Binding of isofraxidin to bovine serum albumin

The binding of isofraxidin to bovine serum albumin (BSA) was studied under physiological conditions with BSA concentration of 1.5 x 10(-6) mol x L(-1) and drug concentration in the range of 1.67 x 10(-6) mol x L(-1) to 2.0 x 10(-5) mol x L(-1). Fluorescence quenching spectra in combination with uv absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and CD spectroscopy was used to determine the drug-binding mode, binding constant, and the protein structure changes in the presence of isofraxidin in aqueous solution. The linearity of Scatchard plot indicates that isofraxidin binds to a single class of binding sites on BSA and the values given for the binding constants agree very closely with those obtained by the modified Stern-Volmer equation. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated to be -17.63 kJ x mol(-1) and 51.38 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to BSA.     

Frontal gel chromatographic analysis of the interaction of a protein with self-associating ligands: aberrant saturation in the binding of flavins to bovine serum albumin

Frontal gel chromatography is an accurate method to obtain the total free ligand concentration of a protein-ligand mixture in which ligands self-associate. The average number of bound ligands per protein molecule is obtained as a function of the total free ligand concentration. The method was applied to the interaction of bovine serum albumin with self-associating flavins. The binding curves for FMN and FAD leveled off at about 0.7 and 0.5, respectively. These data were simulated well by a binding model where flavins undergo isodesmic indefinite self-association and the monomer alone binds to a single binding site of albumin. The isodesmic association constants of FMN and FAD were (1.7 +/- 0.1) x 10(2) and (2.2 +/- 0.3) x 10(2) M(-1), respectively. The binding constants of the monomer of FMN and FAD were (7.6 +/- 0.2) x 10(2) and (3.5 +/- 0.2) x 10(2) M(-1), respectively. FMN competitively inhibited the binding of FAD to albumin. The affinity to flavins was in the following order at pH 5.8: lumiflavin, FMN, riboflavin, and FAD. The SH modification and the binding of palmitate did not affect the FMN binding to bovine albumin at pH 5.8. As pH increased from 5.8 to 9.0, the affinity to FMN of bovine albumin decreased 3-fold, whereas that of human albumin increased about 80-fold. The present study clearly showed how isodesmic self-association of a ligand can cause apparent saturation of the interaction of a protein with the ligand at levels lower than 1.     

Evaluation of the binding of benzo-a-pyrene by blood serum lipoproteins using the cellulose disk method

A simple and convenient method has been developed for evaluation binding capacity of lipophilic ligands with lipoproteins or other transport proteins. For this purpose cellulose discs (Whatman 3 MM) were loaded with the constant quantity of 3H-benzopyrene and increasing amounts of unlabelled benzo[a]pyrene in dimethylsulfoxide, dried up and incubated in solution of lipoproteins. Dissociation constants were deduced from the remained disk radioactivity using the Scatchard plot method. The obtained values of the dissociation constants were: HDL--4.1 x 10(-5) M; LDL--1.5 x 10(-6) M; VLDL--8.8 x 10(-6) M.     

Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities

The interaction between sialosyl cholesterol (- or neuraminyl cholesterol, - or β-SC) and the plasma membrane of astrocytes was investigated by the use of ¹⁴C-labeled - or β-SC. Both - and β-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 × 10⁹ molecules of both -SC/cell (apparent K_d = 2.29 × 10⁻⁵ M) and β-SC/cell (apparent K_d = 5.39 × 10⁻⁵ M) at 37°C. Both the binding of -SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4°C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of -SC with the slightly lower K_d-value (8.03 × 10⁻⁶ M) than that for the binding site on astrocytes. BSA not only suppressed the -SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the -SC-binding, promoted the small release of -SC from the cell surface, and inhibited the morphological changes of astrocytes induced by -SC. The mechanism of -SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.     

Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase.

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.     

Kinetics of bilirubin oxidase and modeling of an immobilized bilirubin oxidase reactor for bilirubin detoxification

The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin-serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin-serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K(1) = 6 x 10(7)M(-1) and K(2) = 4.5 x 10(6)M(-1), respectively. The rate of total bilirubin degradation in bilirubin-serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8-mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350muM and a serum albumin concentration of 500muM.     

Interaction of taxol with human serum albumin

Taxol (paclitaxel) is an anticancer drug, which interacts with microtuble proteins, in a manner that catalyzes their formation from tubulin and stabilizes the resulting structures (Nogales et al., Nature 375 (1995) 424-427). This study was designed to examine the interaction of taxol with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 0.0001-0.1 mM, and HSA (fatty acid free) concentration of 2% w/v. Gel electrophoresis, absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second-derivative resolution enhancement were used to determine the drug binding mode, binding constant and the protein secondary structure in the presence of taxol in aqueous solution. Spectroscopic evidence showed that taxol-protein interaction results into two types of drug-HSA complexes with overall binding constant of K=1.43 x 10(4) M(-1). The molar ratios of complexes were of taxol/HSA 30/1 (30 mM taxol) and 90/1 (90 mM taxol) with the complex ratios of 1.9 and 3.4 drug molecules per HSA molecule, respectively. The taxol binding results in major protein secondary structural changes from that of the alpha-helix 55 to 45% and beta-sheet 22 to 26%, beta-anti 12 to 15% and turn 11 to 16%, in the taxol-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of taxol in aqueous solution.     

Renal plasma membrane receptors for certain modified serum albumins. Evidence for participation of a heparin receptor.

Binding of formaldehyde-treated (f-alb), reduced-carboxymethylated (ac-alb) or reduced-acetamidated (am-alb) bovine serum albumins to purified rat renal plasma membranes was studied. Radioiodinated f-alb or ac-alb bound to kidney membranes while am-alb neither bound significantly nor competed with f-alb binding to kidney membranes. The binding was specific, saturable and heat- and proteinase-sensitive. Competition studies showed that f-alb and ac-alb sites may be the same on these membranes. To determine the role played by charge in binding, competition experiments with polyanions were performed. Polyanions such as nucleic acid or glycosaminoglycans were effective competitors of f-alb binding to cell membranes. Heparin was especially inhibitory, being several-fold more so than chondroitin sulphate. Completely reduced and carboxymethylated albumin was a better competitor than its partially modified counterpart. Furthermore, f-alb was a significant competitor of [35S]heparin binding to kidney membranes. Also, partially purified heparin receptor demonstrated specific binding of 125I-f-alb. These data suggest that a heparin receptor is responsible for binding and internalization of intravenously injected f-alb. A Scatchard plot revealed two classes of receptors with dissociation constants of 3.2 X 10(-6) M and 4.7 X 10(-5) M.     

Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

Paclitaxel is pharmaceutically formulated in a mixture of Cremophor EL and ethanol (1:1, v/v). The unbound fraction of the anticancer drug paclitaxel in plasma is dependent on both plasma protein binding and entrapment in Cremophor EL micelles. We have developed a simple and reproducible method for the quantification of the unbound paclitaxel fraction in human plasma. Human plasma was spiked with [3H]paclitaxel and [14C]glucose (unbound reference) and incubated at 37 degrees C for 30 min. Plasma ultrafiltrate was prepared by a micropartition system (MPS-1) and collected in a sample cup containing 100 microl of plasma to prevent the loss of paclitaxel due to adsorption. The radionuclides were separated after combustion of the biological samples using a sample oxidizer and the radioactivity was determined by liquid scintillation counting. The unbound fraction of paclitaxel was calculated by dividing the ratios of 3H and 14C in plasma ultrafiltrate and in plasma. The method was thoroughly validated using human plasma spiked with pharmacologically relevant concentrations of paclitaxel (10-1000 ng/ml) and Cremophor EL (0.25-2.0%). The method was precise, with a within-day precision ranging from 3.9 to 11.0% and a between-day precision ranging from 5.8 to 13.1%. In patient plasma with low serum albumin values containing 1% of Cremophor EL, the unbound fraction appeared to be significantly higher than that in plasma with normal albumin values. The determination of the unbound fraction of paclitaxel proved to be stable during a 10-week storage at -20 degrees C. Furthermore, the assay was applicable in patient samples. This assay can be used to determine the unbound fraction of paclitaxel in plasma. Moreover, its design should allow the determination of the unbound concentrations of other hydrophobic drugs.     

Occurrence and properties of a prostaglandin F2alpha receptor in bovine corpora lutea.

Prostaglandin F2alpha was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5 X 10(3) M-1 S-1) and dissociation (2.1 X 10-4 S-1) reactions gave a dissociation constant of 2.8 X 10(-8) M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5 X 10(-8) M). The receptor was stable for several hours at 23 degrees C but was rapidly destroyed at 37 degrees C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2alpha and had much lower affinities for other prostaglandins. Luteinizing and follicle-stimulating hormones had no effect on the prostaglandin F2alpha-receptor interaction.     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.     

Epidermal growth factor receptor binding is not a simple one-step process

The binding kinetics of 125I-labeled mouse epidermal growth factor (EGF) to receptors on human fibroblast cells in monolayer culture were measured at 4 degrees C. Initial binding rates as a function of hormone concentration allowed estimation of simple two-state on-off rate constants of 1.2 x 10(6) M-1 s-1 and 4.9 x 10(-3) s-1, respectively. These two-state parameters gave inadequate computer fits to long term kinetic and equilibrium-binding data, suggesting that an additional process(es) was occurring. Nonlinear equilibrium Scatchard plots and transient "pseudo-Scatchard" plots taken at pre-equilibrium times support the idea that at least one other process is occurring during receptor binding. 125I-EGF-receptor dissociation kinetic plots were biphasic, yielding rate constants of 1.5 x 10(-2) s-1 and 5.6 x 10(-5) s-1 with the ratio of the two components changing with the time of initial incubation with 125I-EGF. Application of a ternary complex model which assumed complexation of the bound receptor with a cell surface interaction molecule gave satisfactory fits to all data.     

Aggregation of tyrocidine in aqueous solutions.

The formation of aggregates of tyrocidine B at 4°C and 20°C in aqueous solutions was studied by means of light scattering and fluorescent techniques. The apparent weight molecular weight of tyrocidine B aggregates was found to be 36,000 at 4°C and 28,800 at 20°C. Fluorescence titration experiments with dansyl-chloride resulted in an aggregational number of 31 (4°) and 28 (20°) indicating that one molecule of dye is bound per monomer of molecular weight 1,200. From a Scatchard plot apparent association constants of 1.22 × 10⁵ M (4°) and 0.95 × 10⁵ M (20°) were calculated. From the angular dependence of scattered intensity the radii of gyration were determined to be 60 Å and 58 Å, respectively.     

Characterization of binding of four monoclonal antibodies to the human ovarian adenocarcinoma cell line HEY

Four mouse monoclonal antibodies (mAb) (10B, IgG1; 8C, IgG2a; M2A, IgG2a; M2D, IgG2b) were characterized with respect to their binding to the ovarian adenocarcinoma cell line HEY, using displacement assays and Scatchard plot analyses. The four mAb reacted with different antigens on the surface of HEY cells, with affinity constants ranging from 1 X 10(9) to 3 X 10(9) M-1. The number of binding sites per cell for each antibody was approximately 2 X 10(4). mAb 8C and M2D remained associated with the cell surface following binding to their respective antigens, while mAb 10B was rapidly internalized, with 50% of the bound mAb being lost from the cell surface during 4 h of incubation at 37 degrees C. These different binding characteristics of the mAb may influence their ability to target radioactivity and cytotoxic drugs to HEY cells.     

Initial steps in Haemophilus influenzae transformation. Donor DNA binding in the com10 mutant

The com10 mutant of Haemophilus influenzae binds donor DNA reversibly, but is deficient in uptake. The DNA binding has all the characteristics of interaction with a protein receptor; it is saturable, reversible, and specific. However, binding specificity is 6-fold weaker in com10 than is uptake specificity in wild-type. The binding of small (120 base pairs) and large (14,400 base pairs) DNA molecules were compared. For small molecules, binding data fitted a straight line by Scatchard analysis (Bmax = 4.8 DNA molecules/cell, Kd = 0.5 X 10(-9) M). In contrast, for large DNA molecules, the Scatchard plot was not linear. A high affinity binding (Kd = 0.4 X 10(-12) M) and a lower affinity binding (Kd = 1.2 X 10(-11) M) were found with a total number of 3 molecules bound per cell. In wild-type cells, 3.2 large molecules were taken up per cell, whereas up to 40 small 120-base pair DNA fragments were taken up per cell. Uptake of small DNA molecules followed a Michaelis-Menten function with a Km of 0.5 X 10(-9) M and a maximal initial velocity of 1.5 molecules/cell/min at room temperature. For large DNA molecules, maximal initial velocity was approximately 2 molecules/cell/min at room temperature. The analysis of the binding and uptake data suggest to us that a receptor or a receptor complex is responsible for the uptake of either a single large DNA molecule or, successively, a number of small DNA molecules.     

Identical independent sites for dye ligand on bovine serum albumin demonstrated by multivariate analysis

The most fundamental parameters concerning an interaction between a ligand and a protein are equilibrium constants and the number of binding sites. The Scatchard plot has for a long time been widely used to obtain those parameters. However, controversy in 1982-1983 over the reliability of this plot (the graphical estimation of the number of identical independent sites from the x-intercept) indicated that some methodologies other than the Scatchard plot are expected. Over the past decade, we have developed a method for applying multivariate analysis to the problem of determining spectral features of a ligand associated with a protein molecule. In principle, this method is based mainly on the computer-assisted adjustment of dissociation constants to an assumed reaction model. We discovered in this process that an n-parameter, introduced into an equation for calculating the amount of dye ligand bound to a protein, coincided with the number of identical independent sites, under a certain condition in principal factor analysis calculation. In this study, we established a new methodology for determining the number of identical independent sites using synthesized spectral series, and we then applied this method to a simple reaction system composed of bovine serum albumin (BSA) and bromocresol purple (BCP) anions. BSA was found to have two identical independent sites for BCP anions at pH 8.8.     

Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.     

Interaction of the Laburnum anagyroides lectin with fucoantigens

We studied interaction of the lectin from the bark of Golden Rain shrub (Laburnum anagyroides, LABA) with a number of basic fucose-containing carbohydrate antigens by changes in its tryptophan fluorescence. The strongest LABA binding was observed for the trisaccharide H of type 6 [alpha-L-Fucp-(1-2)-beta-D-Galp-(1-4)-D-Glc, Ka= 4.2 x 10(3) M(-1)]. The following antigens were bound with a weaker affinity: H-disaccharide alpha-L-Fucp-(1-2)-D-Gal, a glucoanalogue of tetrasaccharide Ley alpha-L-Fucp-(1-2)-beta-D-Galp-(1-4)-[alpha-L-Fucp-(1-3)]-D-Glc, and 6-fucosyl-N-acetylglucosamine, a fragment of core of the N-glycans family (Ka 1.1-1.7 x 10(3) M(-1)). The lowest binding was observed for L-fucose (Ka = 2.7 x 10(2) M-1) and trisaccharide Lea, (3-Galp-(1-3)-[a-L-Fucp-(1-4)]-GlcNAc (Ka = 6.4 x 10(2) M(-1)). The Lea, Lea, and Lex pentasaccharides and Leb hexasaccharide were not bound to LABA.     

Presence of ACTH and its receptor on a B lymphocytic cell line: a possible autocrine function for a neuroendocrine hormone

A mouse B lymphocytic cell line, designated BCL1, was found to produce immunoreactive ACTH and to secrete this molecule into culture supernates. The BCL1-derived ACTH induced Y-1 adrenal cells to undergo a steroidogeneic response and was eluted from gel filtration columns at a molecular weight similar to that expected for pituitary-derived ACTH. Furthermore, ACTH receptors were detected on the surface of BCL1 cells using indirect immunofluorescence and 125I-ACTH binding studies. Scatchard analysis demonstrated the presence of high and low affinity binding sites with dissociation constants of 4.5 x 10(-12) M and 2.8 x 10(-9) M, respectively. The production of both ACTH and its receptor by this B lymphocyte cell line suggests that an autocrine mechanism might be important for the maintenance of the transformed phenotype.     

Binding study of desethyloxybutynin using high-performance frontal analysis method

Plasma protein binding of N-desethyloxybytynin (DEOXY), a major active metabolite of oxybutynin (OXY), was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of DEOXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. DEOXY is bound in human plasma strongly and enantioselectively. The unbound drug fraction in human plasma samples containing 5 microM (R)- or (S)-DEOXY was 1.19 +/- 0.001 and 2.33 +/- 0.044%, respectively. AGP plays the dominant role in this strong and enantioselective plasma protein binding of DEOXY. The total binding affinity (nK) of (R)-DEOXY and (S)-DEOXY to AGP was 2.97 x 10(7) and 1.31 x 10(7) M(-1), respectively, while the nK values of (R)-DEOXY and (S)-DEOXY to HSA were 7.77 x 10(3) and 8.44 x 10(3) M(-1), respectively. While the nK value of (S)-DEOXY is weaker than that of (S)-OXY (1.53 x 10(7) M(-1)), the nK value of (R)-DEOXY is 4.33 times stronger than that of (R)-OXY (6.86 x I0(6) M(-1)). This suggests that the elimination of an ethyl group weakens the binding affinity of the (S)-isomer because of the decrease in hydrophobicity, while the binding affinity of the (R)-isomer is enhanced by the decrease in steric hindrance. The total binding affinity of DEOXY to HSA is much lower than that of DEOXY-AGP binding as well as OXY-HSA binding (2.64 x 10(4) and 2.19 x 10(4) M(-1) for (R)-OXY and (S)-OXY, respectively). The study on competitive binding between OXY and DEOXY indicated that DEOXY enantiomers and OXY enantiomers are all bound competitively at the same binding site of AGP molecule.