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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.Binding equilibrium studies for long-chain fatty acids to serum albumin are complicated by very low ligand solubility. We have in previous work failed to demonstrate any solubility of palmitic, stearic and oleic acid at neutral pH and 37°C [l]. It is, in consequence, not possible to describe binding of these acids in terms of equilibria between free and bound ligand, as in usual binding studies. On the other hand, equilibrium with respect to transfer of an insoluble ligand from one carrier to another can be assessed quantitatively, utilizing the reserve carrier concept. The concentration of reserve albumin for binding of a ligand has previously been defined as the concentration of a purified standard albumin preparation which in buffered solution binds a trace amount of the radiolabeled ligand as tightly as it is bound in the sample [2]. The concentration of reserve albumin for binding of palmitate or stearate is thus an inverse measure of how tight the fatty acids are bound and this parameter can be measured in albumin solutions and in serum or plasma even if the fatty acids are insoluble.The aim of the present work is to investigate binding equilibria of long-chain fatty acids in human blood plasma (serum) under varying conditions and to compare the results with previous observations on binding to serum albumin in buffered solutions. MATERIALS AND METHODS Human serum albuminHuman serum albumin was obtained from AB Kabi Vitrum (Stockholm, Sweden; lot no. RFM 57). More than 95% of the total protein of this preparation is serum albumin. Fatty acidsPalmitic, oleic, linoleic and myristic acids were obtained from Fluka AG, Buchs, Switzerland. The purity of all four acids was determined as >99% by gas-liquid chromatography.[l-'4C]Palmitic acid (specific activity 58 Ci/mol) was from Amersham Intern...

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.

Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5-37'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 < 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.Serum albumin, being synthesized in the liver, is the most abundant protein in the extracellular fluid. The concentration in human plasma is about 0.6 mM, whereas in the interstitial fluids it varies in the different tissues between 0.2 -0.5 mM [l].Albumin is known to bind a plethora of different compounds including inorganic ions and a multitude of metabolites, hormones, and drugs [2]. These very flexible binding properties constitute the transport function of albumin.Albumin binding of fatty acids which have a low water solubility is important for the energy-metabolizing muscles. The fatty acids are released into the plasma as a result of mobilization of fat from adipose tissue during fasting, stress or exercise, and in plasma the concentration of fatty acids can reach 2.5 mM, i.e. four times the concentration of albumin Estimation of biological responses to varying concentrations of fatty acids therefore necessitates evaluation of multiple equilibria between fatty acids and albumin. Results, as well as difficulties, from this type of investigation have recently been reviewed by Spector [3, 41. We have introduced a dialysis method allowing determination of the concentration of unbound fatty acid anion by measuring the rate of exchange of labelled fatty acid across a dialysis membrane under conditions of equilibrium [5, 61. With this method we have obtained solubility data for different fatty acids and established binding isotherms for multiple equilibria between albumin and lauric acid [6] as well as myristic acid [7].In the present paper we extend this investigation by studying the effect of temperature on the binding curves in an attempt to estimate changes of enthalpy and entropy on binding of the anions of lauric and myristic acid to human serum albumin.131.

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 0 2 , 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 0 2 . 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.