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R Brodersen

Publications and source records attributed to R Brodersen.

At least 37 records · Page 2Linked to original sources
PubMed1988

Multiple binding of bilirubin to human serum albumin and cobinding with laurate.

Numerical analysis of multiple binding of two ligands to one carrier has been accomplished, using the principle of several sets of acceptable binding constants, with bilirubin-laurate-albumin as an example. Binding of bilirubin to defatted human serum albumin was investigated by a spectroscopic method, based upon a difference of light absorption spectrum for free and bound bilirubin. The observations were supplemented with previous data from an independent technique, measurement of oxidation rates of free bilirubin with hydrogen peroxide and peroxidase. A continuous isotherm was obtained, showing binding of at least 4 mol bilirubin per mole albumin with the following stoichiometric binding constants, 1.11 X 10(8), 1.7 X 10(7), 8 X 10(5), and 4 X 10(4) M-1 at pH 8.2, ionic strength 0.15 M, 25 degrees C. The binding is anticooperative at all steps. A saturation level was not reached. Cobinding of bilirubin and laurate was studied, with up to 2 mol of each ligand per mole albumin, using the peroxidase method for determination of free equilibrium concentrations of bilirubin, and a dialysis rate technique for free laurate. The findings could be described in terms of a stoichiometric model. Heterotropic cooperativity was present among the first bilirubin and the first and second laurate molecules. More than two molecules of either ligand can be bound at the same time.

Bilirubin↗
PubMed1988

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 Förster'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.

Bilirubin↗
PubMed1988

Albumin binding of MADDS--a measure of bilirubin binding--in women during pregnancy and after delivery and in their infants.

The purpose of this study was to investigate the binding potential of MADDS (monoacetyldiaminodiphenyl sulphone) to albumin, a measure for binding of unconjugated bilirubin, in healthy women during pregnancy, during and after delivery, and in their infants. The serum concentrations of unconjugated bilirubin, reserve albumin for binding of MADDS and total albumin were measured in: (a) 21 non-pregnant women; (b) 16 pregnant women in the 16th-24th, 28th-32nd, and 36th-38th gestational weeks, and at the time of delivery from both mother and infant; and (c) 15 women at the time of delivery, and 24 and 72 hours after delivery. The bilirubin concentrations did not change during pregnancy or at delivery and were very small compared with the concentrations of reserve albumin for binding of MADDS and total albumin. Therefore, the ratio of reserve albumin to total albumin was an expression of the binding potential of the albumin for MADDS in women. During pregnancy, the reserve albumin decreased equal to total albumin, so that the ratio was not significantly changed (p greater than 0.05). In contrast, at delivery the reserve albumin was significantly lower (p less than 0.01) than in the 36th-38th gestational weeks, without any significant difference in total albumin, i.e. the ratio was significantly lower than during pregnancy (p less than 0.01). During the first 3 days after delivery the reserve albumin increased significantly (p less than 0.05), also without any significant change in total albumin, so that the ratio increased significantly (p less than 0.05) and was normalized. The binding potential for MADDS to albumin in newborn infants was even lower than that of their mothers. Since the binding-potential is reduced in both mother and infant at delivery, a relation is suggested.

Adolescent↗
PubMed1986

Warfarin binding to plasma albumin, measured in patients and related to fatty acid concentrations.

A method for determination of reserve albumin equivalent for binding of warfarin as previously described [1] has been used for assessing the influence of non-esterified fatty acid concentration (NEFA) on binding of warfarin to human serum albumin (HSA). Reserve albumin concentration can be used for calculation of the expected fraction of bound warfarin in serum. It is shown in vitro that binding of warfarin increases with added oleate up to 4 mol of oleate per mol of albumin and then decreases. Twenty-four patients on permanent warfarin treatment showed no correlation of serum albumin and reserve albumin concentrations (r = 0.10, P greater than 0.50) indicating that warfarin binding is governed by other factors. However, in the same patients there was a significant correlation between reserve albumin concentration and NEFA/HSA (r = 0.54, P less than 0.01). In one human volunteer changes of NEFA were provoked by strenuous work and it was found that reserve albumin concentration increased with NEFA concentration as expected from the in vitro findings (r = 0.90, P less than 0.001). Five uraemic patients on permanent warfarin treatment showed increasing reserve albumin concentration with increasing NEFA concentration induced by heparin. These findings indicate that, both in vitro and in vivo, the reserve albumin concentration for binding of warfarin and hence the free warfarin concentration is markedly influenced by NEFA concentration. This may add to the understanding of warfarin dose requirement during anticoagulant therapy.

Adult↗
PubMed1979

Bilirubin. Solubility and interaction with albumin and phospholipid.

Bilirubin is generally considered a lipophilic substance, and its neurotoxicity is ascribed to an affinity for lipids in the central nervous system. In the present paper, it is shown that the solubility of bilirubin in apolar solvents and in triglycerides is low and increases with solvent polarity. Consequently, bilirubin should not be characterized as lipophilic. The solubility in aqueous buffers was studied under exclusion of light and was found lower than previously reported, about 7 nM at pH 7.4, temperature 37 degrees C, increasing with higher pH, approximately in inverse proportion with the squared hydrogen ion concentration. Binding of bilirubin to human serum albumin was studied by the rate of oxidation with peroxidase of the free ligand at equilibrium. The stoichiometry of proton involvement in the binding process was investigated by acidimetric titration. It is concluded that precipitation of bilirubin in vivo is thermodynamically possible. It was further demonstrated by light absorption spectroscopy that bilirubin forms a complex with phosphatidylcholine in diethyl ether and that an aqueous suspension of phosphatidylcholine enhanced aggregation of bilirubin. Transfer of bilirubin dianion from its complex with plasma albumin and precipitation of bilirubin acid with phosphatidylcholine in membranes of nerve cells is therefore possible and may account for the neurotoxicity.

Bilirubin↗
PubMed1979

Kinetics of bilirubin oxidation with peroxidase, as applied to studies of bilirubin-albumin binding.

In the determination of unbound bilirubin by rate of oxidation with peroxidase, errors may be caused by (1) phenol, propylparaben, and phenothiazines (free radical acceleration), (2) haemoglobin (peroxidase effect), and (3) ascorbate (inhibition). Such errors may be diminished by dilution 1:40, or with an anti-oxidant, tert-butyl-p-hydroxyanisole, and ascorbate oxidase.

Ascorbate Oxidase↗
PubMed1979

Bilirubin acidity. Titrimetric and 13C NMR studies.

Acidimetric titration of bilirubin IX-alpha, dissolved in excess aqueous sodium hydroxide, showed that two protons are dissociated with pK values well below 7 and that one or several additional acidic groups titrate with pK around 12.9. Precipitation of the nearly insoluble acid precluded determination of the two lower pK values by titration in aqueous solution. In dimethyl sulfoxide solution, four acidic protons were demonstrated, titrating two by two without precipitation. 13C NMR spectra of bilirubin IX-alpha were recorded and complete assignments were made by comparison with the spectra of bilirubin XIII-alpha and mesobilirubin etc. Such spectra, recorded after addition of 2 and 4 mol of base per mol of bilirubin IX-alpha, showed that both carboxyl groups are titrated by the first 2 mol of base, and both lactams by the following 2 mol of base. Cotitrations of bilirubin IX-alpha with other acids, o- and m-hydroxybenzoic acid and 2-pyridone, were used to determine relative pK values in dimethyl sulfoxide solution, and pK values for the four acidic protons of bilirubin IX-alpha in aqueous solution were calculated from the Born equation. Both carboxyl groups exhibited pK = 4.4, and both lactams pK = 13.0, in good agreement with values expected from the chemical structure of the bilirubin molecule. The implications of these findings for understanding the mechanism of bilirubin neurotoxicity are discussed.

Bilirubin↗
PubMed1978

Determination of the vacant amount of high-affinity bilirubin binding site on serum albumin.

A principle for determination of the vacant amount of the high-affinity bilirubin binding site on serum albumin in the newborn is proposed: A small amount of monoacetyl-4,4'-diaminodiphenylsulphone (MADDS) is added to the serum sample and the equilibrium of binding of this substance is assessed by measuring the rate of dialysis into another volume of the same serum. It is shown that MADDS is bound selectively to the specific bilirubin site. Results obtained after addition of varying amounts of five drugs and of oleate are compared with those found by the peroxidase method. Good agreement is found although certain deviations are encountered, as expected in the case of allosteric affects. Free bilirubin concentrations can be calculated from the results.

Bilirubin↗
PubMed1977

Binding of bilirubin and long-chain fatty acids to human serum albumin with general remarks on displacement of firmly bound ligands.

Binding of bilirubin to human serum albumin was studied by estimation of the free bilirubin concentration from the rate of oxidation with hydrogen peroxide and peroxidase, and by spectrophotometry: nI = 1, kI = 7 x 10(7) l/mol; nII = 1, kII = 5 x 10)5) l/mol, at pH 7.4, 37 degrees C, ionic strength 0.1. Palmitate or oleate in excess of 4 mol per mol albumin, influences the high-affinity binding of bilirubin as described by an empirical equation. Theoretical consideration of competitive displacement of a biologically active substance, firmly bound in an inactive state to a macromolecular carrier, demonstrates that significant displacement may occur on addition of another ligand with a lower binding constant. Displacement of bilirubin from its high-infinity site by fatty acids and drugs is thermodynamically feasible and probably clinically important.

Bilirubin↗
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↗
PubMed1977

Bilirubin displacing effect of stabilizers added to injectable preparations of human serum albumin.

Stabilizers added to preparations of human serum albumin before heat treatment were tested for bilirubin displacing effect, using the peroxidase method. It was found that N-acetyltryptophan and sodium caprylate displace bilirubin from its complex with human serum albumin in vitro. The quantitative findings were used for a rough estimate of the effect of these substances on the free bilirubin concentration in blood plasma, expected when stabilized albumin preparations are given intravenously for prevention of kernicterus. The calculated effect is a delay of the decrease of free bilirubin concentration, or even a temporary increase. Sodium mandelate displaces less strongly.

Bilirubin↗
PubMed1977

Prevention of kernicterus, based on recent progress in bilirubin chemistry.

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.

Albumins↗
PubMedDate not supplied

Fatty acid-binding protein from human heart localized in native and denaturing two-dimensional gels.

A group of low molecular weight fatty acid-binding cytosolic proteins, FABPc, with high abundance in heart, liver, skeletal muscle, intestine and adipose tissue, are anticipated to play a role in long-chain fatty acid metabolism in these tissues. Recently, a FABPc with Mr 15 kDa has been purified from human heart muscle and found to be present in levels 2-4% of cytosolic proteins of human heart myocytes. In the present study two-dimensional gel electrophoresis under native and denaturing conditions has been used to characterize FABPc from human heart and this protein is found to be a major protein of human heart myocytes. The pI of FABPc from human heart was found to be about 5.3 under native conditions and about 6.5 in the presence of 9 M urea.

Carrier Proteins↗