Differential scanning calorimetric studies on bovine serum albumin IV. Effect of anionic surfactants with various lengths of hydrocarbon chain

Using defatted and SH-blocked bovine serum albumin (BSA), measurements of differential scanning calorimetry (DSC) have been made at pH 7 on the complexes of BSA and a series of sodium alkyl sulfates. Alkyl sulfates used were sodium decyl sulfate (SDeS), sodium octyl sulfate (SOS), sodium hexyl sulfate (SHS) and sodium ethyl sulfate (SES). Results obtained were compared with those on the system BSA-sodium dodecyl sulfate (SDS) studied previously. Two peaks P 1 and P2 existed in the DSC curve of BSA. These peaks originate in the heat-induced transition of BSA. The pattern of DSC curve changed with the amount of the ligand added, i.e. with the molar mixing ratio ligand/BSA (1). The change for systems BSA-SDeS, BSA-SOS and BSA-SHS was qualitatively the same as that for the system BSA-SDS (2). Interestingly, SES, which is not a surfactant, interacts with BSA. The change for the system BSA-SES was qualitatively the same as that for the system BSA-Na₂SO₄. All alkyl sulfates suppressed the heat-induced transition at lower concentrations. A linear relationship was obtained for the plots of log(D/A)₁ versus log CMC, where (D/A)₁ is the molar mixing ratio of anionic surfactant (D) to BSA (A) at which the most heat-stable complex is formed. This suggests that the hydrophobic force has a serious effect on the formation of heat-stable complexes.     

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.     

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.     

Comparison of the binding affinity of chlorogenic acid with two serum albumins

Chlorogenic acid (CA) is a well-known ester of caffeic acid present in some food. It is also an active component in traditional Chinese medicines which are used to treat various diseases, but the molecular basis of CA is not clear. In the present work, the proton selective relaxation rate and the affinity index were used to investigate the interaction of CA with human serum albumin and bovine serum albumin under the same buffer conditions. The results indicated that the binding affinity of chlorogenic acid to BSA was stronger than that to HSA. The binding site of the ligand-protein complex was elucidated by molecular docking, and the specific interaction was observed from those hydrogen bonds formed by the ligand and active residues. Using a combination of TR-NOE detection, the optimal ligand conformation was illustrated. Further conformational analysis of the complex revealed that the ability of hydrogen bond formation by polar side chain residues in the binding site of BSA might contribute to the greater binding affinity. The results provide a better understanding of CA binding and should contribute towards the design of modifications of CA for therapeutic purposes.     

The binding of pyridoxal 5'-phosphate to human serum albumin

Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.     

Formation of the molten globule-like state of cytochrome c induced by n-alkyl sulfates at low concentrations

The molten globule state of cytochrome c is the major intermediate of protein folding. Elucidation of the thermodynamic mechanism of conformational stability of the molten globule state would enhance our understanding of protein folding. The formation of the molten globule state of cytochrome c was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS, at low concentrations. The refolding states of the protein were monitored by spectroscopic techniques including circular dichroism (CD), visible absorbance and fluorescence. The effect of n-alkyl sulfates on the structure of acid-unfolded horse cytochrome c at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of the molten globule state. The addition of n-alkyl sulfates to the unfolded state of cytochrome c appears to support the stabilized form of the molten globule. The m-values of the refolded state of cytochrome c by SOS, SDeS, SDS, and STS showed substantial variation. The enhancement of m-values as the stability criterion of the molten globule state corresponded with increasing chain length of the cited n-alkyl sulfates. The compaction of the molten globule state induced by SDS, as a prototype for other n-alkyl sulfates, relative to the unfolded state of cytochrome c was confirmed by Stokes radius and thermal transition point (T(m)) measured by microviscometry and differential scanning calorimetry (DSC), respectively. Thus, hydrophobic interactions play an important role in stabilizing the molten globule state.     

Allosteric modulation of myristate and Mn(III)heme binding to human serum albumin. Optical and NMR spectroscopy characterization

Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity. HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids. Here, we report myristate binding to the N and B conformational states of Mn(III)heme-HSA (i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal. Myristate binding to a secondary site FAx, allosterically coupled to the heme site, not only increases optical absorbance of Mn(III)heme-bound HSA by a factor of approximately three, but also increases the Mn(III)heme affinity for the fatty acid binding site FA1 by 10-500-fold. Cooperative binding appears to occur at FAx and accessory myristate binding sites. The conformational changes of the Mn(III)heme-HSA tertiary structure allosterically induced by myristate are associated with a noticeable change in both optical absorbance and NMR spectroscopic properties of Mn(III)heme-HSA, allowing the Mn(III)-coordinated water molecule to exchange with the solvent bulk. At pH = 10.0 both myristate affinity for FAx and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme binds to HSA with higher affinity than at pH 7.0 even in the absence of myristate, and the metal-coordinated water molecule is displaced. As a whole, these results suggest that FA binding promotes conformational changes reminiscent of N to B state HSA transition, and appear of general significance for a deeper understanding of the allosteric modulation of ligand binding properties of HSA.     

n-Dodecyl β-D-maltoside specifically competes with general anesthetics for anesthetic binding sites

We recently demonstrated that the anionic detergent sodium dodecyl sulfate (SDS) specifically interacts with the anesthetic binding site in horse spleen apoferritin, a soluble protein which models anesthetic binding sites in receptors. This raises the possibility of other detergents similarly interacting with and occluding such sites from anesthetics, thereby preventing the proper identification of novel anesthetic binding sites. n-Dodecyl β-D-maltoside (DDM) is a non-ionic detergent commonly used during protein-anesthetic studies because of its mild and non-denaturing properties. In this study, we demonstrate that SDS and DDM occupy anesthetic binding sites in the model proteins human serum albumin (HSA) and horse spleen apoferritin and thereby inhibit the binding of the general anesthetics propofol and isoflurane. DDM specifically interacts with HSA (K_d?=?40?μM) with a lower affinity than SDS (K_d?=?2?μM). DDM exerts all these effects while not perturbing the native structures of either model protein. Computational calculations corroborated the experimental results by demonstrating that the binding sites for DDM and both anesthetics on the model proteins overlapped. Collectively, our results indicate that DDM and SDS specifically interact with anesthetic binding sites and may thus prevent the identification of novel anesthetic sites. Special precaution should be taken when undertaking and interpreting results from protein-anesthetic investigations utilizing detergents like SDS and DDM.     

Species-dependent stereoselective drug binding to albumin: a circular dichroism study

Drug binding to albumins from different mammalian species was investigated to disclose evidence of species-dependent stereoselectivity in drug-binding processes and affinities. This aspect is important for evaluating the reliability of extrapolating distribution data among species. The circular dichroism (CD) signal induced by drug binding to the albumins [human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), and dog serum albumin (DSA)] were measured and analyzed. The binding of selected drugs and metabolites to HSA significantly differed from the binding to the other albumins in terms of affinity and conformation of the bound ligands. In particular, phenylbutazone, a marker of site one on HSA, showed a higher affinity for binding to BSA with respect to RSA, HSA, and DSA, respectively. In the case of diazepam, a marker of site two on HSA, the affinity decreased in order from HSA to DSA, RSA, and BSA. The induced CD spectra were similar in terms of energy and band signs, suggesting almost the same conformation for the bound drug to the different albumins. Stereoselectivity was high for the binding of ketoprofen to HSA and RSA. A different sign was observed for the CD spectra induced by the drug to the two albumins because of the prevalence of a different conformation of the bound drug. Interestingly, the same induced CD spectra were obtained using either the racemic form or the (S)-enantiomer. Finally, significant differences were observed in the affinity of bilirubin, being highest for BSA, then decreasing for RSA, HSA, and DSA. A more complex conformational equilibrium was observed for bound bilirubin.     

Stereoselective plasma protein binding of amlodipine

The binding of the (R)‐ and (S)‐enantiomers of amlodipine to bovine serum albumin (BSA), human serum albumin (HSA), α₁‐acid glycoprotein (AGP), and human plasma (HP) was studied by equilibrium dialysis over the concentration range of 75–200 μM at a protein concentration of 150 μM. Unbound drug concentrations were determined by enantioselective capillary electrophoresis using 50 mM phosphate buffer, pH 2.5, containing 18 mM α‐cyclodextrin as background electrolyte. Saturation of the protein binding sites was not observed over the concentration range tested. Upon application of racemic amlodipine besylate, (S)‐amlodipine was bound to a higher extend by HSA and HP compared with (R)‐amlodipine, whereas the opposite binding of the enantiomers was observed for BSA and AGP. Scatchard analysis was used to illustrate the different binding affinities of amlodipine besylate enantiomers to BSA, HSA and AGP. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

On the modulation of photoinduced fluorescence enhancement and conformational stability of albumin-bound bilirubin: effect of epsilon-NH(2) groups blocking and chloroform binding

Photoinduced fluorescence enhancement of bilirubin bound to primary binding site on human serum albumin (HSA) was completely ceased when epsilon-NH(2) groups of its internal lysine residues were covalently blocked by acetylation or succinylation though the pigment bound to these derivatives in a folded conformation akin to that bound to HSA. These photoinduced fluorescence modulations cannot be ascribed to the binding of bilirubin to secondary low affinity sites as the CD spectrum of bilirubin bound to these derivatives showed complete inversion upon addition of chloroform which binds to subdomain IIA in HSA where high affinity bilirubin binding site is located. Presence of chloroform reconciled the photoinduced alterations in the CD spectrum observed in its absence, suggesting that chloroform stabilized the bound ligand against light but the fluorescence properties of bilirubin complexed with acetylated or succinylated derivatives remained unchanged. Guanidination of internal epsilon-NH(2) groups in HSA by O-methylisourea did not alter the spectral properties of the bound ligand. These results suggest that salt linkage(s) existing between epsilon-NH(2) groups of lysine residues in HSA and carboxyl groups of bilirubin, act(s) as a potential barrier during conformational rotation of the bound ligand assisted by photoactivation and their abolishment can alter its dynamics and stereoselectivity, a hitherto unnoticed implication of salt linkage(s) in BR-HSA complex.     

The differences in binding 12‐carbon aliphatic ligands by bovine β‐lactoglobulin isoform A and B studied by isothermal titration calorimetry and X‐ray crystallography

Isoforms A (LGB‐A) and B (LGB‐B) of bovine lactoglobulin, the milk protein, differ in positions 64 (D?G) and 118 (V?A). Interactions of LGB‐A and LGB‐B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12‐carbon ligands possessing differently charged polar groups, were investigated using isothermal titration calorimetry and X‐ray crystallography, to study the proton linkage phenomenon and to distinguish between effects related to different isoforms and different ligand properties. The determined values of ΔS and ΔH revealed that for all ligands, binding is entropically driven. The contribution from enthalpy change is lower and shows strong dependence on type of buffer that indicates proton release from the protein varying with protein isoform and ligand type and involvement of LA and Asp64 (in isoform A) in this process. The ligand affinities for both isoforms were arranged in the same order, DTAC < LA < SDS, and were systematically lower for variant B. The entropy change of the complexation process was always higher for isoform A, but these values were compensated by changes in enthalpy, resulting in almost identical ΔG for complexes of both isoforms. The determined crystal structures showed that substitution in positions 64 and 118 did not influence the overall structure of LGB complexes. The chemical character of the ligand polar group did not affect the position of its aliphatic chain in protein β‐barrel, indicating a major role of hydrophobic interactions in ligand binding that prevailed even with the repulsion between positively charged DTAC and lysine residues located at binding site entrance. Copyright © 2013 John Wiley & Sons, Ltd.     

On the contemporaneous, reversible interaction of different ligands with serum albumins in dilute aqueous solutions: Fluorescein and phenylbutazone

The binding affinity of fluorescein and of phenylbutazone to human serum albumin (HSA) and to bovine serum albumin (BSA), respectively, as well as of the two drugs together to each protein in dilute aqueous solution has been studied by means of gel permeation chromatography, circular dichroism, U.V. absorption and fluorescence spectroscopy. Identity of and/or interdependence between primary binding sites for the two ligands considered on HSA and BSA are evidenced and correlated with a simple theoretical approach to mixed drugs binding.     

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

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

The fatty acid analogue 11-(dansylamino)undecanoic acid is a fluorescent probe for the bilirubin-binding sites of albumin and not for the high-affinity fatty acid-binding sites.

1. The fluorescent fatty acid probe 11-(dansylamino)undecanoic acid (DAUDA) binds with high affinity to bovine and human serum albumin (BSA and HSA) at three sites. 2. The Kd of the primary binding site could not be determined; however, the two secondary sites appeared to be equivalent, with an apparent Kd of 8 x 10(-7) M for both BSA and HSA. 3. The spectral characteristics of DAUDA when bound to the primary site of the two albumins were different, with HSA producing a greater fluorescence enhancement and emission maximum at a shorter wavelength (480 nm) than for BSA (495 nm). 4. Displacement studies indicated that the DAUDA-binding sites were not equivalent to the primary long-chain fatty acid-binding sites on albumin, but corresponded to the bilirubin sites. Fatty acyl-CoAs also bind to the bilirubin sites, as do medium-chain fatty acids. 5. The solubility, stability and spectral properties of DAUDA make it an excellent probe for investigating the bilirubin-binding sites of albumin, particularly HSA.     

Enhanced separation and detection of serum bilirubin species by capillary electrophoresis using a mixed anionic surfactant—protein buffer system with laser-induced fluorescence detection

The four major bilirubin species in serum are separated by capillary electrophoresis and detected using laser-induced fluorescence detection. The optimum buffer system consists of 40 mM sodium dodecyl sulfate (SDS)—0.012 mM bovine serum albumin (BSA). The use of the SDS—BSA mixture in the mobile phase allows for the separation of four major bilirubin species at physiological pH with untreated capillaries. The results show that the use of BSA as a run buffer modifier in SDS solution improves separation efficiency and increases sample solubility via pH changes of the run buffer. The limits of detection for the bilirubin species using laser-induced fluorescence are between 30 and 150 nM, depending on the bilirubin species; not only is this approximately two orders of magnitude lower than with visible-light absorption methods, it allows the bilirubin species in normal sera to be quantitatively measured without sample pretreatment.     

In vitro plasma protein binding and aqueous aggregation behavior of astaxanthin dilysinate tetrahydrochloride

The tetrahydrochloride salt of astaxanthin di-L-lysinate (lys(2)AST) is a highly water-dispersible astaxanthin-amino acid conjugate, with an aqueous dispersibility of > or = 181.6 mg/mL. The statistical mixture of stereoisomers has been well characterized as an aqueous-phase superoxide anion scavenger, effective at micromolar (microM) concentrations. In the current study, the aqueous aggregation behavior and in vitro plasma protein binding [with fatty-acid-free human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP)] were investigated with a suite of techniques, including circular dichroism (CD) and UV-vis spectroscopy, ultrafiltration, competitive ligand displacement, and fluorescence quenching. Induced CD bands obtained in Ringer buffer solution of HSA demonstrated high affinity monomeric binding of the compound at low ligand per protein (L/P) ratios (in aqueous solution alone the carotenoid molecules formed card-pack aggregates). The binding constant ( approximately 10(6)M(-1)) and the binding stoichiometry (approximately 0.2 per albumin molecule) were calculated from CD titration data. CD displacement and ultrafiltration experiments performed with marker ligands of HSA indicated that the ligand binding occurred at a site distinct from the main drug binding sites of HSA (i.e., Sites I and II). At intermediate L/P ratios, both monomeric and aggregated ("chirally complexed") binding occurred simultaneously at distinct sites of the protein. At high L/P ratios, chiral complexation predominantly occurred on the asymmetric protein template. The tentative location of the chirally-complexed aggregation on the HSA template was identified as the large interdomain cleft of HSA, where carotenoid derivatives have been found to bind previously. Only weak binding to AGP was observed. These results suggest that parenteral use of this highly potent, water-dispersible astaxanthin-amino acid conjugate will result in plasma protein association, and plasma protein binding at sites unlikely to displace fatty acids and drugs bound at well-characterized binding sites on the albumin molecule.     

A drug release-facilitating mechanism for human serum albumin in reverse micelles: requirement of a structural switch.

We measured the binding of a drug oxyphenylbutazone to the N-terminal peptic fragment of human serum albumin in 0.1 M Tris buffer, pH 8.0 (Kass = 2.4 10(5) M-1) and in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate and buffer in isooctane (Kass. = 2.7 10(5) M-1). In the absence of any measured change in conformation of the fragment in reverse micelles, the peptide affinity for the drug is not decreased, in contrast to what is observed in intact albumin (HSA) under similar conditions. The interaction and the subsequent unfolding of HSA at the membrane-mimetic interface, constitutes thus a drug release-facilitating mechanism.     

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.