Anders Overgaard Pedersen

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.

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.

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.

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

SummaryA 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 MT 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.

Fatty acid binding to serum albumin in type I insulin-dependent diabetes mellitus.

SummaryEquilibria of binding of long-chain fatty acids to albumin in sera from type I diabetic patients and healthy adults were studied by dialysis exchange rate determinations and described by,p*, thereserve albumin concentration for binding of fatty acid, C*/p*, the,total availability of fatty acids, whereC* is the total concentration of non-esterified fatty acid, andL*, thefatty acid binding property of albumin, which isL*=p*/P+0.05C*/P, whereP is the albumin concentration. Studies in samples from 81 diabetic patients and 99 healthy adults showed that availability of fatty acids increased with increasing fatty acid concentrations, equally in the two groups. Some diabetics had higher fatty acid concentrations, and thus higher fatty acid availabilities, than the normals. It is shown that the fatty acid binding property of serum albumin is individually variable, ranging about the same mean value in normal and diabetic persons but with a larger variation in the latter. The fatty acid binding property of albumin in serum,L*, and sixteen clinical parameters were measured in 42 of the 81 diabetic patients. Regression analysis indicated thatL* was correlated, to serum cholesterol concentration (probability of 0-hypothesis, p=0.01) and to serum triglyceride concentration (p=0.05). Values ofL* were slightly correlated to age, age on diagnosis, duration, body Mass Index (BMI), diastolic blood pressure, albumin excretion rate, serum creatinine concentration, and serum non-esterified fatty acid concentration with p-values varying from 0.10 to 0.50. For sex, retinopathy, hemoglobin A1c, systolic blood pressure, daily insulin dose, and blood glucose concentration no correlation, toL* was found, p-values ranging from 0.56 to 0.96 Non-enzymatic glycosylation of serum albumin did not decrease binding affinity for fatty acidin vitro.

Structural analysis, fatty acid and thyroxine binding properties of Vancouver and Naskapi variants of human serum albumin.

OBJECTIVES To purify and structurally identify two albumin variants found in the Canadian population of native Amerindian origin. To assess the ability of variant albumins to bind lauric acid and L-thyroxine. METHODS The structural characterization of the alloalbumins was performed by conventional protein chemistry methods and by mass spectrometric analysis. Lauric acid and L-thyroxine affinities to variant albumins were assessed by kinetic dialysis and equilibrium dialysis techniques, respectively. RESULTS The sequence investigations proved the two variants to be albumin Naskapi [372Lys --> Glu] and albumin Vancouver [501Glu --> Lys], respectively. Among the carriers of albumin Naskapi, we found a rare case of homozygosity. Furthermore, this is the first reported case of the 501Glu-->Lys mutation in the native North American population. Scatchard plot analysis revealed that the association constants for lauric acid and L-thyroxine to the two variants were indistinguishable from the endogenous form of albumin. CONCLUSION We show that albumin variants Vancouver and Naskapi have normal fatty acid and L-thyroxine binding capabilities. These findings support the assumption that bisalbuminemias associated with these albumin variants are benign conditions.