Dielectric behavior and atomic structure of serum albumin

After 1946, serum albumin was available for studies. Its residue sequence and internal disulfide bonding was developed by 1976. We began to make dielectric dispersion studies and apply Perrin's equations for rotational relaxation times around the two axes of revolution in 1938. These data indicated that albumin should have an elongated shape. In 1992 atomic structure data indicated the molecule was heart-shaped. A similar 1998 study of albumin complexed with fatty acid showed that the molecule was substantially rearranged. We found that the dielectric constant of albumin solutions was sensitive to fatty acid content, making this property an attractive probe in stop–flow kinetic studies. Such studies show that the fatty acid reaction is a two-step process. The fatty acid first binds to exterior sites in a diffusion-limited second order reaction complete in 1 ms. Then a first order rearrangement reaction with 400 ms half-life follows. Thus the highly specialized serum albumin sequence of amino acid residues determines not only the structure of the unligated molecule, but also the distinctive structures of the numerous multiligated molecules.     

Soluble macromolecular complexes involving bacterial teichoic acids.

Cell wall and membrane teichoic acids from several bacteria formed soluble complexes with polysaccharides and bovine plasma in alkyl alcohol solutions. Polysaccharides which contain different monomeric units and anomeric configurations complexed with the teichoic acids, suggesting that the interaction is relatively nonspecific. Teichoic acids complexed glycogen or bovine plasma albumin in 50 to 97% ethanol solutions. The macromolecular association between teichoic acids and polysaccharides or proteins was independent of teichoic acid size over a threefold molecular weight range. Glycerol phosphates or an acid hydrolysate of teichoic acid would not complex to either glycogen or bovine plasma albumin in ethanol. The optimal interaction between glycogen and the Bacillus subtilis lipoteichoic acid occurred between pH 4.5 and 8.2. The ability of teichoic acids to bind polysaccharides and proteins in moderate dielectric constant solvents suggests that these polymers may serve as complexing agents for hydrophilic molecules found in membranes.     

Influence of pH of the medium on free fatty acid utilization by isolated mammalian cells

Studies with Ehrlich ascites tumor cells showed that small decreases in the pH of the incubation medium from 7.4 increase the magnitude of incorporation of free fatty acid (FFA) into the cells from an albumin solution. A similar effect occurred when rabbit erythrocytes, rat heart slices, or rat liver slices were incubated with FFA-bovine albumin solutions and when tumor cells were incubated with FFA in media containing human albumin, -lactoglobulin, or rat plasma. The effect was not seen when the medium contained no protein. When the pH of the albumin-containing medium was lowered from 7.4 to 6.6, oxidation of FFA to CO(2) by the tumor cells increased, esterification of the FFA (mostly into phospholipids and triglycerides) increased, and less esterified radio-active fatty acid was depleted from the cells. Hence, more fatty acid accumulated in the cells in more acid media. These findings suggest that small changes in extracellular pH might regulate FFA utilization and lipid accumulation in mammalian tissues.     

Electric field strength of membrane lipids from vertebrate species: membrane lipid composition and Na+-K+-ATPase molecular activity

Intramembrane electric field strength is a very likely determinant of the activity of ion-transporting membrane proteins in living cells. In the absence of any transmembrane electrical potential or surface potential, its magnitude is determined by the dipole potential of the membrane's lipid components and their associated water of hydration. Here we have used a fluorometric method to quantify the dipole potential of vesicles formed from lipids extracted from kidney and brain of 11 different animal species from four different vertebrate classes. The dipole potential was compared with the fatty acid composition and with the Na(+)-K(+)-ATPase molecular activity of each preparation. The magnitude of the dipole potential was found to be relatively constant across all animal species, i.e., 236-334 mV for vesicles prepared from the total membrane lipids and 223-256 mV for phospholipids alone. The significantly lower value for phospholipids alone is potentially related to the removal of cholesterol and/or other common soluble lipid molecules from the membrane. Surprisingly, no significant dependence of the dipole potential on fatty acid composition was found. This may, however, be due to concomitant compensatory variations in lipid head group composition. The molecular activity of the Na(+)-K(+)-ATPase was found to increase with increasing dipole potential. The fact that the dipole potential is maintained at a relatively constant value over a wide range of animal species suggests that it may play a fundamental role in ensuring correct ion pump conformation and function within the membrane.     

Quantitative analysis of uptake of free fatty acid by mammalian cells: lauric acid and human erythrocytes

Quantitative aspects of the binding of free fatty acid to human erythrocytes were studied by measuring the distribution of various amounts of [1-(14)C]lauric acid between washed human erythrocytes and defatted human plasma albumin. Incubations were done at 37 degrees C in an isotonic phosphate-buffered salt solution. Laurate uptake approached a steady state value within 1 hr of incubation over the range of laurate-albumin molar ratios that were tested. Uptake was due primarily to a transfer of laurate from albumin to the cell, not to incorporation of the intact laurate-albumin complex. The fatty acid binding sites of the erythrocyte are located predominantly on or within the cell membrane. The binding model which best fitted the laurate uptake data consisted of two classes of erythrocyte binding sites. This model contains a small number of sites, 2.0 x 10(-13) moles/10(6) cells, that have an average apparent association constant of 1.8 x 10(6) m(-1) for laurate. Thus, the average strength of these sites is of the same order of magnitude as the stronger laurate binding sites of albumin. The binding model also contains a relatively large number of weaker fatty acid binding sites, 1.3 x 10(-11) moles/10(6) cells, that have an average apparent association constant of 1.3 x 10(4) m(-1) for laurate. These sites are too weak to bind appreciable amounts of laurate unless the fatty acid-albumin molar ratio is elevated.     

Palmitic acid uptake by the rat soleus muscle in vitro.

Abstract: The rate of fatty acid uptake, oxidation, and deposition in skeletal muscles in relation to total and unbound to albumin fatty acids concentration in the medium were investigated in the incubated rat soleus muscle. An immunohistochemical technique was applied to demonstrate whether the albumin-bound fatty acid complex from the medium penetrates well within all areas of the muscle strips. It was found that the percentage of incorporation of palmitic acid into intramuscular lipids was fairly constant, independently of the fatty acid concentration in the medium, and amounted to 63-72% for triacylglycerols, 7-12% for diacylglycerols-monoacylglycerols, and 19-26% for phospholipids. Both palmitic acid incorporation into the muscle triacylglycerol stores and its oxidation to CO2 closely correlated with an increase in both total and unbound to albumin fatty acid concentrations in the incubation medium. Under conditions of increased total but constant unbound to albumin palmitic acid concentrations, the incorporation of palmitic acid into triacylglycerols and its oxidation to CO2 were also increased, but to a lower extent. This supports the hypothesis that the cellular fatty acid metabolism depends not only on the availability of fatty acids unbound to albumin, but also on the availability of fatty acids complexed to albumin.     

A model for the calculation of the dielectric dispersion and the dipole moment of globular proteins in solution

We describe a new procedure whereby the magnitude of the dielectric dispersion of a solution of globular protein molecules can be calculated. The protein molecule is considered to have spherical symmetry and the charged residues are thought to be situated in a medium whose dielectric constant increases continuously as a function of the distance from the centre of mass. The dipole moment of the protein in the solution is made up of two parts: the intrinsic dipole moment due to the charge distribution of the protein and the dipole moment due to polarization of the medium and the ionic cloud. When the model is applied to solutions of cytochrome c it is found that polarization of the medium results in a decrease in the dielectric dispersion amplitude. The mean square dipole moment calculated with the help of this method indicates that the fluctuation of the configurations cannot be responsible for the large dispersion in the megahertz region.     

The binding of L-tryptophan to serum albumins in the presence of non-esterified fatty acids.

Bovine, human and rat serum albumins were defatted and palmitic acid, oleic acid and lauric acid added in various molar ratios. The binding of L-tryptophan to these albumins was measured at 20 degrees C in a 0.138 M salt solution at pH 7.4, by using an ultrafiltration technique, and analysed in terms of n, the number of available tryptophan-binding sites per albumin molecule, with apparent association constant, k. 2. n and k were 0.90 and 2.3x10(-4)M(minus-1) respectively for defatted bovine serum albumin and 0.87 and 9.7x10(-3)M(-minus-1) for human albumin. Addition of palmitic acid did not decrease n until the molar ratio, fatty acid/bovine albumin, approached and exceeded 2. The decrease in k was small and progressive. In contrast, lauric caused a marked decrease in n and k at ratios as low as 0.5. A similar distinction between the effects on n of palmitic acid and oleic acid and those of lauric acid was seen for human albumin. k for human albumin was not significantly affected by fatty acids under the conditions studied. 3. It is concluded that primary long-chain fatty acid sites interact only weakly with the tryptophan site on albumin and that inhibition of tryptophan binding occurs when secondary long-chain sites are occupied. Primary medium-chain fatty acid sites are distinct from primary long-chain sites but may be grouped with secondary long-chain sites. 4. The relationship between free and bound tryptophan in samples of rat plasma (Stoner et al., 1975) is discussed in terms of a similar but limited study of rat albumin.     

Solute complexation degree with human serum albumin: biochromatographic approach

A mathematical model was developed for the study of the D,L-dansylamino acid retention mechanism in reversed-phase liquid chromatography using a C18 column as a stationary phase and human serum albumin (HSA) as an eluent modifier. The solute retention factor is dependent on the HSA concentration in the eluent as well as the binding constant of the guest-HSA complex. A determination of the degree of complexation n(c) (the percent of the complexed guest) could be carried out. Different Van 't Hoff plot shapes of the degree of complexation were observed with different eluent pH, confirming a change in the solute complexation mechanism for physiological pH (between 7-7.5). Enthalpy-entropy compensation was also analysed in relation to this mathematical model to confirm the solute complexation behavior with HSA. These results finally confirmed that at physiological pH and temperature (approximately 35 degrees C) values the HSA was in a favorable structural conformation for its binding with a great majority of drugs.     

pH-controlled hydrogen-bonding (Short Communication)

The pH-dependence of the degree of hydrogen-bonding between a base and its conjugate acid is considered. When only a small proportion of the total base is complexed, the amount complexed is proportional to (1+coshp)(-1) where p=2.303 (pK(a)-pH), pK(a) being the dissociation constant of the conjugate acid. This represents sharp pH-dependence. As the proportion complexed increases, the curve broadens, eventually becoming flat-topped, with more than half the base complexed over the range of pH values pK(a)+/-logKC, approximately. (K is the complex association constant and C is the formal base concentration, including all forms.) There are similarities to the extent of mono-protonation of a dibasic acid.     

Reaction Field Simulations of Monatomic and Diatomic Dipolar Fluids

Firstly, the conditions are explored for which molecular dynamics simulations yield reliable thermodynamic results when the reaction field method is applied for dipolar fluids. The influence of the system size, of the scaling method for avoiding the total energy updrift, and of the reaction field dielectric constant are studied. With appropriate conditions, the results for the Stockmayer fluid when compared to previous results show excellent agreement up to very high dipole moments. In the next step the method is applied to two-centre Lennard-Jones plus ideal axial dipole (2CLJD) molecules of elongation L = 0.505. For one state point, the influence of the molecular shape as well as of the dipolar strength on energy and pressure is studied in detail. Finally, in the project of developing a physically based equation of state for polar fluids, thermodynamic results are presented for 2CLJD fluids of elongation L = 0.505 for four different dipole moments at 29 state points in a temperature and density grid. For these 116 runs which all were performed with vectorized codes on a CYBER 205 also the uncertainties in the results are given as have been estimated from the running averages.     

An in vitro study of interactions between insulin-mimetic zinc(II) complexes and selected plasma components

The speciations of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 1,2-dimethyl-3-hydroxypyridinone with (O,O) and picolinic acid with (N,O) coordination modes, were studied via solution equilibrium investigations of the ternary complex formation in the presence of small relevant bioligands of the blood serum such as cysteine, histidine and citric acid. Results show that formation of the ternary complexes, especially with cysteine, is favoured at physiological pH range in almost all systems studied. Besides these low molecular mass binders, serum proteins among others albumin and transferrin can bind zinc(II) or its complexes. Accordingly, the distribution of zinc(II) between the small and high molecular mass fractions of the serum was also studied by ultrafiltration. Modelling calculations relating to the distribution of zinc(II), using the stability constants of the ternary complexes studied and those of the serum proteins reported in the literature, confirmed the ultrafiltration results, namely, the primary role of albumin in zinc(II) binding among the low and high molecular mass components of the serum.     

Continuous culture of rat C6 glioma in serum-free medium

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.     

Effect of albumin on oleic acid lymphatic absorption in rats

1. The aim of this study was to investigate how fatty acid absorption was affected when exogenous fatty acids were complexed with albumin in absence of bile. Experiments were carried out in vivo, in order to study overall absorption processes. 2. An equimolar mixture of 14C oleic acid, palmitic acid and monopalmitin was infused intraduodenally in bile- and pancreatic juice-diverted rats. 3. Lipids were emulsified with either sodium taurocholate or fatty acids complexed with albumin. 4. Lymphatic lipid output was compared during the 6 hr following infusion of 90 mumol of the radioactive lipid mixture. 5. Lymphatic radioactive lipid recovery was significantly decreased by albumin. 6. Only 17% of the infused radioactivity was recovered in lymph when fatty acids were complexed with albumin against 37% when lipids were emulsified with sodium taurocholate. 7. Unrecovered lymph radioactivity was found at the distal part of intestine. Moreover, albumin significantly decreased lymph flow. 8. We conclude that undigested albumin acted at the luminal level of lipid absorption processes and specifically decreased fatty acid uptake.     

Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.     

Mechanism of proton permeation through chloroplast lipid membranes.

Electrical measurements were carried out to investigate the contribution of chloroplast lipids to the passive proton permeability of both the thylakoid and inner-envelope membranes. Permeability coefficient and conductance to protons were measured for solvent-free bilayers made from monogalactosyldiglyceride:digalactosyldiglycerid: sulfoquinovosyldiglyceride:phosphatidylglycerol (2:1:0.5:0.5, w/w) in the presence of a pH gradient of 7.4/8.1. The permeability coefficient for protons in glycolipids was 5.5 +/- 1.1 x 10(-4) cm s-1 (n = 14). To determine whether this high H+ permeability could be explained by the presence of lipid contaminants such as weak acids, we investigated the effects of (a) bovine serum albumin, which can remove some amphiphilic molecules such as free fatty acids, (b) 6-ketocholestanol, which increases the membrane dipole potential, (c) oleic acid, and (d) chlorodecane, which increases the dielectric constant of the lipid bilayer. Our results show that free fatty acids are inefficient protonophores, as compared with carbonylcyanide-m-chlorphenythydrazone, and that the hypothesis of a weak acid mechanism is not valid with glycolipid bilayers. In the presence of deuterium oxide the H+ conductane was reduced significantly, indicating that proton transport through the glycolipid matrix could occur directly by a hydrogen bond process. The passive transport of H+ through the glycolipid matrix is discussed with regard to the activity of the thylakoid ATP synthase and the inner-envelope H(+)-ATPase.     

Dielectric response of triplex DNA in ionic solution from simulations.

We have analyzed a 1.2-ns molecular dynamics simulation of 51 mM d(CG.G)7 with 21 Na+ counter-ions and 1 M NaCl in water. Via the dipole fluctuations, the dielectric constant for the DNA is found to be around 16, whereas that for the bases and sugars combined is only 3. The dielectric constant for water in this system is 41, which is much smaller than 71 for pure SPC/E water, because of the strong restriction imposed on the motion of water molecules by the DNA and the ions. Also addressed in the present work are several technical issues related to the calculation of the dipole moment of an ionic solution from molecular dynamics simulations using periodic boundary conditions.     

Effect of ionizing radiation on artificial (planar) lipid membranes. II. The ion carriers valinomycin and nonactin as probes for radiation induced structural changes of the membrane

Planar lipid membranes in the presence of the ion carriers valinomycin or nonactin were irradiated with 14 MeV electrons from a linear accelerator. A large increase of the membrane conductance by up to more than two orders of magnitude was found. The effect is virtually abolished either at high pH, or in the absence of oxygen, or in the presence of the radical scavenger ethanol. A further prerequisite for the effect is the presence of unsaturated fatty acid residues. A kinetic analysis of the carrier transport model based on current-voltage curves and on voltage-jump relaxation experiments was performed as a function of radiation dose. Only the translocation rate constant, kMS, of the charged carrier-ion complex was found to be influenced by irradiation. The effect is interpreted as an increase of the polarity (dielectric constant) of the membrane interior induced by the presence of polar products of lipid peroxidation. A combined action of OH- and HO2-radicals seems to be responsible for the phenomena. At large radiation doses (greater than or equal to 10(3) Gy) a reduction of the membrane conductance was observed. This is interpreted as an increased microviscosity, possibly caused by cross-linking of fatty acid residues. Ion carriers represent sensitive probes of radiation induced membrane damage.