Detection of carrier heterogeneity by rate of ligand dialysis: medium-chain fatty acid interaction with human serum albumin and competition with chloride

Binding equilibria for decanoate, octanoate, and hexanoate to defatted human serum albumin were investigated by dialysis exchange rate determinations in 66 mM sodium phosphate buffer, pH 7.4, 37 degrees C. The binding isotherms for decanoate and octanoate could not be fitted by the general binding equation. It was necessary to assume the presence of two albumin components, one with high affinity and one with low affinity, about 0.65 of the albumin having high binding affinity. The first stoichiometric binding constants for the high- and low-affinity albumin components were 1.1 X 10(7) and 1.4 X 10(5) M-1, respectively, for decanoate; 1.6 X 10(6) and 3.5 X 10(4) for octanoate; and 7.1 X 10(4) and 8.0 X 10(2) M-1 for hexanoate. The high-affinity albumin component binds 1 mol decanoate, 1 mol octanoate, or 2 mol hexanoate more than is bound to the low-affinity component. Chloride ions compete with the high-affinity binding of all three ligands. Albumin dimer, present in the commercial human serum albumin, has approximately the same binding properties as the monomer. Mercaptalbumin, isolated from the preparation, also consists of two proteins, with first stoichiometric binding constants 8.0 X 10(6) and 1.4 X 10(5) M-1 for decanoate, approximately 0.5 of the mercaptalbumin having high affinity.     

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.     

Cobinding of bilirubin and laurate to human serum albumin: spectroscopic characterization of stoichiometric complexes

Light absorption and CD spectra of bound bilirubin and albumin fluorescence spectra have been recorded from mixtures containing albumin, A, bilirubin, B, and laurate, L, in Tris-NaCl buffer at pH 8.2, 25 degrees C. Concentrations of the corresponding stoichiometric complexes, ABiLj, for i = 0/3 and j = 0/3, have been calculated from previously determined stoichiometric cobinding constants (H. Sato et al. (1988) Arch. Biochem. Biophys. 260, 811-821). Spectral data of the complexes have finally been found by iterative computer fitting using the principle of several acceptable solutions (R. Brodersen et al. (1987) Eur. J. Biochem. 169, 487-495). The results were utilized at the microscopic level to investigate ligand-induced conformational changes. When laurate was bound to AB, a decrease of the distance between Trp-214 and the bound bilirubin occurred, as measured according to F?rster's principle. The distances were 21.9 +/- 0.3 A in AB, 19.7 +/- 0.3 A in ABL, and 17.9 +/- 0.2 A in ABL2.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

Cobinding of bilirubin and sulfonamide and of two bilirubin molecules to human serum albumin: a site model

Differential light absorption spectra of the bilirubin-albumin 1:1 complex, obtained on addition of 20 different sulfonamides, differ with respect to shape and amplitude. This finding seems to indicate that the sulfonamide molecule is bound in direct touch with the bilirubin. The light absorption spectrum of bilirubin-albumin 1:1 undergoes changes on cobinding of a fatty acid anion, laurate, and on variation of pH, previously explained by a change of dihedral angle between the two chromophores of the bilirubin molecule. In bilirubin-albumin 2:1, binding of laurate and variation of pH cause little change of the spectrum. This is best explained by binding of the two bilirubin molecules in close proximity, preventing conformational changes in the complex. From measurements of fluorescence of the lone tryptophan group in albumin and quenching on binding of bilirubin, we calculated the distance of 22 A from tryptophan to the first bound bilirubin molecule, and of 18 A to the second. Mutual quenching of the bilirubin fluorescence from two bound bilirubin molecules seemed to indicate that the two are bound closely together. A model of bilirubin-albumin with a binding site capable of accommodating one bilirubin and one sulfonamide molecule, or two molecules of bilirubin, is compatible with our findings.     

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.     

Spectroscopic studies of the binding of bilirubin by ligandin and aminoazo-dye-binding protein A.

Ligandin and aminoazo-dye-binding protein A both bind bilirubin at a single site. Quantitative studies of the interactions using difference spectrophotometry show that at pH 7.0, protein A binds the tetrapyrrole with an association constant (K) greater than or equal to 2 X 10(7) litre/mol, whereas binding by ligandin is slightly weaker (K = 7 X 10(6) litre/mol) at this pH. The protein-bilirubin complexes give rise to absorption and fluorescence spectra quite different from those of unbound bilirubin and also to large Cotton effects. It appears that on binding to both proteins, the ligand is forced into a rigid twisted configuration in a hydrophobic environment. Ligandin and protein A resemble serum albumin in their interactions with bilirubin.     

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.     

Phospholipid-binding properties of bovine factor V and factor Va.

Factor V and factor Va binding to single bilayer phospholipid vesicles was investigated by light-scattering intensity measurements. This technique allows the measurement of free and phospholipid-bound protein concentrations from which equilibrium constants can be obtained. As controls, the Ca2+-dependent phospholipid binding of prothrombin and factor X were also studied. The average values obtained for the dissociation constants (Kd) and lipid to protein ratio at saturation, moles/mole (n), for prothrombin (Kd = 2.3 X 10(-6) M, n = 104) and factor X (Kd = 2.5 X 10(-6) M, n = 46) binding to vesicles containing 25% Folch fraction III and 75% phosphatidylcholine in the presence of 2 mM Ca2+ were in agreement with those reported in the literature. The average factor V and factor Va values for the dissociation constants and lipid to protein ratio at saturation (moles/mole) were Kd = 7.2 X 10(-8) M and n = 270 for factor V and Kd = 4.4 X 10(-7) M and n = 76 for factor Va. In contrast to prothrombin and factor X, factor V and factor Va demonstrated Ca2+-independent lipid binding. In addition, the number of factor V and factor Va molecules bound per vesicle was found to be dependent both on the phosphatidylserine content of the vesicle and the ionic strength of the buffer.     

Cooperativity of binding of anilinonaphthalenesulfonate to serum albumin induced by a second ligand.

When a ligand X is multiply bound to energetically identical, noninteracting sites of a protein, cooperative binding of this ligand can be induced by the presence of a second ligand Y. This effect should appear whenever multiple interactions exist between the bound X and Y ligands, and vanish when the concentration of Y is made sufficiently large to ensure Y saturation at all concentrations of X. These predictions have been verified for the binding of 8-anilino-1-naphthalenesulfonate to serum albumin, when Y, the effector ion, is 3,5-dihydroxybenzoate. In the presence of 2mM dihydroxybenzoate, the Hill coefficient for anilinonaphthalenesulfonate binding rose steadily from 1 to 1.5 as the number of molecules of ligand bound increased from 1 to 3.3 per albumin molecule. The theory of interactions between isolated ligands, applied in the previous paper (D. A. Kolb and G. Weber (1975), Biochemistry, preceding paper in this issue), is extended to cases of multiple interactions, and applied here to show that the experimental results are tolerably well reproduced for a model in which four anilinonaphthalensulfonate molecules are homogeneously coupled to four molecules of dihydroxybenzoate by free energies of 3.0 and 3.5 thermal units.     

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.     

Oestriol binding to plasma proteins

Simple diffusion experiments indicated that oestriol was retained by human pregnancy plasma more effectively than by albumin solutions of a corresponding concentration. Oestriol bound (Ka = 6 X 10(6) l/mol at 4 degrees C) to a glycoprotein which had been isolated from plasma by adsorption to Concanavalin A. The free energy of binding at 37 degrees C was -38 kJ/mol. Competition experiments indicated that the oestriol binding glycoprotein had properties expected of sex hormone binding globulin. The distribution of oestriol among the protein fractions of human pregnancy plasma--glycoprotein bound 7.8%, albumin bound 78.6%, unbound 13.6%--suggests that this glycoprotein plays little part in the transport of oestriol.     

Binding and endocytosis of glycoproteins by isolated chicken hepatocytes

The binding and endocytosis of glycoproteins containing different terminal sugars by isolated chicken hepatocytes were studied. At 2 degrees C, where there is no endocytosis, the hepatocyte surface bound 30 800 GlcNAc44-AI-BSA molecules [a bovine serum albumin (BSA) derivative which contains 44 residues of N-octylglucosamine (GlcNAc)] [Lee, Y.C., Stowell, C.P., & Krantz, M.J. (1976) Biochemistry 15, 3956-3963] and 32 900 asialoagalactoorosomucoid (AGOR) molecules per cell with estimated dissociation constants of 5 X 10(-10) and 4 X 10(-9) M, respectively. In the presence of digitonin or Triton X-100, each hepatocyte bound 7-18 times more ligand than in the absence of these detergents. Bound 125I-AGOR could be dissociated from the cell surface by 5.5 X 10(-5) M GlcNAc44-AI-BSA with a t 1/2 of 30 min, while GlcNAc (10 mM) or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (4 mM) could dissociate over 98% of the surface-bound radioactivity within 10 min. Several neoglycoproteins inhibited the binding of 125I-AGOR, requiring for 50% inhibition 2.1 X 10(-9), 4.0 X 10(-7), 1.6 X 10(-6), and 2 X 10(-6) M for GlcNAc44-, Glc37-, Man43-, and L-Fuc28-AI-BSA, respectively. The bound AGOR and neoglycoproteins were internalized and degraded at 37 degrees C. [125I]Iodide was the only labeled degradation product found. When the hepatocytes were exposed to 250 nM AGOR at 37 degrees C, ca. 100 000 molecules of AGOR were associated with the cell surface at the steady state of endocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids.

A structural protein of Rauscher oncovirus of about 8,000 to 10,000 daltons (p10), encoded by the gag gene, has been purified in high yield to apparent homogeneity by a simple three-step procedure. The purified protein was highly basic, with an isoelectric point of more than 9.0, and its immunological antigenicity was chiefly group specific. A distinctive property of the protein was the binding to nucleic acids. The stoichiometry of p10 binding to Rauscher virus RNA was analyzed using both 125I-labeled p10 and 3H-labeled RNA. The protein-RNA complex, cross-linked by formaldehyde, was separated from free RNA and free protein by velocity sedimentation and density gradient centrifugation. A maximum of about 140 mol of p10 was bound per mol of 35S RNA, or about one molecule of p10 per 70 nucleotides. This protein-RNA complex banded at a density of about 1.55 g/ml. The number of nucleic acid sites bound and the affinity of p10 binding differed significantly among the other polynucleotides tested. The protein bound to both RNA and DNA with a preference for single-stranded molecules. Rauscher virus RNA and single-stranded phage fd DNA contained the highest number of binding sites. Binding to fd DNA was saturated with about 30 mol of p10 per mol of fd DNA, an average of about one p10 molecule per 180 nucleotides. The apparent binding constant was 7.3 X 10(7) M(-1). The properties of the p10 place it in a category with other nucleic acid binding proteins that achieve a greater binding density on single-stranded than on double-stranded molecules and appear to act by facilitating changes in polynucleotide conformation.     

The kinetics of interactions of bilirubin with lipid bilayers and with serum albumin.

Rate constants for the hydration of bilirubin bound to unilamellar bilayers of dioleoylphosphatidylcholine and albumin were measured by stopped-flow methods. Rate constants for association of bilirubin with these vesicles and albumin were calculated from measured rate constants for dissociation and the equilibrium binding constants of bilirubin and lipids or albumin. Rate constants for hydration (dissociation) for bilirubin bound to dioleoylphosphatidylcholine and albumin were 71 s-1 and 1.8 s-1 respectively. Rate constants for association were 4.0 10(7) s-1 and 1.1 10(9) M-1 s-1, respectively. Both rates for interactions of bilirubin with bilayers were essentially independent of temperature in the range 0-40 degrees C, indicating that barriers to entry and exit of bilirubin from bilayers were entropic. Rates of transbilayer movement of bilirubin in dioleoylphosphatidylcholine were too fast to resolve by measuring rates of hydration of bilirubin. Rate constants for hydration of bilirubin bound to bilayers with less avidity for bilirubin as compared with dioleoylphosphatidylcholine also were too fast to measure with stopped-flow methods. In addition to providing details of the energetic basis for interactions between bilirubin and membranes, the data allow for calculating the maximal rates at which bilirubin could transfer spontaneously from sites on albumin in blood to the interior of cells. The data show, in this regard, that this rate is 10-50 fold faster than measured rates of uptake of bilirubin by intact liver.     

Binding of histamine and histamine analogs to lymphocyte subsets analyzed by flow cytometry

The binding of histamine, 4-methylhistamine (a histamine type 2 receptor agonist), cimetidine (a histamine type 2 receptor antagonist), and telemethylhistamine (an inactive analog) to human peripheral blood mononuclear cell subsets was investigated by flow cytometry by using conjugates of these ligands coupled to fluorescein-labeled human serum albumin. Our results indicate that binding of fluorescent protein conjugates of histamine and its analogs does not selectively identify a lymphocyte subset(s) that mediates the immunomodulatory effects of histaminergic ligands. Conjugates with both low (2.5 to 2.8:1) and high (28 to 57:1) ligand to protein coupling ratios were used. No binding above background could be detected for the low mole ratio reagents. The high mole ratio reagents were bound by 95 to 99% of all lymphocytes when used at ligand concentrations of 50 microM or greater. At lower ligand concentrations, the number of lymphocytes exceeding a set fluorescence threshold was decreased, but fluorescence distributions remained unimodal at all concentrations used (1 to 500 microM). Monocytes also bound the high mole ratio reagents and gave rise to a second high-intensity peak in the fluorescence distribution unless they were excluded by other means. Levels of conjugate binding detected by flow cytometry did not parallel ligand potencies at classical histamine type 2 receptors; at equivalent ligand concentrations, approximately equal amounts of histamine or 4-methylhistamine conjugate were bound per lymphocyte, and only 30% less telemethylhistamine conjugate was bound. Competition with free ligands (10(2)- to 10(4)-fold excess histamine, 4-methylhistamine, cimetidine, or telemethylhistamine) did not significantly decrease the level of binding observed for the high mole ratio reagents at bound ligand concentrations of 1 to 25 microM. Dual staining with fluorescein-labeled conjugate and phycoerythrin-labeled monoclonal antibodies Leu-3ab (anti-helper T), Leu-2a (anti-suppressor T), Leu-M3 (anti-monocyte), or anti-HLA-DR (B cells and monocytes) was also carried out. The extent of conjugate binding to helper and suppressor cells was identical for each of the ligands used, but higher levels of conjugate binding were seen for monocytes and B cells than for T cells in every case. Our data do not exclude the possibility of enhanced conjugate binding to small numbers of activated (HLA-DR positive) T cells that might be involved in mediation of histamine effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyanate binding to the ferric heme octapeptide from cytochrome c. A model for anion binding to high spin ferric hemoproteins

Equilibrium constants for the binding of cyanate to the ferric heme c octapeptide in 50% ethylene glycol, 50% aqueous buffer were measured spectrophotometrically. Equilibrium constants measured at several temperatures from -20 degrees C to 0 degrees C exhibited an apparent van't Hoff relationship yielding thermodynamic values of delta Ho = -1.3 X 10(3) +/- 0.9 X 10(3) J/mol (-3.1 X 10(2) +/- 2 X 10(2) cal/mol), delta So = -3 +/- 3 J/K X mol (-0.6 +/- 0.8 cal/K X mol). The equilibrium constant for cyanate binding at 25 degrees C and pH 7.4 is 1.21 which is approximately 2 to 3 orders of magnitude lower than that observed for cyanate binding to methemoglobin and metmyoglobin. Krel, the ratio of the hemoprotein to model heme octapeptide binding constants, for NCO- is smaller than Krel for N3- suggesting that hydrogen bonding between the terminal ligand atoms and the distal histidine in hemoglobin and myoglobin does not contribute to the increased protein ligand stabilization observed for these anions relative to the model. A donor-acceptor interaction between the distal histidine and the electrophilic middle atoms of these bound ligands is proposed.     

Enthalpy-driven apolipoprotein A-I and lipid bilayer interaction indicating protein penetration upon lipid binding

The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K(a)), the number of protein molecules bound per vesicle (n), enthalpy change (DeltaH degrees), entropy change (DeltaS degrees ), and the heat capacity change (DeltaC(p) degrees). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 degrees C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the alpha-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of POPC/FC at a molar ratio of 6/1 at (2.8-4) x 10(6) M(-1) and 2.4 apoA-I molecules bound per vesicle or 1.4 x 10(2) phospholipids per bound apoA-I. A plot of DeltaH degrees against temperature displayed a linear behavior, from which the DeltaC(p) degrees per mole of bound apoA-I was calculated to be -2.73 kcal/(mol x K). These results suggested that binding of apoA-I to POPC vesicles is characterized by nonclassical hydrophobic interactions, with alpha-helix formation as the main driving force for the binding to cholesterol-containing vesicles. In addition, comparison to literature data on peptides suggested a cooperativity of the helices in apoA-I in lipid interaction.     

The non-convalent binding of small molecules by ligandin. Interactions with steroids and their conjugates, fatty acids, bromosulphophthalein carcinogens, glutathione and realted compounds.

1. Equilibrium dialysis studies have been made of the binding of a number of small molecules by rat ligandin. Direct measurements of binding together with competition experiments indicated that bromosulphophthalein, oestrone sulphate and dehydroepiandrosterone sulphate each bind at the same single primary binding site with association constants of 1.1 X 10(7), 6.6 X 10(5) and 2.6 X 10(5) 1/mol respectively at pH 7.0,IO.16M,4 degrees C. As well as bromosulphophthalein and dehydroepiandrosterone sulphate, a number of strucurally similar organic anions including 2-hydroxyoestradiol-glutathione oestrone glycyronide, N-methyl-4-aminoazobenzene-glutathione and several bile acids, were able to displace oestrone sulphate from ligandin in a manner consistent with competition at a single binding site. From these experiments association constants for the competing ligands were derived; these were inthe range 1 X 10(4)-1 X 10(6) 1/mol. 2. Ligandin was found to bind a number of compounds for which, because of their low aqueous solubilities relative to their binding affinities complete binding isotherms could bot be obtained. These included several steroids (but not cortisol), 20-methylcholanthrene, diethylstilboestrol, oleate and palmitate. Oestrone sulphate was able to compete with these ligands for binding and the results of the competition experiments were interpretable in terms of 1:1 competition at a single binding site. 3. In general the conjugation of non-polar ligands with sulphate or glutathione resulted in increased affinities, but such increases were relatively small (approximately 15% in therms of free energy) implying that the main driving force for the binding of both the conjugated and unconjugated species was the hydrophobic effect. This conclusion is borne out by the observations that both oestrone and its sulphate showed slight increases in affinity with increase in ionic strength, as would be expected for hydrophobic interactions. 4. As well as non-polar compounds and organic anions, ligandin was also found to bind sulphate and glucuronate to a measurable degree, and to interact quite strongly with glutathione. For the latter compound a single binding site was found with an association constant of 1 X 10(5) 1/mol. Glutathione was able to cause the dissociation of the ligandin-oestrone sulphate complex, but this effect was not explicable in terms of simple 1:1 competition. 5. Both oestrone and oestrone sulphare were bound most strongly at pH 6-7, the affinity of the protein for these ligands falling off quite sharply on either side of this maximum. 6. The affinities of ligandin for bromosulphophthalein, steroids and their conjugates, diethylstilboestrol and N,N-dimethyl-4-aminoazobenzene are similar in magnitude to those of serum albumin and aminoazodye-binding protein A (B. Ketterer, E. Tipping, J.F. Hackney and D. Beale, 1976).