Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography.

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component.     

Linoleic acid passage through the blood-brain barrier and a possible effect of age

It has already been established that the blood-brain barrier is readily crossed by unsaturated fatty acids, while saturated fatty acid transport appears to be protein mediated. When the passage of the fatty acids is tested in vivo by using perfusion buffers containing both linoleate and palmitate in different concentrations, linoleate is able to decrease the palmitate passage, while palmitate increases the linoleate passage. These results could be related to the effect of two fatty acids on the ratio between the fatty acids bound to the serum albumin and the free fatty acid pool, which is only available for transport through membranes. However, on the basis of some results obtained with aged rats, the possibility that a relationship may exist between palmitate and linoleate during their passage through the BBB is discussed. Moreover, it seems likely that in aged rats a moderate modification for fatty acids takes place in the BBB.     

Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling

Affinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin. [1-14C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin. Activated [14C]palmitic acid coupled covalently to albumin producing [14C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin. The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin. Albumin carrying 1 eq. of [14C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin. Radioactive peptides were isolated by high pressure liquid chromatography. Three peptides accounted for greater than 90% of the label. Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10, 45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.     

High affinity of alpha-foetoprotein for arachidonate and other fatty acids.

Fatty acid analysis of purified bovine alpha-foetoprotein showed it to contain 2.7 mol of fatty acid/mol of alpha-foetoprotein. Purified alpha-foetoprotein focused at isoelectric point 4.8. Removal of bound ligands from alpha-foetoprotein by charcoal treatment changed its isoelectric point to 5.2. This change could be reversed by addition of exogenous fatty acids to the defatted alpha-foetoprotein. Albumin isolated from the same foetal calf serum source as alpha-foetoprotein contained 1.4 mol of fatty acid/mol of protein. alpha-Foetoprotein and albumin contained comparable amounts of fatty acids with 14 to 18 carbon atoms, but alpha-foetoprotein contained 16 times as much of the long-chain polyunsaturated fatty acids as albumin. alpha-Foetoprotein was found to have slightly higher affinity for palmitate and linoleate and severalfold higher affinity for arachidonate than albumin. These findings suggest that alpha-foetoprotein may play a role in the foetal metabolism of the long-chain polyunsaturated fatty acids.     

Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.     

The Antarctic toothfish (Dissostichus mawsoni) lacks plasma albumin and utilises high density lipoprotein as its major palmitate binding protein

Plasma from the Antarctic toothfish, Dissostichus mawsoni, a member of the advanced teleost Nototheniidae family, was analysed. Agarose gel electrophoresis showed a major diffuse anionic protein that bound [14C]palmitic acid but not 63Ni2+, and two more cationic proteins that bound 63Ni2+ but not palmitate. Oil Red O staining following cellulose acetate electrophoresis indicated that the palmitate binding protein was a lipoprotein. Two-dimensional electrophoresis showed that this palmitate binding band was composed of three proteins with M(r) of 11, 30, and 42 kDa, without any trace of material at approximately 65 kDa, the mass of albumin. N-terminal sequencing of the palmitate binding band gave a major sequence of DAAQPSQELR-, indicating a high degree of homology to apolipoprotein A-I (apo-AI), the major apolipoprotein of high density lipoprotein (HDL). N-terminal sequencing of the major nickel binding band produced a sequence with no homology to albumin. When ultracentrifugation was used to isolate the lipoproteins from Antarctic toothfish plasma, the palmitate binding protein was found solely in the lipoprotein fraction. In competitive binding experiments, added human albumin did not prevent palmitate binding to toothfish HDL. In conclusion, there is no evidence for albumin in Antarctic toothfish plasma and HDL assumes the role of fatty acid transport.     

Distribution of pyridoxal-5-phosphate between proteins and low molecular weight components of plasma: effect of acetaldehyde

It is found that approximately 65-70% of pyridoxal-P at physiological concentrations is bound to plasma proteins; 15% of its amount is bound to amino acids and peptides as a result of the Schiff base formation. Over 85% of pyridoxal-P associated with plasma proteins is bound to serum albumin. Inorganic phosphate and NaCl decrease the affinity of pyridoxal-P for albumin or other proteins. Acetaldehyde interacts with the alpha-amino group of the aspartic acid residue of the N-end of the polypeptide chain of the albumin molecule and with two epsilon-amino groups of the lysine residues having anomalously low value of pKa and deprotonated at physiological values of pH of the medium. Acetaldehyde competes with pyridoxal-P for the first (of the highest affinity) binding site of the coenzyme on serum albumin. Acetaldehyde is not bound at the second site of high affinity for pyridoxal-P on serum albumin.     

Fatty acid uptake and metabolism in a human intestinal cell line (Caco-2): comparison of apical and basolateral incubation

Free fatty acids can enter the enterocyte via the apical or basolateral plasma membrane. We have used the Caco-2 intestinal cell line to examine the polarity of free fatty acid uptake and metabolism in the enterocyte. Differentiated Caco-2 cells form polarized monolayers with tight junctions, and express the small intestine-specific enzymes sucrase and alkaline phosphatase. Cells were grown on permeable polycarbonate Transwell filters, thus allowing separate access to the apical and basolateral compartments. Total uptake of [3H]palmitate bound to bovine serum albumin (palmitate-BSA 4:1) was twofold higher (P less than 0.05 or less) at the apical surface than at the basolateral surface. The relative apical and basolateral membrane surface areas of the Caco-2 cells, as measured by partition of the fluorophore trimethylammonium-diphenylhexatriene TMA-DPH), was found to be 1:3. Thus, apical fatty acid uptake was sixfold higher than basolateral uptake per unit surface area. Analysis of metabolites after incubation with submicellar concentrations of [3H]palmitate showed that the triacylglycerol to phospholipid (TG:PL) ratio was higher for fatty acid added to the apical as compared to the basolateral compartment (20% at 60 min, P less than 0.025). Little fatty acid oxidation was observed. Preincubation with albumin-bound palmitate, alone or with monoolein, increased the incorporation of both apical and basolateral free fatty acids into TG. The results suggest that the net uptake of long-chain free fatty acids across the apical plasma membrane is greater than uptake across the basolateral membrane. In addition, a small increase in the TG:PL ratio for apically, compared to basolaterally, added free fatty acids suggests that polarity of metabolism occurs to a limited extent in Caco-2 enterocytes.     

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.     

Binding of Streptococcal Lipoteichoic Acid to Fatty Acid-Binding Sites on Human Plasma Fibronectin

The ability of Streptococcus pyogenes lipoteichoic acid and palmitic acid to bind to purified human plasma fibronectin was investigated. Initial studies indicated that intact fibronectin formed soluble complexes with lipoteichoic acid, resulting in a change in the mobility of fibronectin in an electrical field. Fibronectin covalently linked to agarose beads bound radiolabeled lipoteichoic acid in the acylated form but not in the deacylated form. An 18-M excess of fibronectin inhibited binding of lipoteichoic acid to the immobilized protein by 92%. Fibronectin-bound [(3)H]lipoteichoic acid could be specifically eluted with unlabeled lipoteichoic acid, as well as by fatty acid-free serum albumin. Serum albumin, which is known to contain fatty acid-binding sites capable of binding to the lipid moieties of lipoteichoic acid, inhibited the binding of lipoteichoic acid to fibronectin in a competitive fashion. The fibronectin-bound lipoteichoic acid could be eluted by 50% ethanol and various detergents but not by 1.0 M NaCl, various amino acids, or sugars. Similarly, radiolabeled palmitic acid adsorbed to fibronectin could be eluted with 50% ethanol but not with 1.0 M NaCl. Fibronectin adsorbed to a column of palmityl-Sepharose was eluted with 50% ethanol in 0.5% sodium dodecyl sulfate but not with 1.0 M NaCl or 1% sodium dodecyl sulfate alone. The binding of lipoteichoic acid to fibronectin followed first-order kinetics and was saturable. A Scatchard plot analysis of the binding data indicated a heterogeneity of lipoteichoic acid-binding sites similar to that previously found for serum albumin. Nevertheless, fibronectin contains at least one population of high-affinity binding sites for lipoteichoic acid. The binding affinity (nKa approximately 250 muM(-1)) is 2 orders of magnitude greater than the binding affinity of serum albumin. These data suggest that human plasma fibronectin contains specific binding sites for fatty acids and that lipoteichoic acid binds to these sites by way of its glycolipid moiety.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine

Alpha-1 acid glycoprotein (AGP, orosomucoid), a major acute phase protein in plasma, displays potent cytoprotective and anti-inflammatory activities whose molecular mechanisms are largely unknown. Because AGP binds various exogenous drugs, we have searched for endogenous ligands for AGP. We found that AGP binds lysophospholipids in a manner discernible from albumin in several ways. First, mass spectrometric analyses showed that AGP isolated from plasma and serum contained lysophosphatidylcholine (LPC) enriched in mono and polysaturated acyl chains, whereas albumin contained mostly saturated LPC. Second, AGP bound LPC in a 1:1 molar ratio and with a higher affinity than free fatty acids, whereas albumin bound LPC in a 3:1 ratio but with a lower affinity than that of free fatty acids. Consequently, free fatty acids displaced LPC more avidly from albumin than from AGP. Competitive ligand displacement indicated the highest affinity for AGP to LPC20:4, 18:3, 18:1, and 16:0 (150-180 nM), lysophosphatidylserine (Kd 190 nM), and platelet activating factor (PAF) (Kd 235 nM). The high affinity of AGP to LPC in equilibrium was verified by stopped-flow kinetics, which implicated slow dissociation after fast initial binding, being consistent with an induced-fit mechanism. AGP also bound pyrene-labeled phospholipids directly from vesicles and more efficiently than albumin. AGP prevented LPC-induced priming and PAF-induced activation of human granulocytes, thus indicating scavenging of the cellular effects of the lipid ligands. The results suggest that AGP complements albumin as a lysophospholipid scavenging protein, particularly in inflammatory conditions when the capacity of albumin to sequester LPC becomes impaired.     

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.     

Determination of the distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein by concanavalin A affinity chromatography

The distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein was measured in vitro by a new method based on the separation of the two proteins by virtue of the binding specificity of concanavalin A for the carbohydrate moiety of alpha-fetoprotein. Human and bovine proteins were investigated. It was found that palmitate and oleate were distributed almost equally between albumin and alpha-fetoprotein, while docosahexaenoate and diethylstilbestrol bound preferentially to alpha-fetoprotein even at an albumin: alpha-fetoprotein ratio of 10:1. The results confirm the binding specificity of alpha-fetoprotein for polyunsaturated fatty acids and also show that alpha-fetoprotein binds diethylstilbestrol much more strongly than albumin does. This suggests that alpha-fetoprotein may play a role in the fetal uptake of diethylstilbestrol.     

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.     

A theoretical analysis of the binding of palmitate by human serum albumin

The distribution of palmitate between the form bound by human serum albumin and the free form in plasma was calculated by use of 12 stepwise equilibrium constants and a computer program. Computations were carried out for molar ratios of palmitate to serum albumin of 0.5, 1,2,3, and 4. At most 0.0003% of the palmitate would be in the unbound form, and the remainder distributed among different complexes with albumin. At low molar ratios, the complexes with 1 to 2 moles of palmitate/albumin would predominate while at the highest ratio the complexes of 3 and 4 moles of palmitate/albumin would be most abundant. In the delivery of palmitate to tissues the relative contribution of the different complexes would change, as the molar ratio of fatty acid to albumin changed.     

Determining molecular binding sites on human serum albumin by displacement of oleic acid

An NMR method was developed for determining binding sites of small molecules on human serum albumin (HSA) by competitive displacement of (13)C-labeled oleic acid. This method is based on the observation that in the crystal structure of HSA complexed with oleic acid, two principal drug-binding sites, Sudlow's sites I (warfarin) and II (ibuprofen), are also occupied by fatty acids. In two-dimensional [(1)H,(13)C]heteronuclear single quantum coherence NMR spectra, seven distinct resonances were observed for the (13)C-methyl-labeled oleic acid as a result of its binding to HSA. Resonances corresponding to the major drug-binding sites were identified through competitive displacement of molecules that bind specifically to each site. Thus, binding of molecules to these sites can be followed by their displacement of oleic acids. Furthermore, the amount of bound ligand at each site can be determined from changes in resonance intensities. For molecules containing fluorine, binding results were further validated by direct observations of the bound ligands using (19)F NMR. Identifying the binding sites for drug molecules on HSA can aid in determining the structure-activity relationship of albumin binding and assist in the design of molecules with altered albumin binding.     

Crystal structure analysis of warfarin binding to human serum albumin: anatomy of drug site I

Human serum albumin (HSA) is an abundant transport protein found in plasma that binds a wide variety of drugs in two primary binding sites (I and II) and can have a significant impact on their pharmacokinetics. We have determined the crystal structures at 2.5 A-resolution of HSA-myristate complexed with the R-(+) and S-(-) enantiomers of warfarin, a widely used anticoagulant that binds to the protein with high affinity. The structures confirm that warfarin binds to drug site I (in subdomain IIA) in the presence of fatty acids and reveal the molecular details of the protein-drug interaction. The two enantiomers of warfarin adopt very similar conformations when bound to the protein and make many of the same specific contacts with amino acid side chains at the binding site, thus accounting for the relative lack of stereospecificity of the HSA-warfarin interaction. The conformation of the warfarin binding pocket is significantly altered upon binding of fatty acids, and this can explain the observed enhancement of warfarin binding to HSA at low levels of fatty acid.