Binding of phthalate plasticizers to human serum albumin in vitro: a multispectroscopic approach and molecular modeling

As endocrine-disrupting chemicals, a few frequently used phthalate plasticizers were banned or restricted for use as additives in food in some countries. The interaction mechanisms between three phthalate plasticizers with human serum albumin (HSA) were studied by fluorescence (quenching, synchronous, and three-dimensional), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy, in combination with molecular modeling under simulative physiological conditions, respectively. The results obtained from fluorescence quenching data revealed that the plasticizers-HSA interaction altered the conformational strcture of HSA. Meanwhile, the alterations of HSA secondary structure in the presence of phthalate plasticizers were investigated. The binding distances for the plasticizers-HSA system were provided by the efficiency of fluorescence resonance energy transfer. Furthermore, the thermodynamic analysis implied that hydrophobic forces were the main interaction for the plasticizers-HSA system, which agreed well with the results from the molecular modeling study.     

Study on the binding of propiconazole to protein by molecular modeling and a multispectroscopic method

Propiconazole (PCZ) is an N-substituted triazole used as a fungicide on fruits, grains, seeds, hardwoods, and conifers. Although the triazole fungicides have shorter half-lives and lower bioaccumulation than the organochlorine pesticides, possible detrimental effects on the aquatic ecosystem and human health also exist. To evaluate the toxicity of PCZ at the protein level, its effects on human serum albumin (HSA) were characterized by molecular modeling and multispectroscopic method. On the basis of the fluorescence spectra, PCZ exhibited remarkable fluorescence quenching, which was attributed to the formation of a complex. The thermodynamic parameters ΔH and ΔS were calculated to be -14.980 KJ/mol and 26.966 J/(mol K), respectively, according to the van't Hoff equation, which suggests hydrophobic and electrostatic interactions are the predominant intermolecular forces in stabilizing the PCZ-protein complex. Furthermore, HSA conformation was slightly altered in the presence of PCZ. These results indicated that PCZ indeed affected the conformation of HSA.     

Deactivation of ferrylmyoglobin by vanillin as affected by vanillin binding to β-lactoglobulin

Vanillin was found to be efficient as a deactivator of ferrylmyoglobin with a second-order rate constant of k(2) = 57 ± 1 L mol(-1) s(-1) for reduction to metmyoglobin with ΔH(?) = 58.3 ± 0.3 kJ mol(-1) and ΔS(?) = -14 ± 1 J mol(-1) K(-1) in aqueous pH 7.4 solution at 25 °C. Binding to β-lactoglobulin (βLG) was found to affect the reactivity of vanillin at 25 °C only slightly to k(2) = 48 ± 2 L mol(-1) s(-1) (ΔH(?) = 68.4 ± 0.4 kJ mol(-1) and ΔS(?) = 17 ± 1 J mol(-1) K(-1)) for deactivation of ferrylmyoglobin. Binding of vanillin to βLG was found to have a binding stoichiometry vanillin/βLG > 10 with K(A) = 6 × 10(2) L mol(-1) and an apparent total ΔH° of approximately -38 kJ mol(-1) and ΔS° = -55.4 ± 4 J mol(-1) K(-1) at 25 °C and ΔC(p, obs) = -1.02 kJ mol(-1) K(-1) indicative of increasing ordering in the complex, as determined by isothermal titration microcalorimetry. From tryptophan fluorescence quenching for βLG by vanillin, approximately one vanillin was found to bind to each βLG far stronger with K(A) = 5 × 10(4) L mol(-1) and a ΔH° = -10.2 kJ mol(-1) and ΔS° = 55 J mol(-1) K(-1) at 25 °C. The kinetic entropy/enthalpy compensation effect seen for vanillin reactivity by binding to βLG is concluded to relate to the weakly bound vanillin oriented through hydrogen bonds on the βLG surface with the phenolic group pointing toward the solvent, in effect making both ΔH(?) and ΔS(?) more positive. The more strongly bound vanillin capable of tryptophan quenching in the βLG calyx seems less or nonreactive.     

Optical spectroscopic exploration of binding of Cochineal Red A with two homologous serum albumins

Cochineal Red A is a negatively charged synthetic azo food colorant and a potential carcinogen. We present here the study of binding of Cochineal Red A with two homologous serum albumins, human (HSA) and bovine (BSA), in aqueous pH 7.4 buffer by optical spectroscopic techniques. Protein intrinsic fluorescence quenching by Cochineal Red A occurs through ground-state static interaction and its binding with BSA is stronger than with HSA. The magnitudes of thermodynamic parameters suggest that dye binding occurs principally via electrostatic complexation. Site-marker competitive binding shows that Cochineal Red A binds primarily to site I of serum albumins. Circular dichroic spectra indicate that dye binding results in some conformational modification of serum albumins. Increased ionic strength of the medium results in lowering of binding. This study provides an important insight into possible means of removal of dye toxicity.     

二苯-α-吡喃酮类链格孢霉毒素和人血清白蛋白的相互作用及机理探究

为探究链格孢酚单甲醚(AME)和链格孢酚(AOH)这两种二苯-α-吡喃酮类链格孢霉毒素与人血清白蛋白(HSA)之间的分子相互作用及机理,本研究模拟血液生理pH值,采用稳态荧光光谱、同步荧光光谱、3D荧光光谱以及圆二色光谱等方法研究两者之间的相互作用。结果表明,AME和AOH与HSA相互作用均发生了静态猝灭,具有较高亲和力,以氢键和范德华力(ΔH<0,ΔS<0)结合形成1:1复合物,互作过程中这两种类毒素破坏了人血清白蛋白中稳定二级结构的氢键网络,使蛋白二级结构展开,且AME和AOH使人血清白蛋白中的α-螺旋结构含量从48.93%分别减少至39.41%和44.01%,并使色氨酸残基极性降低、疏水性增加,但酪氨酸残基极性变化不大,即结合位点可能位于色氨酸残基所在的空腔(即结合位点I);AME对HSA的猝灭程度、猝灭常数(K_sv)及结合常数(K_a)均大于AOH,结合距离也更小(r_AME=2.56 nmAOH=2.60 nm)。本研究结果为进一步探究二苯-α-吡喃酮类链格孢霉毒素的毒代动力学和药代动力学,完善丰富其毒理学相关资料,以及进行风险评估和在食品中限量标准的制定提供了参考依据。     

Molecular spectroscopic studies of farrerol interaction with calf thymus DNA

The interaction between farrerol and calf thymus DNA in a pH 7.4 Tris-HCl buffer was investigated with the use of neutral red (NR) dye as a spectral probe by UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy, as well as viscosity measurements and DNA melting techniques. It was found that farrerol molecules could intercalate into the base pairs of DNA as evidenced by decreases in iodide quenching effect and single-stranded DNA (ssDNA) quenching effect, induced CD spectral changes, and significant increases in relative viscosity and denaturation temperature of DNA. Furthermore, the spectral data matrix of the competitive reaction between farrerol and NR with DNA was resolved with an alternative least-squares (ALS) algorithm, and the concentration profiles in the reaction and the corresponding pure spectra for three species (farrerol, NR, and DNA-NR complex) were obtained. This ALS analysis demonstrated the intercalation of farrerol to the DNA by substituting for NR in the DNA-NR complex. Moreover, the thermodynamic parameters enthalpy change (ΔH°) and entropy change (ΔS°) were calculated to be -16.49 ± 0.51 kJ mol(-1) and 32.47 ± 1.02 J mol(-1) K(-1) via the van't Hoff equation, which suggested that the binding of farrerol to DNA was driven mainly by hydrophobic interactions and hydrogen bonds.     

Sensitive determination of DNA based on the interaction between norfloxacin-Tb3+ complex and DNA

The fluorescence intensity of the norfloxacin (NFX)-Tb3+ complex enhanced by DNA was studied. Therefore, a sensitive fluorescence method for the determination of DNA was developed. The optimal conditions of the method were as follows: the hexamethylenamine (HMA)-HCl buffer was adopted for adjusting the pH to 6.5 +/- 0.1, the concentrations of NFX and Tb3+ were both fixed in 1.0 x 10(-6) mol L(-1), and the excitation and emission wavelengths were selected at 290 and 545 nm, respectively. Under the optimal conditions, the enhanced fluorescence intensity was in proportion to the concentration of DNA in the same range of 5.0 x 10(-9) - 1.0 x 10(-6) g mL(-1) for hsDNA and thermally denatured ctDNA. The detection limits (S/N = 3) were 0.9 and 0.6 ng mL(-1), respectively. In addition, the interaction between NFX-Tb3+ and DNA was discussed in detail. The experimental results from UV absorption spectra, fluorescence spectra, and the salt effect study indicated that the interaction between norfloxacin-Tb3+ complex and DNA had at least two different binding modes: the electrostatic binding and the intercalation binding. The mechanism of the fluorescence enhancement effect was also discussed.     

Characterizing the Interaction between tartrazine and two serum albumins by a hybrid spectroscopic approach

Tartrazine is an artificial azo dye commonly used in food products. The present study evaluated the interaction of tartrazine with two serum albumins (SAs), human serum albumin (HSA) and bovine serum albumin (BSA), under physiological conditions by means of fluorescence, three-dimensional fluorescence, UV-vis absorption, and circular dichroism (CD) techniques. The fluorescence data showed that tartrazine could bind to the two SAs to form a complex. The binding process was a spontaneous molecular interaction procedure, in which van der Waals and hydrogen bond interactions played a major role. Additionally, as shown by the UV-vis absorption, three-dimensional fluorescence, and CD results, tartrazine could lead to conformational and some microenvironmental changes of both SAs, which may affect the physiological functions of SAs. The work provides important insight into the mechanism of toxicity of tartrazine in vivo.     

Screening, identification, and potential interaction of active compounds from Eucommia ulmodies leaves binding with bovine serum albumin

The aqueous extract of Eucommia ulmoides leaves has been commonly known as Du-zhong tea as a functional health food for the treatment of hypertension, hypercholesterolemia, and fatty liver. This study developed a centrifugal ultrafiltration-high-performance liquid chromatography (HPLC) method for screening and identification of bioactive compounds in E. ulmoides leaves binding with bovine serum albumin (BSA). Six active compounds were screened, isolated, and elucidated by their ultraviolet (UV), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) data as geniposidic acid (1), caffeic acid (2), chlorogenic acid (3), quercetin-3-O-sambubioside (4), rutin (5), and isoquercitrin (6). The interaction between active compounds and BSA was investigated in the absence and presence of other compounds by quenching the intrinsic BSA fluorescence. The results indicated that the structures significantly affected the binding process. The values of binding constants for compounds 2-6 were in the range of 10(5)-10(6) mol L(-1), while geniposidic acid (1) hardly quenching the BSA intrinsic fluorescence. However, the quenching process of geniposidic acid was easily affected in the presence of other active compounds. The formation of the geniposidic acid-phenylpropanoid (flavonoid) complex could increase the binding affinity of geniposidic acid with BSA; however, the increased steric hindrance of the complex may make phenylpropanoid or flavonoid dissociate from BSA and then decrease their affinities.     

Influence of B-ring hydroxylation on interactions of flavonols with bovine serum albumin

The B-ring substitution pattern of flavonols is a significant structural feature for their function as free radical scavengers and antioxidants. In this paper, four differently substituted B-ring hydroxylation flavonols (galangin, kaempferol, quercetin, and myricetin) and a flavonol glycoside (quercitrin) were studied for their ability to bind BSA by quenching the protein intrinsic fluorescence. From the spectra obtained, the biomolecular quenching constants, the apparent static binding constants, and the binding site values were calculated. The B-ring hydroxylation of flavonols significantly affected the binding/quenching process; in general, the binding affinity increased with the number of hydroxyl groups on the B-ring. The binding constants ( Ka) were determined as myricetin (4.90 x 10(8) L/mol) > quercetin (3.65 x 10(7) L/mol) > kaempferol (2.57 x 10(6) L/mol) > galangin (6.43 x 10(5) L/mol). The glycoside substitute at the C-ring position decreased the binding affinity. The chromatographic retention factor ( K') and logarithms of apparent partition coefficient (log Kow) were linear to the logarithms of apparent binding constants (log Ka) for flavonols with increasing hydroxyl groups on the B-ring. These results showed that the hydrogen bond force play an important role in binding flavonols to BSA. These results are also in agreement with the generally accepted structure-dependent free radical scavenger and antioxidant abilities of flavonols.     

Bioevaluation of human serum albumin-hesperidin bioconjugate: insight into protein vector function and conformation

Hesperidin is a flavanone glycoside widely available for dietary intake in citrus fruits or citrus fruit derived products; however, exhaustive and reliable data are scarcely available for biological activity when it exerts protective health effects in humans. The principal intent of this work is to check binding domain and structural changes of human serum albumin (HSA), the primary carrier of flavonoids, in blood plasma association with hesperidin by employing molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) methods. From molecular modeling simulations, subdomains IIA and IIIA, which correspond to Sudlow's sites I and II, respectively, were earmarked to possess affinity for hesperidin, but the affinity of site I with flavanone is greater than that of site II. This corroborates the site-specific probe and hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANS) displacement results placing the hesperidin at warfarin-azapropazone and indole-benzodiazepine sites. Steady state and time-resolved fluorescence manifested that static type, due to HSA-hesperidin complex formation (1.941 × 10(4) M(-1)), is the operative mechanism for the diminution in the tryptophan (Trp)-214 fluorescence. Moreover, via alterations in three-dimensional fluorescence and CD spectral properties, we can securely draw the conclusion that the polypeptide chain of HSA is partially destabilized after conjugation with hesperidin. We anticipate that this study can provide better knowledge of bioavailability such as absorption, biodistribution, and elimination, of hesperidin in vivo, to facilitate the comprehension of the biological responses to physiologically relevant flavanones.     

Interaction of different polyphenols with bovine serum albumin (BSA) and human salivary alpha-amylase (HSA) by fluorescence quenching

Phenolic compounds are responsible for major organoleptic characteristics of plant-derived food and beverages; these substances have received much attention, given that the major function of these compounds is their antioxidant ability. In the context of this study, our major aim was study the binding of several phenolic compounds such as (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, malvidin-3-glucoside, tannic acid, procyanidin B4, procyanidin B2 gallate, and procyanidin oligomers to different proteins (bovine serum albumin and human alpha-amylase) by fluorescence quenching of protein intrinsic fluorescence. From the spectra obtained, the Stern-Volmer, the apparent static, and the bimolecular quenching constants were calculated. The structure of polyphenols revealed to significantly affect the binding/quenching process; in general, the binding affinity increased with the molecular weight of polyphenol compounds and in the presence of galloyl groups. For catechin monomer and procyanidin dimer B4, the K(SV) was 14,100 and 13,800 M(-1), respectively, and for galloyl derivatives, the K(SV) was 19,500 and 21,900 M(-1), respectively. Tannic acid was shown to be the major quenching molecule for both proteins. However, comparing different proteins, the same polyphenol showed different quenching effects, which are suggested to be related to the three-dimensional structure of the proteins studied. For (+)-catechin and BSA, the K(SV) was 8700 M(-1), and with alpha-amylase, it was 14,100 M(-1); for tannic acid, the K(SV) was 10,0548 and 11,0674 M(-1), respectively. From the results obtained, besides the main binding analysis performed, we conclude that this technique is more sensitive than thought because we can detect several interactions that have not been proven by other methods, namely, nephelometry. Overall, fluorescence quenching has proven to be a very sensitive technique with many potentialities to analyze the interaction between polyphenols and proteins.     

Investigation on the interaction between ilaprazole and bovine serum albumin without or with different C-ring flavonoids from the viewpoint of food-drug interference

The interaction between ilaprazole and bovine serum albumin (BSA) has been investigated in the absence and presence of four popular flavonoids with different C-ring structures, quercetin, luteolin, taxifolin, and (+)-catechin, by means of fluorescence spectroscopy. The results indicated that ilaprazole had a strong ability to quench the intrinsic fluorescence of BSA, and site marker competitive experiments indicated that the binding of ilaprazole to BSA primarily took place in subdomain IIA. The quenching process of ilaprazole with BSA was easily affected by flavonoids,; however, they did not change the quchenching mechanism of ilaprazole with BSA, whereas all of the fluorescence quenching was initiated by a static quenching procedure combining with nonradiative energy transfer. The presence of flavonoids decreased the quenching constants of ilaprazole with BSA from 2.2 to 23.7% and decreased the binding constants from 73.7 to 98.3%, which depended on the different flavonoids' structures. The decreased binding constants and unchangeable spatial distance of ilaprazole with BSA by the introduction of quercetin, luteolin, and taxifolin may result from the competition of flavonoids and ilaprazole binding to BSA, whereas in the presence of (+)-catichin, decreased binding constants and increased spatial distance possibly resulted from the formation of a ternary ilaprazole-BSA-(+)-catechin complex. All of these results may have relevant consequences in rationalizing the interferences of common food to gastric ulcer treatments.     

Crocetin, dimethylcrocetin, and safranal bind human serum albumin: stability and antioxidative properties

Crocetin (CRT) and dimethylcrocetin (DMCRT) are derived from crocins which are found in the stigmas of saffron (Crocus sativus L.), while safranal is the main component of saffron's essential oil. The aim of the present study was to examine their interaction with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various ligand contents. FT-IR and UV-visible spectroscopic methods were used to determine the ligands' binding mode, the binding constant, and the effects of ligand complexation on protein secondary structure. Structural analysis showed that crocetin, dimethylcrocetin, and safranal bind nonspecifically (H-bonding) via protein polar groups with binding constants of Kcrt =2.05 (+/-0.30) x 103 M-1, Kdmcrt = 9.60 (+/-0.35) x 104 M-1, and Ksaf = 2.11 (+/-0.35) x 103 M-1. The protein secondary structure showed no major alterations at low ligand concentrations (1 microM), whereas at higher content (1 mM), decrease of alpha-helix from 55% (free HSA) to 43-45% and increase of beta-sheet from 17% (free HSA) to 18-22% and random coil 7% (free HSA) to 10-14% occurred in the ligand-HSA complexes. The results point to a partial unfolding of protein secondary structure at high ligand content. The antioxidant activity of CRT, DMCRT, and safranal was also tested by the DPPH* antioxidant activity assay, and their IC50 values were compared to that of well-known antioxidants such as Trolox and butylated hydroxy toluene (BHT). The IC50 values of CRT and safranal were 17.8 +/- 1 microg/mL and 95 +/- 1 microg/mL, respectively, while the inhibition of DMCRT reached a point of 38.8%, which corresponds to a concentration of 40 microg/mL, and then started to decrease. The IC50 values of Trolox and BHT were 5.2 +/- 1 microg/mL and 5.3 +/- 1 microg/mL, respectively.     

Interaction of alkylsulfonate ligands with beta-lactoglobulin AB from bovine milk.

The interaction of alkyl sulfonate ligands (AL) with bovine beta-lactoglobulin AB was measured using Trp fluorescence enhancement. One binding site per protein molecule was observed. The location of this site was related with the dimer formation and could be coincident with the fatty acids and SDS binding site. The apparent binding constants for AL were in the range of 10(-)(6) M, at pH 6.8. At pH 3.0 no binding was observed by this fluorescence method. The strength of the interaction was decreasing in the following way: AL16 > AL12 > AL14 > AL10. Other sites on the monomer were evidenced by the protective action of the AL toward the urea unfolding of the protein.     

Interaction of flavonoids with bovine serum albumin: a fluorescence quenching study

The interaction between four flavonoids (catechin, epicatechin, rutin, and quercetin) and bovine serum albumin (BSA) was investigated using tryptophan fluorescence quenching. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between the flavonoids and BSA. The binding affinity was strongest for quercetin and ranked in the order quercetin > rutin > epicatechin = catechin. The pH in the range of 5-7.4 does not affect significantly (p < 0.05) the association of rutin, epicatechin, and catechin with BSA, but quercetin exhibited a stronger affinity at pH 7.4 than at lower pH (p < 0.05). Quercetin has a total quenching effect on BSA tryptophan fluorescence at a molar ratio of 10:1 and rutin at approximately 25:1. However, epicatechin and catechin did not fully quench tryptophan fluorescence over the concentration range studied. Furthermore, the data suggested that the association between flavonoids and BSA did not change molecular conformation of BSA and that hydrogen bonding, ionic, and hydrophobic interaction are equally important driving forces for protein-flavonoid association.     

Biological relevance of the interaction between procyanidins and trypsin: a multitechnique approach

The interactions between the digestive protease trypsin type IX-S from porcine pancreas and grape seed procyanidins were monitorized by fluorescence quenching, dynamic light scattering, nephelometry, circular dichroism, and enzymatic inhibition assay. This work reports that the inhibition of trypsin activity by grape seed procyanidins and the respective quenching of intrinsic protein fluorescence are closely related. These two phenomena increase with the molecular weight of the tested procyanidins. The interaction between procyanidins and enzyme was shown to involve a specific interaction as inferred from the fluorescence assays. It was also shown by fluorescence spectroscopy that the binding of procyanidin molecules to the enzyme does not induce significant structural modifications. A relationship between aggregate formation, using dynamic light scattering and nephelometry, and fluorescence quenching was observed with maxima achieved for similar stoichiometric ratios. The binding of procyanidins to trypsin affects only slightly protein structure as seen by circular dichroism.     

Preparation and characterization of durum wheat (Triticum durum) straw cellulose nanofibers by electrospinning

Cellulose nanofibers from durum wheat straw ( Triticum durum ) were produced and characterized to study their potential as reinforcement fibers in biocomposites. Cellulose was isolated from wheat straw by chemical treatment. Nanofibers were produced via an electrospinning method using trifluoroacetic acid (TFA) as the solvent. The nanofibers were 270 ± 97 nm in diameter. Analysis of the FT-IR spectra demonstrated that the chemical treatment of the wheat straw removed hemicellulose and lignin. XRD revealed that the crystallinity of the cellulose was reduced after electrospinning, but nanofibers remained highly crystalline. The glass transition temperature (T(g) value) of the fibers was 130 °C, higher than that of cellulose (122 °C), and the degradation temperature of the fibers was 236 °C. Residual TFA was not present in the nanofibers as assessed by the FT-IR technique.     

Ethanol effect on the structure of beta-lactoglobulin b and its ligand binding

The changes of structure and ligand binding properties of beta-LG B have been studied by fluorescence and circular dichroism spectroscopy in ethanolic solutions. Fluorescence measurements of retinol/beta-LG interactions at 480 nm in various ethanol concentrations show that the maximal fluorescence intensity induced by this interaction between retinol and beta-LG is observed around 20% v/v of ethanol. It is reduced to zero at 40% and 50% of ethanol. These results suggest that there are two distinct structural changes in beta-LG occurring between 20% and 30% and around 40% of ethanol. The first transition, which increases affinity and the apparent number of binding sites for retinol, may be related or similar to the Tanford transition. The strong quenching of retinol emission at 480 nm in 40% of ethanol indicates the radical transformation of beta-LG tertiary structure and the release of retinol. CD spectra at the aromatic region show that secondary and tertiary structures of beta-LG are not significantly affected between 0% and 20% of ethanol. In 30% of ethanol, beta-sheet percentage of beta-LG decreases with respect to native beta-LG (from 55% to 46%). beta-Sheet percentage in beta-LG increases in 40% and 50% alcohol (51% and 53%) relative to 30% of ethanol, which also indicates the strong rearrangement of the secondary structure of beta-LG, while its tertiary structure and beta-LG interactions are radically changed.     

Binding of the pepper alkaloid piperine to bovine beta-lactoglobulin: circular dichroism spectroscopy and molecular modeling study

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.