby competitively displacing DS. Longchain FFA (C12–C20), however, occupy their binding sites far from the benzodiazepine binding site. Inhibition occurs via an allosteric mechanism. Capric acid is unique in showing as well as competitive as well as allosteric inhibition characteristics.Abbreviations k ₂ Dissociation velocity constant - k ₂ association velocity constant - K _A affinity constant - K _A affinity constant of the intermediate complex - DS dansylsarcosine - FFA free fatty acids - HSA human serum albumin This work has been presented in part at the Spring Meeting of the German Pharmacological Society in Mainz, March 1988 (Menke et al. 1988) Send offprint request to N. Rietbrock at the above address     

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.     

The Determination of Naproxen by Spectrofluorometry and its Binding to Serum Proteins

Abstract A fluorometric method for the determination of naproxen in serum, albumin solutions and protein free buffer solutions is described. The detection limit is about 10 ng/ml. Furosemide, thiopental and salicylic acid did not show any disturbing fluorescence while phenprocoumon did. The in vitro binding of naproxen in albumin solutions and serum was studied by equilibrium dialysis. A small but significant increase was found in the percentual binding in albumin solutions as compared to serum. The percentual binding was not affected by changes in the pH from 5-8. By fitting the binding data to a model assuming two classes of binding sites, association constants and binding capacities were determined. A very high affinity and a high capacity were found. The association constant for the first class of binding sites was higher for human serum albumin than for Serum (8.5 versus 5.3 · 10⁶ M^-1). The difference in the protein binding to the first class of binding sites in human serum albumin and serum can be explained by the existence of a competitive inhibitor in serum.     

High-Performance Frontal Analysis of the Binding of Thyroxine Enantiomers to Human Serum Albumin Binding of Thyroxine Enantiomers to Human Serum Albumin Kimura

No HeadingPurpose. The aim of this study was to characterize the binding property between thyroxine and human serum albumin (HSA) qualitatively and enantioselectively using high-performance frontal analysis (HPFA).Methods. An on-line HPLC system consisting of an HPFA column, an extraction column, and an analytical HPLC column was developed to be used to determine the unbound concentrations of thyroxine enantiomers.Results. Both enantiomers were bound to human serum albumin at two high-affinity sites with similar affinities. The binding constant (K) and the number of binding sites on an HSA molecule (n) evaluated from Scatchard plot analysis were K = 1.01 × 10⁶m^-1 and n = 1.90 for l-thyroxine, and K = 9.71 × 10⁵ m^-1 and n = 1.97 for d-thyroxine. The binding sites were identified using phenylbutazone and diazepam as site-specific probes for sites I and II, respectively, and each enantiomer was found to bind to both sites. Incorporation of a chiral HPLC column into the on-line system permitted the investigation of enantiomer-enantiomer interactions, which revealed that both enantiomers competitively bind to the same binding sites without significant allosteric effects.Conclusions.     

Biophysical study on the interaction of etomidate and the carrier protein in vitro

Etomidate is a unique drug used for induction of general anesthesia and sedation, and is usually used through intravenous injection clinically. Before targeting to the receptor, etomidate binds proteins in blood when it comes into veins. Thus to study the interaction of etomidate and serum albumin would be of great toxicological and pharmacological importance. In this study, the interaction between etomidate and human serum albumin (HSA) was studied using fluorescence spectroscopy, UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) spectroscopy, site maker displacement and molecular modeling methods. Investigations of the binding constant (K?=?3.55?×?10^5?M^?1, 295?K), the number of binding sites (n?=?1.16), thermodynamic parameters (ΔG?=?3.13?×?10^4?J·mol^?1, ΔS?=?364?J·mol^?1·K^?1 and ΔH?=??6.85?×?10^5?J·mol^?1) for the reaction and changes to the binding sites and conformation in HSA in response to etomidate were presented. Results show that etomidate can bind HSA tightly through electrostatic forces, and the protein skeleton conformation and secondary structure changes thereby. This is the first spectroscopic report for etomidate–HSA interactions which illustrates the complex nature of this subject.     

Effects of a perfluorochemical blood substitute on diazepam binding by human albumin

The binding of 0.4 microgram mL-1 diazepam and 0.75 mol diazepam mol-1 of albumin by a perfluorochemical (PFC) emulsion, Fluosol-DA, 20%, by human serum albumin (HSA), and by their mixtures, has been examined at ambient temperature. The concentration of free diazepam was determined by standard centrifugation followed by supernatant ultrafiltration. Non-specific loss of diazepam occurred to the ultrafiltration device. This loss was independent of drug concentration and a correction factor was employed to calculate the true free diazepam concentration. Diazepam was extensively bound by the PFC emulsion. The percent free diazepam increased as the emulsion concentration decreased, while the binding of diazepam appeared to be independent of drug concentration. Diazepam did not partition into the pure PFC liquids, indicating that emulsion-bound diazepam is only associated with the emulsifiers of the droplets. Diazepam was extensively bound by HSA and the percent free diazepam increased as drug concentration increased or as HSA concentration decreased. The PFC emulsion significantly displaced HSA bound diazepam in all mixtures examined. Studies with the individual and combined components of the emulsion indicated that this displacement is largely attributed to the oleic acid component and, to a much smaller degree, the Pluronic F-68 component of the emulsion.     

Design,Synthesis, and Biological Evaluation of Catecholamine Vehicle for Studying Dopaminergic System

Catecholamine mimetic EDTA‐bis(tyramide) was synthesized and characterized by various spectroscopic techniques (NMR, mass spectroscopy) and λ_em 310 nm for the excitation at 270 nm. Molecular docking studies were performed with human serum albumin (PDB 1E78), showing binding pattern with amino acid residues Arg218, Arg222, and Lys444, identifies the ligand‐human serum albumin interaction for the transportation affinity of the ligand at the specific site of the target. Subsequently, binding study with human serum albumin at λ_ex = 350 nm found to be 5.847 × 10⁴ m ^?1 shows effective quenching effect. Additionally, to go more insight, acetylcholinesterase binding affinity was investigated, which shows 90% binding affinity for the 10 mm concentration. IC50 value was found 18.60 μm for MAO‐B inhibition. Finally, EDTA‐bis(tyramide) labeled with ^99mTc to investigate its in vivo radiopharmaceutical efficiency having 97% binding affinity with 98% radiochemical purity. In vivo studies were carried out for ^99mTc‐EDTA‐bis(tyramide) included blood kinetics showed a quick wash out from the circulation via renal route, and biodistribution revealed that maximum %ID/g was found in kidney at 1 h, and its scintigraphy image shows 3.96% brain uptake with respect to whole body.     

Antifungal efficiency of miconazole and econazole and the interaction with transport protein: A comparative study

Abstract

Context: Miconazole (MIZ) and econazole (ECZ) are clinically used as antifungal drugs.

Objective: The drug effect and binding property with transport protein human serum albumin of MIZ and ECZ were studied.

Materials and methods: The antifungal efficiency was investigated by microdiluting drug solutions from 0 to 48?μmol?L^?1 through microcalorimetry and voltammetry studies. Transmission electron microscopy was used for morphological observations of C. albicans. The interaction with HSA was studied by electrochemical methods, fluorescence spectrometry, electron microscopy, and molecular simulation.

Results: IC₅₀ of MIZ and ECZ for C. albicans were obtained as 19.72 and 29.90?μmol?L^?1. Binding constants of MIZ and ECZ with HSA of 2.36?×?10⁴ L?mol^?1 and 3.73?×?10⁴ L?mol^?1 were obtained. After adding MIZ solution of 12 and 40?μmol?L^?1, the peak currents increased to 4.887 and 6.024?μA. The peak currents of C. albicans in the presence of 20 and 48?μmol?L^?1 ECZ were 4.701 and 5.544?μA. The docking scores for MIZ and ECZ of the best binding conformation in site I and site II were 5.60, 4.79, 5.63, and 5.85.

Discussion and conclusion: Strong inhibition to the metabolism of C. albicans and destructive effect was proved for both drugs. The lower IC₅₀, growth rate constant of C. albicans, and higher peak current, reveal stronger antifungal activity of MIZ. Both drugs show an efficient quenching effect to intrinsic fluorescence residues of protein. MIZ mainly binds on site I while ECZ on site II. Molecular modeling experiments give further insight of the binding mechanism.     

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.     

Kinetic analysis of covalent binding between N-acetyl-L-cysteine and albumin through the formation of mixed disulfides in human and rat serum in vitro

Purpose. Covalent binding between N-acetyl-L-cysteine (NAC) and albumin was evaluated kinetically by conducting in vitro experiments. Methods. After ¹⁴C-NAC was incubated with human or rat serum, the solution was analyzed by anion-exchange HPLC. The albumin-bound ¹⁴C-NAC was quantified by measuring the radioactivity in the albumin fraction. Results. Ultraviolet chromatograms and/or radiochromatograms indicated the presence of a stable covalent bond between ¹⁴C-NAC and either human or rat albumin. By analyzing the time dependence of this protein binding in serum, the first-order binding and dissociation rate constants (k_on and k_off) were obtained. The serum was treated in a CO₂ incubator to avoid oxidative interference, and the initial rates were determined separately. The k_on values obtained were 0.33 (h^–1) and 0.48 (h^–1) for human and rat serum, respectively. L-Cysteine was required to initiate the dissociation of ¹⁴C-NAC bound to albumin. Following the addition of appropriate amounts of L-cysteine, the k_off values were determined to be 0.30-1.0 h^–1 and 0.54-1.4 h^–1 for human and rat serum, respectively. Conclusions. The k_on and k_off values obtained for rat serum were in good agreement with the in vivo plasma protein binding kinetics of NAC in rats, indicating the reliability of this in vitro method for evaluating protein binding. No species differences in protein binding kinetics were found between human and rat serum.     

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.     

Unraveling the Energetics and Mode of the Recognition of Antibiotics Tetracycline and Rolitetracycline by Bovine Serum Albumin

An understanding of the detailed energetics and mechanism of the binding of drugs with target proteins is essential for devising guidelines to synthesize new drugs. Binding of the antibiotic drugs tetracycline and rolitetracycline with serum albumin has been studied by a combination of isothermal titration calorimetry, differential scanning calorimetry, steady‐state and time‐resolved fluorescence, and circular dichroism spectroscopies. Both tetracycline and rolitetracycline bind to bovine serum albumin in a sequential manner with first binding being the major binding event with an association constant of the order of 10⁴ for tetracycline and 10³ for rolitetracycline, respectively. Ionic strength dependence and binding in the presence of tetrabutylammonium bromide and sucrose indicate involvement of a mix of hydrophobic, ionic, and hydrogen bonding interactions. The isothermal titration calorimetry results for the binding of these drugs to bovine serum albumin in the presence of warfarin and in the presence of each other indicate that both these drugs share binding site 2 on bovine serum albumin. The differential scanning calorimetry results provide quantitative information on the effect of drugs on the stability of bovine serum albumin. A comparison of isothermal titration calorimetry and fluorescence results demonstrates that the former technique has been able to explain the sequential binding events that can be missed by the fluorescence measurements.