Heat denaturation of human serum albumin. Migration of bound fatty acids.

Prolonged heat treatment of solutions of human serum albumin at 60 degrees C resulted in formation of one aggregate fraction and one fraction that was stable against further heat treatment. Fatty acid analyses of these fractions indicate that the heat stable fraction is formed by migration of fatty acids from the aggregating molecules to the remaining monomer thereby stabilizing the latter against heat denaturation. Disulphide and SH groups are involved in the aggregation process.     

Long-chain fatty acids bound to alpha-fetoprotein and to serum albumin from fetal and adult pig

1. Pig alpha-fetoprotein (AFP) and albumin were isolated from fetal serum by DEAE-Sephadex ion exchange chromatography combined with Cibacron Blue-Sepharose and trypsin-Sepharose adsorptions. 2. AFP, fetal albumin and adult albumin carried 2.6, 2.4, and 1.9 moles of fatty acids per mole of protein, respectively. 3. Most of fatty acids bound to AFP were polyunsaturated: mainly arachidonic (20:4, n-6) and docosahexaenoic (22:6, n-3) acids, which accounted respectively for 21.7 and 18.8% of the total fatty acids. 4. By contrast, the fatty acids found in the albumins (fetal and adult) were preferentially saturated and monounsaturated. 5. Arachidonic acid was a minor component in both albumins, and no docosahexaenoic acid was detected.     

Using bound fatty acids to disclose the functional structure of serum albumin

Background

Serum albumin is a major transport protein in mammals and is known to have at least seven binding sites for long-chain fatty acids (FAs).

Scope of review

We have devised a new electron paramagnetic resonance (EPR) spectroscopic approach to gain information on the functional structure of serum albumin in solution in a “coarse-grained” manner from the ligands' point of view. Our approach is based on using spin labeled (paramagnetic) stearic acids self-assembled with albumin and subsequent nanoscale distance measurements between the FAs using double electron–electron resonance spectroscopy (DEER). Simple continuous wave (CW) EPR spectroscopy, which allows for quantification of bound ligands, complements our studies.

Major conclusions

Based on DEER nanoscale distance measurements, the functional solution structure of human serum albumin (HSA) has remarkably been found to have a much more symmetric distribution of entry points to the FA binding sites than expected from the crystal structure, indicating increased surface flexibility and plasticity for HSA in solution.In contrast, for bovine serum albumin (BSA), the entry point topology is in good agreement with that expected from the crystal structure of HSA. Changes in the solution structures between albumins can hence be revealed and extended to more albumins to detect functional differences at the nanoscale.Going beyond fundamental structural studies, our research platform is also excellently suited for general studies of protein–solvent interactions, temperature effects and ligand binding.

General significance

We discuss how our research platform helps illuminate protein dynamics and function and can be used to characterize albumin-based hybrid materials. This article is part of a Special Issue entitled Serum Albumin.     

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.     

Fatty acids bound to serum albumin potentiate platelet prostaglandin biosynthesis.

Utilization of arachidonic acid by human platelets is increased when the fatty acid content of serum albumin is increased as well. Platelet aggregation induced by arachidonic acid and by low concentrations of thrombin is thus potentiated, suggesting that platelet responsiveness to aggregating agents is influenced by the plasma content in free fatty acids.     

Removal of fatty acids from serum albumin by Lipidex 1000 chromatography

Fatty acids can be effectively removed from serum albumin preparations by a single passage through a column of Lipidex 1000 at 37 degrees C. The procedure is easier and milder and shows a better (nearly quantitative) recovery of protein than charcoal treatment. The ability for fatty acid binding by the protein is not affected by either procedure.     

Binding of 3-carbethoxipsoralen to human serum albumin and human serum: influence of free fatty acids

Characteristics of the binding of 3-carbethoxipsoralen (3CPS) to human serum albumin (HSA) and serum proteins have been studied. An electrophoretic study showed that the predominant binding protein fraction was albumin, with small binding to globulins. Binding to HSA, studied by equilibrium dialysis, is 75% and characterized by a small saturable number of binding sites (N = 0.27) with a moderate affinity constant (K = 8 X 10(4) M-1). Free fatty acids were shown to decrease 3CPS binding to HSA by a non competitive process.     

Binding of long-chain fatty acids to bovine serum albumin

We have studied the binding of long-chain free fatty acids (FFA) to crystalline bovine serum albumin (BSA) that had been extracted with charcoal to remove endogenous fatty acids. The data were analyzed in terms of a model consisting of six high-energy binding sites and a large number of weak binding sites. The high-energy sites were resolved into two distinct classes, each containing three sites. At 37 degrees C and pH 7.4, k'(1) (the apparent association constant of a class of binding sites) was about 10(6) m(-1) for binding to the three primary sites, and k'(2) was about 10(5) m(-1) for binding to the three secondary sites. The number of weak (tertiary) sites was estimated to be 63 with a k'(3) of 10(3) m(-1). In general, palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, or myristate, and much more tightly than laurate. The association of palmitate with human and rabbit albumin also was analyzed in terms of this model. Palmitate was bound less firmly by human or rabbit albumin than by BSA. Palmitate binding to BSA was dependent upon the pH and temperature of the incubation medium. Long-chain hydrocarbons that did not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexadecane) were bound to a limited extent and weakly. The presence of positively charged protein sites and native protein tertiary structure were required for maximal binding of palmitate to BSA. Of nine other proteins tested, only -lactoglobulin exhibited a significant capacity to bind palmitate.     

Crystal structures of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids.

The primary ligands of human serum albumin (HSA), an abundant plasma protein, are non-esterified fatty acids. In vivo, the majority of fatty acids associated with the protein are unsaturated. We present here the first high-resolution crystal structures of HSA complexed with two important unsaturated fatty acids, the monounsaturated oleic acid (C18:1) and the polyunsaturated arachidonic acid (C20:4). Both compounds are observed to occupy the seven binding sites distributed across the protein that are also bound by medium and long-chain saturated fatty acids. Although C18:1 fatty acid binds each site on HSA in a conformation almost identical with that of the corresponding saturated compound (C18:0), the presence of multiple cis double bonds in C20:4 induces distinct binding configurations at some sites. The observed restriction on binding configurations plausibly accounts for differences in the pattern of binding affinities for the primary sites between polyunsaturated fatty acids and their saturated or monounsaturated counterparts.     

Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma.

The dye Cibacron Blue F-3-GA was conjugated to Sepharose to provide an affinity column for serum albumin. Passage of whole human plasma through a column of Cibacron Blue-Sepharose results in the removal of approx. 98% of the albumin. The latter can be quantitatively recovered by desorption with NaSCN. Albumin-depleted plasma can be readily resolved into discrete fractions by a combination of conventional biochemical techniques. In particular, the resolution of plasma proteins with properties similar to those of native human plasma albumin can readily be accomplished by ion-exchange chromatography of the Sepharose-dye-treated plasma on DEAE-cellulose.     

Steroid-protein interactions. 40. The effect of fatty acids on progesterone binding to human serum albumin

Human serum albumin was delipidated by solvent extraction or by treatment with charcoal. Progesterone complexes formed with these albumin preparations had higher association constants than those formed with the untreated samples. The charcoal method of delipidation resulted in somewhat higher affinity constants than extraction with chloroform/methanol. Addition of 5 mol lauric acid per mol albumin reduced the association constant of the progesterone complex by approx. 50%. Studies with lauric, myristic, and palmitic acid showed that the decrease of binding affinity for progesterone was proportional to the amount of fatty acid added to albumin, and to its chain length. These results confirm and extend our previous findings of inhibition of progesterone binding to human albumin by long-chain fatty acids.     

Effects of aliphatic fatty acids on the binding of Phenol Red to human serum albumin.

Binding of Phenol Red to human serum albumin at pH 7.0 was studied by ultrafiltration (n1 = 1, K1 = 3.9 X 1-(4) M-1, n2 = 5, K2 = 9.6 X 10(2) M-1). The presence of 1 mol of octanoate or decanoate per mol of albumin caused a decrease in dye binding (dye/protein molar ratio 1:1), which, in contrast with additional fatty acid, was very pronounced: 1-8 mol of palmitate or stearate resulted in a small, and apparently linear, displacement of Phenol Red. The displacement effect of 1-5 mol of oleate, linoleate or linolenate per mol of albumin was comparable with that of the equimolar concentrations of palmitate or stearate. A higher molar ratios the unsaturated acids caused a drastic decrease in dye binding. The different Phenol Red-displacement effects of low molar ratios of medium-chain and long-chain fatty acids indicate that these acids have different high-affinity binding sites. In accordance with this proposal, low concentrations of stearate had only a small effect on the Phenol Red-displacement effect of octanoate. Phenol Red-binding curves in the presence of 1 mol of octanoate, 8 mol of stearate and 6 or 7 mol of linolenate per mol of albumin respectively indicated that the dye and the fatty acids do not complete for a common primary binding site. In contrast, a secondary Phenol Red-binding site could be identical with the primary octanoate-binding site. Furthermore, the primary Phenol Red-binding site could be the same as a secondary linolenate-binding site. Assignment of the different primary binding sites for Phenol Red and for medium-chain and long-chain fatty acids to a model of the secondary structure of albumin is attempted.     

Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin. II. Electrostatic interactions in individual fatty acid binding sites

13C NMR chemical shift results as a function of pH for a series of carboxyl 13C-enriched saturated fatty acids (8-18 carbons) bound to bovine serum albumin (BSA) are presented. For octanoic acid bound to BSA (6:1, mol/mol), the chemical shift of the only FA carboxyl resonance (designated as peak c), plotted as a function of pH, exhibited a complete sigmoidal titration curve that deviated in shape from a corresponding theoretical Henderson-Hasselbach curve. However, FA carboxyl chemical shift plotted as a function of added HCl yielded a linear titration curve analogous to those obtained for protein-free monomeric fatty acid (FA) in water. The apparent pK of BSA-bound octanoic acid was 4.3 +/- 0.2. However, the intrinsic pK (corrected for electrostatic effects resulting from the net positive charge on BSA) was approximately 4.8, a value identical to that obtained for monomeric octanoic acid in water in the absence of protein. For long-chain FA (greater than or equal to 12 carbons) bound to BSA (6:1, mol/mol), chemical shift titration curves for peak c were similar to those obtained for octanoic acid/BSA. However, the four additional FA carboxyl resonances observed (designated as peaks a, b, b', and d) exhibited no change in chemical shift between pH 8 and 3. For C14.0 X BSA complexes (3:1 and 6:1, mol/mol) peaks b' and a exhibited chemical shift changes between pH 8.8 and 11.5 concomitant with chemical shift changes in the epsilon-carbon (lysine) resonance. In contrast, peaks c and d exhibited no change and peak b only a slight change in chemical shift over the same pH range. We conclude: the carboxyl groups of bound FA represented by peaks a, b, b', and d were involved in ion pair electrostatic interactions with positively charged amino acyl residues on BSA; the carboxyl groups of bound FA represented by peak c were not involved in electrostatic interactions with BSA; the similarity of the titration curves of peak c for BSA-bound octanoic acid and long-chain FA suggested that short-chain and long-chain FA represented by peak c were bound to the same binding site(s) on BSA; bound FA represented by peaks b' and a (but not d or b) were directly adjacent to BSA lysine residues. We present a model which correlates NMR peaks b, b', and d with the putative locations of three individual high-affinity binding sites in a three-dimensional model of BSA.     

Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin. I. The filling of individual fatty acid binding sites

13C NMR chemical shift and intensity results for a series of carboxyl 13C-enriched saturated fatty acids (8-18 carbons) bound to bovine serum albumin (BSA) are presented as a function of increasing fatty acid (FA)/BSA mole ratio. Spectra for long-chain (greater than or equal to 12 carbons) FA X BSA complexes exhibited up to five FA carboxyl resonances, designated a, b, b', c, and d. Only three resonances (peaks b, b', and d) were observed below 3:1 FA X BSA mole ratio, and at greater than or equal to 3:1 mole ratio, two additional resonances were observed (peaks c and a). In a spectrum of 5:1 stearic acid X BSA complexes, peaks b, b', and d each represented approximately one-fifth, and peak c approximately two-fifths, of the total FA carboxyl intensity. Plots of total carboxyl/carbonyl intensity ratio as a function of FA X BSA mole ratio were linear up to 7-9 mole ratio. Deviation from linearity at mole ratios greater than or equal to 7 was accompanied by the detection of crystalline unbound FA (as 1:1 acid/soap) by X-ray diffraction. In contrast to long-chain FA X BSA complexes, 13C NMR spectra of octanoic acid X BSA complexes yielded only one FA carboxyl resonance (peak c) at FA X BSA mole ratios between 1 and 20. We conclude: peaks b, b', and d represent FA bound to three individual high affinity (primary) long-chain FA binding sites on BSA; peak c represents FA bound to several secondary long-chain (or primary short-chain) FA binding sites on BSA; peak a represents long-chain FA bound to an additional lower affinity binding site. We present a model that correlates the observed 13C NMR resonances with individual binding site locations predicted by a recent three-dimensional model of BSA.     

The displacement of albumin bound bilirubin by free fatty acids in vivo

Variations in bilirubinaemia in response to alterations of the free fatty acid level was studied in conscious and unstressed Gunn rats. When only one molecule of bilirubin is bound to albumin (without bilirubin overload), no displacement of bilirubin is observed, even if the protein binds as much as 6 molecules of free fatty acids. After overloading with exogenous unconjugated bilirubin, the second site of fixation of bilirubin on albumin is partly occupied; in this situation, a displacement is observed, but only when more than 3.5 molecules of free fatty acids are simultaneously bound to the protein. In vivo, free fatty acids do not spontaneously reach such levels as those responsable for the observed displacement of bilirubin. In the ranges of bilirubin and free fatty acids concentrations likely to be encountered clinically, free fatty acids might not represent an effective agent of displacement for bilirubin, as it is commonly thought.     

Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.     

Interaction of human serum albumin with fatty acids. Role of anionic group studied by affinity partition.

The interaction of human serum albumin with fatty acids has been determined using the method of affinity partitioning in aqueous biphasic systems containing dextran, poly(ethylene glycol) and esters of dicarboxylic acids with poly(ethylene glycol). The difference in the partition of albumin in phase systems with and without the poly(ethylene glycol)-bound fatty acid group provides a measure of the interaction of fatty acids with the protein. The relative contribution of the polar and non-polar interaction to the binding of fatty acids to albumin has been estimated by comparing the present data with that obtained earlier using poly(ethylene glycol)-bound straight chain aliphatic hydrocarbons. In both cases, the aliphatic chain should contain a minimum of 8 carbon atoms to affect the partition of albumin and that the maximum effect is obtained with chains containing 16 carbon atoms. The effect of the polymer-bound fatty acid group is higher than the corresponding hydrocarbon only when the number of carbon atoms in it exceeds 12. The relative effect of polymer-bound 16-carbon chains, with and without a carboxyl group in the terminal position is independent of pH in the range 5--9.     

Quantitative comparison of lipoprotein fractions derived from human plasma and serum by liquid chromatography-tandem mass spectrometry

Background  

Lipoproteins are complex, globular molecules which play essential roles in the transport and metabolism of cholesterol. Their implication in the development of cardiovascular diseases, such as atherosclerosis, has sustained a great deal of interest in these particles. Their various functions, and their contribution to the development of atherosclerosis, are often attributed to their protein constituents, which vary greatly among the different lipoprotein classes. Recent advances in the field of mass spectrometry have provided insight into the array of proteins associated with low-density lipoproteins (LDLs) and, even more so, with high-density lipoproteins (HDLs). Plasma and serum are the most commonly used samples for the analysis of lipoproteins. Although these lipoprotein sources are unique, it was our goal to determine whether or not their inherent differences would ultimately affect a quantitative analysis of the LDL and HDL proteomes. To this end, we isolated LDL and HDL fractions with fast protein liquid chromatography-size exclusion chromatography (FPLC-SEC) from both human plasma and serum samples from the same individuals. After delipidating these samples, we performed a quantitative proteomic analysis to compare the lipoprotein-associated proteins derived from both plasma and serum.