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A O Pedersen

Publications and source records attributed to A O Pedersen.

At least 19 recordsLinked to original sources
PubMed1996

High-affinity binding of laurate to naturally occurring mutants of human serum albumin and proalbumin.

Binding of laurate (n-dodecanoate) to genetic variants of albumin or its proprotein and to normal albumin isolated from the same heterozygous carriers was studied by a kinetic dialysis technique at physiological pH. The first stoichiometric association constant for binding to proalbumin Lille (Arg-2-->His) and albumin (Alb) Roma (Glu321-->Lys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys541-->Glu) was decreased to 80%. In contrast, normal laurate-binding properties were found for as many as nine other albumin variants with single amino acid substitutions. Because the net charges of all these mutants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long-range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. The most pronounced effect was observed for the glycosylated Alb Casebrook, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total blocking of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes induced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using different preparations of normal albumin as controls in the binding experiments, it was also possible to compare the effect of various methods for isolation and defatting on laurate binding.

Amino Acid Sequence↗
PubMed1995

Effects of ionic strength and pH on the binding of medium-chain fatty acids to human serum albumin.

Binding equilibria for the interactions of the medium-chain fatty acid anions, laurate and myristate, with defatted human serum albumin have been investigated under varying environmental conditions such as ionic strength and pH. Since these ligands bind strongly to albumin (Kass approximately 10(7) M-1), conventional equilibrium dialysis is not a feasible method for these investigations. Consequently, we employed a dialysis method, allowing determination of very low concentrations of unbound ligand by measuring the rate of exchange of labelled ligand across a dialysis membrane under conditions of chemical equilibrium. Over a range of ionic strength, 8-68 mM, the binding of the first few molecules of laurate to albumin was weakened with increasing ionic strength, whereas the binding of subsequent molecules seemed to proceed independently of ionic strength. The binding of myristate by albumin, however, appeared to be independent of ionic strength in the observed range of concentrations. The influence of pH in the range 5.1-9.0 on the binding of the two fatty acid anions by albumin was more complicated. The first molecule of laurate appeared to bind with a slightly weaker affinity to albumin at low pH, compared to pH 7 and high pH, while the trends for the following molecules varied. The binding of myristate (irrespective of concentration) seemed to strengthen monotonously with pH, but this conclusion depends critically on the interpretation of the kinetic behaviour of the myristate anion. We have previously shown [Pedersen, A. O., Honoré, B. & Brodersen, R. (1990) Eur. J. Biochem. 190, 497-502] that the strength of binding of the first few molecules of the two fatty acid anions to albumin decreases with increasing temperature, whereas binding of subsequent molecules seems to proceed independently of temperature. We explain these findings as follows. The binding of the first few (3 or 4) molecules of the C12 laurate anion is clearly driven by formation of ionic bonds between the fatty acid anion and positively charged groups, such as lysine residues, in the albumin molecule, whereas the binding of subsequent molecules of laurate seems to depend more on hydrophobic interactions. In the case of the C14 myristate anion, the binding of the first few (only 1 or 2) molecules may depend on ionic forces, but binding of the following molecules of myristate seems to depend on hydrophobic interactions only.(ABSTRACT TRUNCATED AT 250 WORDS)

Dialysis↗
PubMed1995

Calcium ion binding to clinically relevant chemical modifications of human serum albumin.

Calcium binding to glycated, penicilloylated, acetylated, and normal defatted human serum albumin as well as to mercapt- and nonmercaptalbumin was studied by equilibrium dialysis of radioactive Ca2+. Binding was quantified by five Scatchard constants [ni = 1, (i = 1-4) and n5 = 10]. Glycation resulted in increased k1- and k2-values and unchanged k3-k5-values, whereas penicilloylation increased all five association constants. The increments were greater the more pronounced the modification, and the enhancements caused by penicilloylation were, for the same degree of modification, greater than those produced by glycation. In contrast, acetylation by acetylsalicylate did not affect calcium binding. Likewise, binding to mercapt- and nonmercaptalbumin was the same, a finding showing that the thiol group of cysteine 34 is not important for calcium binding. D-Glucose and penicillin G are known to react with lysine residues of albumin, and the enhancement of binding resulting from glycation or penicilloylation is probably brought about by unspecific electrostatic effects, possibly supplemented by conformational changes of the protein molecule. The relative importance of the three domains of human serum albumin for calcium binding is discussed.

Acetylation↗
PubMed1995

In vivo and in vitro binding of fatty acids to genetic variants of human serum albumin.

The effect of genetic variation on the fatty-acid binding properties of human serum albumin was studied by two methods involving the use of sequenced albumin variants isolated from bisalbuminaemic persons. First, the amount of total fatty acid and of several individuals fatty acids bound to eighteen different variants and to their normal counterpart (Alb A) were determined by a gas-chromatographic micromethod. Pronounced effects on total fatty acid binding were found for the glycosylated variants Alb Redhill (modified in domain II) and Alb Casebrook (domain III) in which cases a 1.7- and 8.6-fold increment, respectively, was found. By contrast, Alb Malm0 (glycosylated in domain I) carried the same amount of fatty acid as Alb A. The fatty acid loads on three chain-termination variants were normal. Finally, eight albumins with single amino-acid substitutions bound normal amounts of fatty acid, whereas one bound increased (1.7-fold) and three albumins bound diminished amounts (0.5-0.6-fold). Information on nineteen individual fatty acids was also obtained. It was possible, based on the type of changes in their relative amounts, to group the fatty acids as follows: (a) = C6:0 - C14:0, (b) = C15:0 - C18:0, (c) = C16:1 - C18:1, and (d) a group composed of essential and conditionally essential fatty acids. For nine variants, in most cases modified in domain III, large changes in one or more of these groups were observed. The changes were not related to any changes in total fatty acid load. Second, the binding of laurate, as a representative of the group (a) fatty acids, to delipidated albumin preparations was studied at pH 7.4 by a kinetic dialysis technique. The first stoichiometric association constant for binding to Alb Redhill (0.7-fold) and Alb Casebrook (0.6-fold) was diminished as compared with binding to their corresponding Alb A, whereas binding to one chain-termination variant and three single amino-acid substitutions were all unaffected by the mutation.

Chromatography, Gas↗
PubMed1992

Solubility of long-chain fatty acids in phosphate buffer at pH 7.4.

The solubility of the saturated fatty acids lauric, myristic, palmitic, and stearic acid and the unsaturated oleic acid at 37 degrees C in phosphate buffer (pH 7.4) was estimated by using two independent methods. The one was a conventional solubility technique measuring the concentration of dissolved fatty acid in buffer by using radioactive compounds. The other was a dialysis exchange technique monitoring possible aggregation of solvated fatty acid anions by measuring the rate of diffusion of labelled compound across a dialysis membrane under conditions of chemical equilibrium. It was found that the results were strongly dependent on the radiochemical purity of the fatty acids. Using highly purified samples of radioactively labelled fatty acids, the solubility of monomeric laurate was shown to be greater than 500 microM, whereas the solubility of monomeric myristate was found to be 20-30 microM. Palmitate, stearate, and oleate solutions, on the other hand, showed a tendency to aggregation even at concentrations below 1 microM. Special attention was given to palmitate, as a reference compound for long-chain fatty acids, and the solubility of monomeric palmitate was estimated to be lower than 10(-10) M.

Autoradiography↗
PubMed1992

Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography.

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component.

Chromatography, Affinity↗
PubMed1991

Binding of long-chain fatty acids to serum albumin in healthy humans. Relationship to obesity.

Equilibria of the binding of palmitate to serum albumin in adults are studied by dialysis-exchange-rate determinations. The results are used for a description of binding equilibria of fatty acids in general, as follows. 1. The reserve albumin concentration, p, for binding of palmitate is used as an approximate measure of p*, the reserve albumin concentration for binding of mixed fatty acids present in serum. 2. The total availability of fatty acids is defined as C*/p*, where C* is the total concentration of non-esterified fatty acid. 3. The fatty-acid-binding property of albumin is described by L* = p*/P = alpha C*/P, where P is the albumin concentration. The numerical value of alpha is -0.05. The above parameters are measured in sera from four healthy volunteers, in whom large variations of serum fatty acid concentration occurred. A group of 64 healthy students showed considerable variation of L* from one individual to another. It is found that L* decrease significantly with increasing body mass index (body mass divided by the square of the body length). In 42 patients with diabetes type I, L* was independent of body mass index. These findings are consistent with a previously formulated hypothesis of mechanism of obesity.

Adult↗
PubMed1990

Thermodynamic parameters for binding of fatty acids to human serum albumin.

Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5-37 degrees C, at pH 7.4. The binding curves indicate that the strength of binding of the first few molecules of fatty acid to albumin (r less than 5) decreases with increasing temperature, whereas binding of the following molecules seems to proceed independently of temperature. Binding data were analyzed according to the general binding equation yielding several sets of acceptable binding constants within a probability limit of 0.75. From the temperature dependence of the first step constant, it was possible to calculate values for the changes in enthalpy and entropy during the initial binding step. For the medium-chain fatty acids, laurate and myristate, binding of the first molecule to albumin appeared to be enthalpic, with a tendency to an increasing contribution of entropy to binding energy with increasing chain length of the fatty acid.

Humans↗
PubMed1990

Multiple fatty acid binding to albumin in human blood plasma.

Binding equilibria of long-chain fatty acids to human serum albumin, in serum or plasma, were studied by a dialysis exchange rate technique. Palmitate was added to citrated plasma in vitro and it was observed that between six and ten palmitate molecules were bound to albumin with nearly equal affinity. Observations in vivo gave similar results in the following series: (a) in two volunteers with increased fatty acid concentrations after fasting, exercise, and a cold shower: (b) in three male volunteers in whom high concentrations of non-esterified fatty acids, up to 4.6 mM, were induced by intravenous administration of a preparation of lecithin/glycocholate mixed micelles, and (c) in 81 patients with diabetes mellitus, type I. The binding pattern of palmitate in serum or plasma is essentially different from that observed with palmitate added to buffered solutions of pure albumin when two molecules are tightly bound and about four additional molecules with lower affinity. The differences may partly be explained by the presence of chloride ions in blood plasma, reducing the affinity for binding of the first two fatty acid molecules, and partly by facilitated binding of several molecules of mixed fatty acids, as found in plasma.

Diabetes Mellitus, Type 1↗
PubMed1989

Serum albumin binding of palmitate and stearate. Multiple binding theory for insoluble ligands.

In usual studies of ligand binding to a carrier, free and bound ligand concentrations are measured in equilibrium mixtures with varying carrier and ligand concentrations. The observed data are then analyzed by a binding equation such as Scatchard's or the general binding equation. With palmitic, stearic and oleic acids as ligands we found that the aqueous solubility is too low to allow this procedure. We have consequently transformed the general binding equation so that it does not contain parameters related to aqueous solutions of the ligand. While the classical binding equations describe affinities of transfer of a ligand from an aqueous solution to the carrier, the new equation is valid for transfer of a ligand from one bound state to another, i.e. for relative binding description. The relative binding constants, L1, L2, L3 ... Li, in the new equation thus define the transfer affinity for the ligand from a 1:1 complex with a standard carrier to an i:1 complex of the ligand with the carrier investigated. Binding of palmitate and stearate to human serum albumin was studied by determination of dialytic exchange rates between identical fatty acid/albumin solutions. The results were analyzed by the new equation without reference to ligands in aqueous solution.

Chlorides↗
PubMed1989

Conformational changes in human serum albumin studied by fluorescence and absorption spectroscopy. Distance measurements as a function of pH and fatty acids.

pH- and fatty acid-induced conformational changes in human serum albumin were investigated by fluorescence-energy transfer, determining the distance between Trp-214 and bound bilirubin at 25 degrees C. This distance changes significantly with the pH, being 2.52 +/- 0.01 nm at pH 6, 2.31 +/- 0.04 nm at pH 9, 2.13 +/- 0.07 nm at pH 11.0 and 2.77 nm at pH 11.9. The influence of different fatty acids on the distance was also determined. At pH 7.4 medium-chain fatty acids seem to increase this distance, whereas long-chain fatty acids, at low concentrations, decrease the distance between the two chromophores. The contraction of the protein carrying long-chain saturated fatty acids is even more pronounced at pH 9.

Bilirubin↗
PubMed1988

Myristic acid binding to human serum albumin investigated by dialytic exchange rate.

Dialysis rate determinations of several fatty acids in the absence of albumin revealed that the myristate anion, like that of laurate, in aqueous solution, pH 7.5, is present as a monomer anion when the concentration is below 25 microM. Palmitate and oleate solutions, on the other hand, show a tendency to aggregation even at concentrations below 0.5 microM. Multiple binding of myristate to human serum albumin in phosphate buffer, at pH 7.5, 37 degrees C, was investigated by exchange of 14C-labeled myristate across a dialysis membrane under conditions of binding equilibrium. A binding isotherm was established by least squares fitting of the stoichiometric binding constants in the stepwise binding equation to the experimental data. The best-fit solution was supplemented with 30 acceptable solutions within a probability limit of 0.95. A concept of one or two distinct high-affinity sites for binding of fatty acids could not be verified; the observations allow a variety of binding mechanisms ranging from cooperativity of the first two myristates to a model with four equal and independent sites.

Dialysis↗
PubMed1987

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.

Bilirubin↗
PubMed1986

Laurate binding to human serum albumin. Multiple binding equilibria investigated by a dialysis exchange method.

Multiple binding of laurate (n-dodecanoate) to human serum albumin was studied by a kinetic dialysis method. With this method the concentration of unbound ligand can be determined by measuring the rate of exchange of radioactive label across a dialysis membrane under conditions of equilibrium. Determination of the rate constant of exchange, in the absence of albumin, revealed that laurate does not change its state of aggregation over a concentration range from 0.1 microM to 500 microM, indicating the prevalence of a monomer. Binding of laurate to albumin, at pH 7.5, 37 degrees C, was investigated through 220 determinations of binding equilibria in the range of 0-10 mol laurate/mol albumin, corresponding to a concentration range of unbound laurate from 1 nM to 0.1 mM. Binding data were analyzed in terms of stepwise binding. Considering the stochastic errors of the experimental data, we generated a variety of possible (equal goodness of fit) solutions to the stoichiometric binding equation. This analysis resulted in reasonably well-defined values for the first two step constants, with an indication of negative interaction. The variation of the higher step constants excluded any mechanistic conclusions, although the binding isotherms, defined by these varying sets of binding constants, fitted the experimental data well within a chosen probability limit.

Dialysis↗
PubMed1986

Spectroscopic properties of bilirubin-human serum albumin complexes: a stoichiometric analysis.

Light absorption spectra, fluorescence of bound bilirubin, fluorescence of albumin as quenched by bilirubin, and circular dichroism spectra have been studied in mixtures of bilirubin and defatted human serum albumin in variable proportions at 25 degrees C and at pH 7.4, 8.2, and 9.0. Corresponding spectral data have been calculated for the stoichiometric bilirubin-albumin complexes, 1:1, 2:1, and 3:1. Light absorption spectra as well as the bound bilirubin fluorescence indicate that all three bound bilirubin dianions are internalized. These data were obtained by curve fitting to least sum of squared deviations. In addition to the best fit we obtained 30 acceptable curves, located within an F contour, thus producing a rough estimate of the variation of the resulting spectral data.

Bilirubin↗
PubMed1980

Reactivity of the thiol group in human and bovine albumin at pH 3--9, as measured by exchange with 2,2'-dithiodipyridine.

The kinetics for exchange between an aromatic disulphide and the thiol group in human and bovine albumin as well as in glutathione were investigated in the pH range 2.5--9.8. For both albumins the rate constants exhibit a maximum near pH 3, confirming the results of Svenson and Carlsson's investigation of bovine albumin [A. Svenson and J. Carlsson (1975) Biochim. Biophys Acta, 400, 433--438]. This was related to the well known N--F conformational change of the protein. At pH 5--8 the reactivity of the thiol group in both albumins and glutathione changes sharply, probably due to ionization of the thiol group. At pH above 8, however, the reactivity of the thiol group in albumins, but not in glutathione, becomes nearly independent of pH. In addition, a conformational change at pH 6.5--8.5 was studied by means of differential spectroscopy of bilirubin, liganded to human albumin. This neutral transition appeared to proceed identically in mercaptalbumin and nonmercaptalbumin. It is concluded that (a) the pK of the thiol group in albumin is significantly below that of SH in glutathione, and (b) ionization of this thiol group, Cys-34, is independent of the neutral transition.

2,2'-Dipyridyl↗
PubMed1977

Photooxidation of human serum albumin and its complex with bilirubin.

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole O2, and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen. Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non-oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole O2. Parallel photooxidation of the protein carrier could not be detected. It is considered possible that the fast, anaerobic process is operative in phototherapy of hyperbilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.

Bilirubin↗