Bilirubin binding by variant albumins in Yanomama Indians

Sera of Yanomama Indians homozygous for the common albumin allele exhibited greater total effective-binding capacities for bilirubin than did sera of individuals homozygous for the Yan-2 albumin variant in the in vitro experiments described herein. Total effective binding capacities of heterozygous samples were close to those of homozygotes for AlA. Individuals homozygous for Yan-2 might experience a higher risk of bilirubin toxicity and related disorders during the neonatal period. Further studies of binding and displacement of bilirubin by competitors, such as dietary or medicinal coumarins, might help explain the existence of these polymorphisms and the significance of phenotypic differences in binding to bilirubin.     

Characterization of inhibitor binding sites of mitochondrial complex I using fluorescent inhibitor

Recent progress in complex I research suggests that a wide variety of complex I inhibitors share a common large binding domain with partially overlapping sites. To verify this concept, we carried out real-time displacement tests of a fluorescent ligand with various competitors using a novel quinazoline-type inhibitor (aminoquinazoline, AQ). In the presence of an excess amount of the competitors, the binding of AQ to the enzyme was completely suppressed, being in line with the concept mentioned above. However, AQ bound to the enzyme was not displaced by subsequent addition of an increasing amount of competitors in the concentration range expected from the relative magnitude of the K(d) values of AQ and competitors, rather, much higher concentrations of the competitors were needed to displace bound AQ. These results cannot be explained merely by the premise of a common or partially overlapping binding site(s) between AQ and competitors. On the other hand, double-inhibitor titration of steady state complex I activity suggested that additivity of inhibition is not necessarily observed for all pairs of complex I inhibitors. Our results are discussed in light of the cooperativity of the inhibitor binding sites.     

Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli

MacB is an ABC-type membrane protein that exports only macrolide compounds containing 14- and 15-membered lactones, cooperating with a membrane fusion protein, MacA, and a multifunctional outer membrane channel, TolC. We determined the membrane topology of MacB by means of site-specific competitive chemical modification of single cysteine mutants. As a result, it was revealed that MacB is composed of four transmembrane (TM) segments with a cytoplasmic N-terminal nucleotide binding domain of about 270 amino acid residues and a periplasmic large hydrophilic polypeptide between TM segments 1 and 2 of about 200 amino acid residues.     

Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin

Previous results (Brouwer and van den Berg, Toxicol. Appl. Pharmacol., 85 (1986) 301) indicated preferential binding of a hydroxylated metabolite of tetrachlorobiphenyl to transthyretin (TTR) a carrier of thyroxine (T4). In the present study it was investigated whether the T4 binding site of TTR could be occupied specifically by hydroxylated chlorinated aromatic compounds using chlorinated phenol congeners as model compounds in a competition assay with [125I]T4. Chlorinated aromatics such as 2,3-dichlorobenzene and 3,4,3',4'-tetrachlorobiphenyl, and phenols such as 4-hydroxybiphenyl and phenol were inefficient competitors. All chlorinated phenols tested were competitors for the T4 binding site of TTR. The ranking in competition was pentachlorophenol (PCP) greater than trichlorophenols greater than dichlorophenols greater than monochlorophenols. Structures with chlorine in both ortho positions to the hydroxyl group were more efficient competitors. The relative affinity of binding of pentachlorophenol (PCP) to TTR was about twice that of T4. Scatchard analysis showed that PCP mainly decreased the affinity constant K11 while the binding capacity R1 was not altered, indicating a competitive type of inhibition. PCP was also able to compete with T4 sites on albumin with a relative affinity of 0.25. T4 binding to thyroid binding globulin (TBG) was much less affected by interference of PCP (relative affinity 0.001). The results indicate a specific interaction of chlorophenols with the T4 binding site of TTR.     

Binding and transcytosis of glycoalbumin by the microvascular endothelium of the murine myocardium: evidence that glycoalbumin behaves as a bifunctional ligand

The binding and transport of glycoalbumin (gA) by the endothelium of murine myocardial microvessels were studied by perfusing in situ 125I-gA or gA-gold complexes (gA-Au) and examining the specimens by radioassays and EM, respectively. After a 3-min perfusion, the uptake of radioiodinated gA is 2.2-fold higher than that of native albumin; it is partially (approximately 55%) competed by either albumin or D-glucose, and almost completely abolished by the concomitant administration of both competitors or by gA. D-mannose and D-galactose are not effective competitors. Unlike albumin-gold complexes that bind restrictively to plasmalemmal vesicles, gA-Au labels the plasma-lemma proper, plasmalemmal vesicles open on the lumen, and most coated pits. Competing albumin prevents gA-Au binding to the membrane of plasmalemmal vesicles, while glucose significantly reduces the ligand binding to plasmalemma proper. Competition with albumin and glucose gives additive effects. Transcytosis of gA-Au, already detected at 3 min, becomes substantial by 30 min. No tracer exit via intercellular junctions was detected. gA-Au progressively accumulates in multivesicular bodies. The results of the binding and competition experiments indicate that the gA behaves as a bifunctional ligand which is recognized by two distinct binding sites: one, located on the plasma membrane, binds as a lectin the glucose residues of gA; whereas the other, confined to plasmalemmal vesicles, recognizes presumably specific domains of the albumin molecule.     

Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB

Periplasmic membrane fusion proteins (MFPs) are essential components of the type I protein secretion systems and drug efflux pumps in Gram-negative bacteria. Previous studies suggested that MFPs connect the inner and outer membrane components of the transport systems and by this means co-ordinate the transfer of substrates across the two membranes. In this study, we purified and reconstituted the macrolide transporter MacAB from Escherichia coli. Here, MacA is a periplasmic MFP and MacB is an ABC-type transporter. Similar to other MFP-dependent transporters from E. coli, the in vivo function of MacAB requires the outer membrane channel TolC. The purified MacB displayed a basal ATPase activity in detergent micelles. This activity conformed to Michaelis-Menten kinetics but was unresponsive to substrates or accessory proteins. Upon reconstitution into proteoliposomes, the ATPase activity of MacB was strictly dependent on MacA. The catalytic efficiency of MacAB ATPase was more than 45-fold higher than the activity of MacB alone. Both the N- and C-terminal regions of MacA were essential for this activity. MacA stimulated MacB ATPase only in phospholipid bilayers and did not need the presence of macrolides. Our results suggest that MacA is a functional subunit of the MacB transporter.     

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.     

Role of ATP binding and hydrolysis in assembly of MacAB–TolC macrolide transporter

MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP‐Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram‐positive and Gram‐negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram‐negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC‐like channel. In this study, we report a real‐time analysis of concurrent ATP hydrolysis and assembly of MacAB–TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on‐ and off‐rates. In contrast, MacA–MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA‐dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans‐envelope complex.     

Enantioselective plasma protein binding of propafenone: mechanism, drug interaction, and species difference

The interaction of propafenone (PPF) enantiomers with human plasma, human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), as well as with plasma from rat, rabbit, and cow was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. The stronger binding of the S-PPF found in human plasma was due to AGP. Two classes of binding sites in AGP were identified: one with high-affinity and small binding capacity (K(1(S)) = 7.65 x 10(6) M(-1), n(1(S)) = 0.50; K(1(R)) = 2.81 x 10(6) M(-1), n(1(R)) = 0.46), which revealed stereoselectivity; the other with low-affinity and high-binding capacity (n(2(S)) K(2(S)) = 9.95 x 10(3) M(-1); n(2(R)) K(2(R)) = 9.74 x 10(3) M(-1)). The binding to HSA was found to be weak and not enantioselective (nK(S) = 2.08 x 10(3) M(-1), nK(R) = 2.05 x 10(3) M(-1)). The interaction between enantiomers observed in human plasma was confirmed as a competitive type interacting at the high-affinity site in AGP. The binding mode of both enantiomers with AGP was mainly hydrophobic bond. PPF enantiomers had higher-binding affinity for the F-S variant of human AGP. Drug-drug binding interaction studies showed that verapamil, diazepam, nifedipine, furosemide, nitrendipine, and nimodipine did not affect the binding of PPF enantiomers except quinidine and aprindine at the therapeutic concentration. Comparative studies indicated considerable species-dependent binding stereoselectivity between plasma of the four species investigated.     

Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.     

Method dependence of apparent stoichiometry in the binding of salicylate ion to human serum albumin: a comparison between equilibrium dialysis and fluorescence titration.

The binding of salicylate ion to human serum albumin (HSA) was studied in 100 mM potassium phosphate buffer (pH 7.4, 25 degrees C), using equilibrium dialysis and fluorescence titration methods. The protein samples tested were (a) dialyzed human plasma and (b) a commercial preparation of HSA, essentially free of globulin and fatty acids. Independent of the analytical method used, Scatchard and nonlinear regression analyses of the data pointed to a single class of high-affinity salicylate binding sites. On the other hand, the binding parameters were found to be method dependent. K(d) ranged between 25 +/- 2.4 and 62 +/- 15 microM in equilibrium dialysis and between 10 +/- 1.3 and 40 +/- 3.0 microM in fluorescence titration. (The higher limits refer to plasma samples at high [HSA]). Following the same pattern, the apparent stoichiometry of binding (though independent of sample identity and concentration) was higher in equilibrium dialysis (n(app) = 3.2 +/- 0.10) than in fluorescence titration (n(app) 1.9 +/- 0.30). The difference between the two methods could be reconciled by invoking two distinct classes of binding sites (I and II), which had identical (or marginally different) K(d) values, while differing in the magnitude of the fluorescence signal (Deltaf) generated upon ligand binding (Deltaf, PL(I) = Deltaf(I); Deltaf, PL(II) = 0). Further, it was assumed that the state of occupation of class II sites affected the fluorescence efficiency of class I sites, such that Deltaf, PL(I,II) = betaDeltaf(I) (beta = interaction factor). A random binding scheme involving P, PL(I), PL(II), and PL(I,II) was formulated. The model adequately predicted the behavior of the system when monitored through the change in protein fluorescence: Taking K(d) = 25 microM and n(T) = 3, the interaction factor beta was found to be 0.62 +/- 0.10. It was concluded that the correct parameters for the binding of salicylate ion to HSA are K(d) = 25 +/- 2.4 microM and n(T) = 3.2 +/- 0.10, as indicated by equilibrium dialysis of purified HSA. Besides updating information relating to the salicylate binding potential of HSA, this study serves to illustrate a likely complication in the study of protein-ligand interactions by fluorometric methods.     

Histone h1(0) and its carboxyl-terminal domain bind in the major groove of DNA

The binding of histone H1(0) to T4 bacteriophage DNA was investigated using thermal denaturation of the DNA titrated with varying concentrations of protein. The H1(0) used was expressed in and purified from a strain of E. coli and is therefore homogeneous with respect to H1 subtype and posttranslational modifications. Two types of T4 DNA were used: wild-type, which contains a modification of the cytosine residues that projects into the major groove: and a mutant type, which lacks the modification of the cytosines. Data were compared to simulated thermal denaturation curves to determine estimates for binding affinity and binding site size in base pairs of the protein. Analysis of the data yielded values of 10(8) M(-1) for K, the binding affinity, and 10 base pairs for n, the number of base pairs covered by one protein, for the mutant T4 DNA. Analysis of the wild-type DNA data suggested that the glucose projecting into the major groove of this DNA decreases the number of sites to which the H1(0) protein can bind, indicating that there are interactions between the protein and the major groove of DNA. The binding site size on this DNA is 10 base pairs, the same as on the unmodified DNA. The affinity for wild-type DNA is slightly higher, 10(9) M(-1). Data were collected and analyzed for binding of two domains of the protein as well, the carboxyl-terminal domain and the central globular domain. Binding of the carboxyl-terminal domain was quantitatively and qualitatively similar to that of the full-length protein. In contrast, binding of the globular domain was quite different: it binds much more weakly, with a K of 6 x 10(4) M(-1), and covers fewer base pairs, with an n of 3. Also, there was no evidence that the globular domain interacts with the major groove of DNA.     

Effects of aliphatic fatty acids on the binding of Phenol Red to human serum albumin.

Binding of Phenol Red to human serum albumin at pH 7.0 was studied by ultrafiltration (n1 = 1, K1 = 3.9 X 1-(4) M-1, n2 = 5, K2 = 9.6 X 10(2) M-1). The presence of 1 mol of octanoate or decanoate per mol of albumin caused a decrease in dye binding (dye/protein molar ratio 1:1), which, in contrast with additional fatty acid, was very pronounced: 1-8 mol of palmitate or stearate resulted in a small, and apparently linear, displacement of Phenol Red. The displacement effect of 1-5 mol of oleate, linoleate or linolenate per mol of albumin was comparable with that of the equimolar concentrations of palmitate or stearate. A higher molar ratios the unsaturated acids caused a drastic decrease in dye binding. The different Phenol Red-displacement effects of low molar ratios of medium-chain and long-chain fatty acids indicate that these acids have different high-affinity binding sites. In accordance with this proposal, low concentrations of stearate had only a small effect on the Phenol Red-displacement effect of octanoate. Phenol Red-binding curves in the presence of 1 mol of octanoate, 8 mol of stearate and 6 or 7 mol of linolenate per mol of albumin respectively indicated that the dye and the fatty acids do not complete for a common primary binding site. In contrast, a secondary Phenol Red-binding site could be identical with the primary octanoate-binding site. Furthermore, the primary Phenol Red-binding site could be the same as a secondary linolenate-binding site. Assignment of the different primary binding sites for Phenol Red and for medium-chain and long-chain fatty acids to a model of the secondary structure of albumin is attempted.     

Interaction of pirprofen enantiomers with human serum albumin.

The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule.     

The E288K Colon Tumor Variant of DNA Polymerase β Is a Sequence Specific Mutator

DNA polymerase β (pol β) is the main polymerase involved in base excision repair (BER), which is a pathway responsible for the repair of tens of thousands of DNA lesions per cell per day. Our recent efforts in sequencing colon tumors showed that 40% of the tumors sequenced possessed a variant in the coding region of the POLB gene; one of these variants is E288K. Expression of the E288K variant in cells leads to an increase in the frequency of mutations at AT base pairs. In vitro, the E288K variant is as active as and binds one-base-gapped DNA with the same affinity as wild-type pol β. Single-turnover kinetic data for the E288K variant show that its mutator phenotype is specific for misincorporating opposite template A up to 6-fold more than the wild-type enzyme and that this is due to a decrease in the degree of discrimination in nucleotide binding. Molecular modeling suggests that the substitution of Lys at position 288 causes the polymerase to adopt a more open conformation, which may be disrupting the nucleotide binding pocket. This may explain the reduced degree of discrimination at the level of nucleotide binding. The enhanced mutagenesis of the E288K variant could lead to genomic instability and ultimately a malignant tumor phenotype.     

Cytogenetic characterization and description of an XX/XY1Y2 sex chromosome system in catfish Harttia carvalhoi (Siluriformes, Loricariidae)

Karyotypic and cytogenetic characteristics of catfish Harttia carvalhoi (Paraíba do Sul River basin, S?o Paulo State, Brazil) were investigated using differential staining techniques (C-banding, Ag-staining) and fluorescent in situ hybridization (FISH) with 18S and 5S rDNA probes. The diploid chromosome number of females was 2n = 52 and their karyotype was composed of nine pairs of metacentric, nine pairs of submetacentric, four pairs of subtelocentric and four pairs of acrocentric chromosomes. The diploid chromosome number of males was invariably 2n = 53 and their karyotype consisted of one large unpaired metacentric, eight pairs of metacentric, nine pairs of submetacentric, four pairs of subtelocentric, four pairs of acrocentric plus two middle-sized acrocentric chromosomes. The differences between female and male karyotypes indicated the presence of a sex chromosome system of XX/XY1Y2 type, where the X is the largest metacentric and Y1 and Y2 are the two additional middle-sized acrocentric chromosomes of the male karyotype. The major rDNA sites as revealed by FISH with an 18S rDNA probe were located in the pericentromeric region of the largest pair of acrocentric chromosomes. FISH with a 5S rDNA probe revealed two sites: an interstitial site located in the largest pair of acrocentric chromosomes, and a pericentromeric site in a smaller metacentric pair of chromosomes. Translocations or centric fusions in the ancestral 2n = 54 karyotype is hypothesized for the origin of such multiple sex chromosome systems where females are fixed translocation homozygotes whereas males are fixed translocation heterozygotes. The available cytogenetic data for representatives of the genus Harttia examined so far indicate large kayotype diversity.     

Characterization of binding sites for acetylated low density lipoprotein in the rat liver in vivo and in vitro.

Acetylated low density lipoprotein (acetyl-LDL) binding to hepatic membrane proteins of rats was analysed in vitro by ligand blotting. Specific binding could be demonstrated to two hepatic proteins with an apparent mol. wt. of 250 kd and 220 kd. Polyanionic competitors and maleylated bovine serum albumin inhibited the binding of acetyl-LDL effectively. To determine the sites of the catabolism of acetyl-LDL, [131I]-acetyl-LDL was injected intravenously into control rats and rats pre-treated with the known competitors of the acetyl-LDL binding. Distribution of the radiolabelled acetyl-LDL was followed by a scintillation camera. Six minutes after injection, the radioactivity was concentrated in the liver. The competitors and unlabelled acetyl-LDL but not native LDL reduced the hepatic uptake of [131I]acetyl-LDL dramatically. Thus, the sensitivity of the 220- and 250-kd membrane binding sites to the competitors for the acetyl-LDL binding resembled that of the hepatic compartment in vivo. Finally, an application of scintigraphy with radiolabelled low density lipoproteins for diagnostic evaluation of tumor compartments is presented.     

A review on structure–affinity relationship of dietary flavonoids with serum albumins

Flavonoids are a class of plant secondary metabolites and among thousands of flavonoids few are considered as dietary flavonoids. Serum albumin (SA), the most abundant protein in plasma, functions as the most important carrier of vital drugs, including dietary flavonoids. The binding affinity of dietary flavonoids to SA is demonstrated to be governed by structure–affinity relationship (SAR) and its bioavailability. The present review summarizes the interactions of flavonoids categorized as flavanol, flavonol, flavone, isoflavone, flavanones, and anthocyanidins with SAs (bovine serum albumin and human serum albumin) in light of SAR. The key findings are: (1) the position and degree of hydroxylation highly influence the affinity of flavonoids to SAs, (2) glycosylation decreases and substitution of methoxy group increases the affinity of flavonoids for SAs, (3) catechin gallates have higher binding affinity to SAs than catechins and gallocatechins, (4) inorganic metal ions modulate the binding affinity of flavonoids to SAs, and (5) hydrophobic interaction plays a major role in the interactions of all flavonoids with SAs.     

Serum prostacyclin binding and half-life in the umbilical circulation

Serum prostacyclin binding and half-life was measured in twenty pairs of maternal and umbilical venous samples and in twenty non-pregnant controls. When compared to non-pregnant values both umbilical and maternal samples demonstrated significantly lower albumin concentrations, percentage of prostacyclin binding and shorter prostacyclin half-life.     

Analysis of binding interaction between the natural apocarotenoid bixin and human serum albumin by circular dichroism and fluorescence spectroscopy

Bixin is an important, pharmacologically active dietary cis-carotenoid, but its interaction with potential macromolecular targets is completely unexplored. This work was aimed to study the binding of bixin to human serum albumin (HSA), the most abundant protein in blood plasma. Circular dichroism (CD) spectroscopy in combination with UV/VIS absorption spectroscopy and fluorescence quenching techniques were applied. Appearance of induced CD bands in the UV- and VIS-absorption spectral regions indicated the formation of non-covalent carotenoid-albumin complexes. Shape and spectral position of the extrinsic Cotton effects suggested the binding of a single bixin molecule to HSA in chiral conformation. Scatchard and non-linear regression analyses of CD titration data resulted in similar values for the association constant (Ka = 6.6 and 4.6x10(5) M(-1), resp.) and for the number of binding sites (n = 1). The binding interaction was independently confirmed by fluorescence-quenching experiment from which the binding parameters were also calculated. CD Displacement measurements performed with marker ligands established that the main drug binding sites of HSA are not involved in binding of bixin. Palmitic acid decreased the amplitude of the induced CD bands suggesting a common albumin binding site for bixin and long-chain fatty acids. The above data indicate that HSA plays a significant role in the plasma transportation of bixin and related dietary carboxylic acid carotenoids.