Rolf Brodersen

Binding of bilirubin to albumin.

(1980). Binding of Bilirubin to Albumin. CRC Critical Reviews in Clinical Laboratory Sciences: Vol. 11, No. 4, pp. 307-399.

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.

DISPLACEMENT OF ALBUMIN‐BOUND BILIRUBIN BY FATTY ACIDS

Kernicterus may be precipitated in icteric newborns by displacement of bilirubin from binding to albumin by various drugs (11, 14, 15, 17). Several investigators (12, 16, 19, 20) have considered the possibility that fatty acids might have a similar effect, competing with bilirubin for the binding sites on albumin. Non-esterified fatty acids are bound reversibly to albumin (8, 18) and are present in the blood plasma of newborn children in relatively high concentrations (6, 13). The present paper deals with displacement of bilirubin by fatty acids from its binding to human plasma albumin, measured by a sensitive technique, recently described by Jacobsen & Fedders (lo), based on determination of the velocity of oxidation of bilirubin with peroxidase and ethyl hydroperoxide.

Determination of reserve albumin-equivalent for ligand binding, probing two distinct binding functions of the protein

Abstract A method is reported for determination of albumin binding capacity for various ligands in 50-μl sample volumes. A small amount of a radioactively labeled test ligand is added to the undiluted sample and the rate of dialysis of the free ligand into an identical sample without added ligand is measured. The reserve albumin-equivalent concentration is defined as the concentration of a standard albumin preparation which in buffered solution gives the same rate of dialysis and hence the same ratio of free/bound concentrations of the added ligand. It is shown that the reserve albumin-equivalent concentration, thus defined, is identical with the sum of concentrations of carrier species, each multiplied by the first stoichiometric binding constant of the test ligand to the carrier and divided by its first stoichiometric binding constant to the standard albumin. Determinations of this parameter are suitable for studies of the chemical potential and transfer affinities of individual ligands and for determination of interaction among several binding substances. Two test ligands have been used, monoacetyldiaminodiphenyl sulfone and diazepam. The former is bound competitively with bilirubin while diazepam engages another, independent binding function. The method can thus be used for separate determinations of the degree of saturation of two distinct binding functions of albumin. Complex mixtures of several carrier proteins with interacting ligands can be studied.

Deposition of Bilirubin Acid in the Central Nervous System–A Hypothesis for the Development of Kernicterus

ABSTRACT. On the basis of the concentration of unconjugated bilirubin and available albumin for the binding of bilirubin it is possible to calculate the level of unbound bilirubin in a serum sample. The solubility of bilirubin can further be calculated when the pH is known. In cases of threatened kernicterus the free bilirubin concentration in serum samples from newborn infants surpasses the solubility by a factor close to one hundred. It is hypothesized that deposition of bilirubin in tissues takes place as an ongoing event, the deposited pigment being eliminated by bilirubin oxidase in healthy infants. Kernicterus results when the rate of deposition becomes overwhelming as a result of high bilirubin concentration, low albumin reserve, low pH, after administration of a displacing drug, or if the bilirubin oxidase system has been compromised by preceding birth asphyxia or other forms of central nervous system injury.

Binding constants for ligand-carrier complexes

Abstract Most descriptions of ligand binding by carriers such as serum albumin in terms of two or more classes of sites with specific affinities are misleading or incorrect. Errors arise because of conceptual misunderstandings of the meaning of ligand binding constants . Rolf Brodersen and colleagues describe the proper analysis of ligand binding affinities for carrier proteins or other receptors. They illustrate this analysis graphically and numerically using fatty acid-serum albumin and sulfamethizole-serum albumin as examples .

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.

PREVENTION OF KERNICTERUS, BASED ON RECENT PROGRESS IN BILIRUBIN CHEMISTRY

Abstract. A review is presented of recent progress in bilirubin chemistry, its binding to albumin, displacement by drugs, and the mechanism of phototherapy. Quantitative formulations of the effect of albumin dosage, of varying pH, and of fatty acids result in a diagram which may be tried as an aid to indications for therapy.

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.

Covalent binding of bilirubin to agarose and use of the product for affinity chromatography of serum albumin

Abstract A method is described for covalent coupling of bilirubin to agarose through an aminohexylamino group, using a carbodiimide reagent. The product contained 1.1 μmoles bilirubin/ml of the packed gel (250 mg dry wt) and could reversibly bind 0.08 μmole rat serum albumin. Elution was effected by salicylate and sulfisoxazole, substances competing for the high-affinity bilirubin site on albumin, and by 8 M urea. Albumin could be separated from human serum by affinity chromatography on this material. Lengthening of the aminoalkylamino “arm” increased the capacity for albumin, but also increased binding of other serum proteins.