Hydrophobic interaction determined by partition in aqueous two-phase systems. Partition of proteins in systems containing fatty-acid esters of poly(ethylene glycol).

In this report we describe a new method which is useful for measuring hydrophobic interactions between aliphatic hydrocarbon chains and proteins in aqueous environment. The method is based on partition of proteins in an aqueous two-phase system containing dextran and poly(ethylene glycol) and different fatty acid esters of poly(ethylene glycol). The partition is measured under conditions where contributions from electrostatic interactions are eliminated. The difference in partition of proteins in phase systems with and without hyrocarbon groups bound to poly(ethylene glycol), deltalog K, where K is the partition coefficient, is taken as a measure of hydrophobic interaction. Deltalog K varies with size of hydrocarbon chain and type of protein. The length of the aliphatic chain should be greater than 8 carbon atoms in order to get a measurable effect in terms of deltalog K. Bovine serum albumin, beta-lactoglobulin, hemoglobin and myoglobin have been shown to have different affinities for palmitic acid ester of poly(ethylene glycol). No hydrophobic effect could be observed for ovalbumin, cytochrome c or alpha-chymotrypsinogen A.     

Changes in structure and hydrophobic surface properties of beta-lactoglobulin determined by partition in aqueous two-phase polymeric systems.

The non-polar surface properties of beta-lactoglobulin and especially its interaction with poly(ethylene glycol)-bound palmitate has been studied as a function of pH, temperature and protein concentration. The maximum interaction between beta-lactoglobulin and polymer-bound palmitate occurs at pH 4.3 and pH 7.8. The change in conformation of beta-lactoglobulin around pH 7.5 seems to involve exposure of apolar amino acids to the solvent which results in an increased affinity for hydrocarbons. This is contrary to the situation at pH 4.8--6.0 where the corresponding change in conformation does not affect the protein-hydrocarbon interaction. The results suggest that partition studies in an aqueous two-phase system is a very useful tool to detect changes in conformation and aggregation and to characterize the corresponding hydrophobic surface properties of a protein.     

Fatty acid binding to plasma albumin.

A review of the available information about fatty acid binding to plasma albumin is presented. Albumin is composed of a single polypeptide chain, folded so as to form three or four spherical units. The strong fatty acid binding sites probably are located in crevices between these spherical regions. The anionic form of the fatty acid binds to albumin. Most of the binding energy comes from nonpolar interactions between the fatty acid hydrocarbon chain and uncharged amino acid side chains that line the binding sites. The binding sites are somewhat pliable, and their configuration can adapt to fit the incoming fatty acid. Stepwise association constants for binding to human albumin of fatty acids containing 6-18 carbon atoms are presented. These data indicate that each mole of fatty acid binds with a different affinity and that the association constants for multiple binding diminish sequentially, i.e., kappa 1 greater than kappa 2 greater than kappa 3 greater ... greater kappan. Because of uncertainties concerning fatty acid association in aqueous solutions, the constants for the 14-18 carbon acids probably are not definitive. In the usual physiological concentration range, free fatty acids do not displace appreciable amounts of a second organic compound from albumin. Sensitive spectrophotometric analyses revealed, however, that even small increases in free fatty acid concentration alter the molecular interaction between human albumin and another organic compound.     

Coupling of poly(ethylene glycol) to albumin under very mild conditions by activation with tresyl chloride: characterization of the conjugate by partitioning in aqueous two-phase systems

Poly(ethylene glycol) activated with tresyl chloride has been covalently linked to albumin as a result of a 2-h incubation in 0.05 M sodium phosphate buffer, pH 7.5, containing 0.125 M sodium chloride (0.344 OSM). The coupling of poly(ethylene glycol) to albumin was demonstrated by the increase in the partition coefficient of the protein in poly(ethylene glycol)-dextran aqueous two-phase systems. A linear relationship between the log of the partition coefficient of the poly(ethylene glycol)-albumin conjugate and the degree of modification (measured as the amino groups consumed during the coupling step) has been demonstrated. Countercurrent distribution in the two-phase system showed that poly(ethylene glycol)-albumin was heterogeneous with respect to its partitioning behavior, indicating that the albumin was not uniformly modified with poly(ethylene glycol).     

The effect of poly(ethyleneglycol) esters on the partition of proteins and fragmented membranes in aqueous biphasic systems

The hydroxyl groups of poly(ethyleneglycol) have been esterified (partly) with a number of carboxylic acids. When these esters are included in dextranpoly(ethyleneglycol)-water biphasic systems the partitions of proteins and membranes between the two phases (and the interface) are in some cases strongly affected. The affinity of serum albumin for the poly(ethyleneglycol)-rich phase is strongly increased when the fatty acid group consists of more than 10 carbon atoms. The partition also depends on the number of double bonds in the fatty acid. A corresponding relationship is found for membranes from spinach chloroplasts. The partitions of ovalbumin, lysozyme (EC 3.2.1.17) and ribonuclease (EC 3.1.4.22) are not influenced by the fatty acid esters. Esters of dibasic carboxylic acids show a minute but marked effect on the partition of proteins in general while malate and tartrate esters affect strongly the partition of chloroplast membranes. The partitions of both proteins and membranes are influenced by poly(ethyleneglycol) deoxycholate. Experiments with malate dehydrogenase (EC 1.1.1.37), lactate dehydrogenase (EC 1.1.1.27), fumarase (EC 4.2.1.2), enolase (EC 4.2.1.11) and glutamate-oxaloacetate transaminase (EC 2.6.1.1) show that their partitions, measured on enzymic activity basis, is changed when esters of benzoic, linolenic, tartaric or deoxycholic acid are included in the biphasic system. The mechanism behind the effect of the esterified poly(ethyleneglycol) on the partition of biomaterial, in this type of aqueous biphasic systems, is discussed in terms of a direct binding of the esters to the partitioned material.     

The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection

Epicuticular lipids in many terrestrial arthropods consist of vast numbers of polar and non-polar aliphatic compounds, which are mainly responsible for the water balance in these animals but can also affect conidia germination of entomopathogenic fungi. In this work the qualitative and quantitative profiles of cuticular fatty acids from three insect species differing in their susceptibility to fungal infection were studied. In an innovative approach, laser light scattering detection was coupled with HPLC in order to identify the non-chromophoric chemicals usually present in cuticular extracts. The acids identified contained from 5 to 20 carbon atoms in the alkyl chain and included unsaturated entities such as C(16:1), C(18:1), C(18:2), C(18:3) and C(20:1). There was a marked dominance of acids containing 16-18 carbon atoms. The relative contents of fatty acids in the extracted waxes varied from trace amounts to 44%. Cuticular fatty acids profile of Calliphora vicina (species resistant to fungal infection) significantly differs from profiles of Dendrolimus pini and Galleria mellonella (both species highly susceptible to fungal infection). The major difference is the presence of C(14:0), C(16:1) and C(20:0) in the cuticle of C. vicina. These three fatty acids are absent in the cuticle of D. pini while G. mellonella cuticle contains their traces. The concentrations of four fatty acids dominating in the G. mellonella larval cuticle (C(16:0), C(18:0), C(18:1) and C(18:2)) were found to fluctuate during the final larval instar and correlate with fluctuations in the susceptibility of larvae to fungal infection. The possible role of cuticular fatty acids in preventing fungal infection is discussed.     

Purification and characterization of mitochondrial thioesterase from rabbit myocardium and its inhibition by palmitoyl carnitine

The inhibitory effects of saturated fatty acids with 4 to 18 carbon atoms on ADP-induced aggregation of bovine platelets were investigated. The inhibitory effects of the acids increased with increase of their alkyl chain length up to C14. On the other hand, from C16 the inhibitory effects tended to decrease with increase of chain length, and stearic acid (C18) was not inhibitory. There was a linear relationship between the inhibitory effects and alkyl chain lengths up to C12. This linear relation and the slope of the linear regression line suggested that the inhibitory effects of the acids depended on their partition into the membrane. The fatty acids decreased the fluorescence of the surface charge probe 2-p-toluidinylnaphthalene-6-sulfonate, indicating that they increased the negative charge on the membrane surface. The relative effects of the acids on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that the inhibition of platelet aggregation by saturated fatty acids is due to a change in the membrane surface charge of the platelet plasma membrane.     

Microbial Incorporation of Fatty Acids Derived From n-Alkanes Into Glycerides and Waxes

When n-alkanes with 13 to 20 carbon atoms were fed to a Nocardia closely related to N. salmonicolor, the produced cellular triglycerides and aliphatic waxes invariably contained fatty acids with an even or an odd number of carbon atoms subject to this feature of the n-alkane substrate. Beta-oxidation and C₂ addition are both operative, as evidenced by the spectra of fatty acids incorporated into the cellular lipid components. There is no distinction in the rate of microbial incorporation of the odd-or even-numbered carbon chains. The fatty acids are apparently directly derived from the long chain n-alkanes, rather than synthesized via the classic C₂-condensation route. The alcohol component of waxes produced by the Nocardia is invariably of the same chain length as the n-alkane substrate.     

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family.     

The effects of short-chain fatty acids on the neuronal membrane functions ofHelix pomatia. III.22Na efflux from the cells

The effect of short-chain fatty acids on both ouabain-sensitive and ouabain-insensitive fractions of 22Na efflux from the neurons of Helix pomatia was studied. Fatty acids, having fewer than 10 carbon atoms in the hydrocarbon chain, increased the ouabain-sensitive 22Na efflux from the neurons, while fatty acids, having more than 9 carbon atoms, inhibited the 22Na efflux in comparison with that in normal physiological solution. All the fatty acids used had an inhibiting effect on the ouabain-insensitive 22Na efflux from the cells independent on the number of carbon atoms in the hydrocarbon chain. These studies indicate that these short-chain fatty acids can be effective modulators of both ouabain-sensitive and ouabain-insensitive fractions of Na efflux from the cells.     

Synthesis in vitro of very long chain fatty acids in Vibrio sp. strain ABE-1

The activity of fatty acid synthetase (FAS) from Vibrio sp. strain ABE-1 required the presence of acyl carrier protein and was completely inhibited by thiolactomycin, an inhibitor specific for a type II FAS. These observations indicate that this enzyme is a type II FAS. Analysis by gas-liquid chromotography of the reaction products synthesized in vitro from [2-¹⁴C]malonyl-CoA by the partially purified FAS revealed, in addition to 16-and 18-carbon fatty acids which are normal constituents of this bacterium, the presence of fatty acids with very long chains. These fatty acids were identified as saturated and mono-unsaturated fatty acids with 20 up to as many as 30 carbon atoms. The longest fatty acids normally found in this bacterium contain 18-carbon atoms. These results suggest that the FAS from Vibrio sp. strain ABE-1 has potentially the ability to synthesize fatty acids with very long chains.Abbreviations ACP acyl carrier protein - FAME fatty acid methyl ester - FAS fatty acid synthetase - FID flame ionization detection - GLC gas-liquid chromatography - TLC thin-layer chromatography - In designations of fatty acids, such as 16:0, 16:1, etc the colon separates the number that denotes the number of carbon atoms and the number that denotes the number of double bonds, respectively, in the molecule - 16:0-CoA CoA ester of 16:0     

Ca2+ and pH dependence of hydrophobicity of alpha-lactalbumin: affinity partitioning of proteins in aqueous two-phase systems containing poly(ethylene glycol) esters of fatty acids.

Hydrophobic affinity partitioning in an aqueous two-phase system, composed of dextran and poly(ethylene glycol), has been used to study the hydrophobic binding capacity of bovine alpha-lactalbumin. The hydrophobicity of the poly(ethylene glycol)-containing phase was adjusted by including varying amounts of fatty acids bound to the polymer via an ester linkage. The change in the logarithmic partition coefficient of the protein in such systems was used as a measure of the hydrophobic binding. This value was strongly influenced by the amount of Ca2+ present as well as the pH value. The results are discussed in terms of the exposure of hydrophobic binding sites on alpha-lactalbumin and their relation to the conformational change in this protein due to Ca(2+)-binding, chelation of Ca2+ and pH dependence.     

Synthesis and bioactivities of poly(ethylene glycol)–chitosan hybrids

Poly(ethylene glycol)–chitosan hybrids of various molecular weights having different degree of substitution were synthesized, by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde, to study their bioactivities. The influence of these chitosan derivatives on the reactive oxygen species generation from canine polymorphonuclear leukocyte cells was investigated in vitro by chemiluminescence response. Reactive oxygen species generation by the influence of poly(ethylene glycol)–chitosan hybrids was decreased with the increase of degree of substitution. The reduction of interaction of poly(ethylene glycol)–chitosan hybrids with polymorphonuclear leukocyte cells might be caused by the decrease of amino group in chitosan main chain and increase of the steric hindrance by poly(ethylene glycol) chain. The influence of the poly(ethylene glycol)–chitosan hybrids on complement component C3 activation was investigated by single radial immunodiffusion method. Influence on complement component C3 activation by poly(ethylene glycol)–chitosan hybrids was almost same as chitosan.     

Relative rates of hydrolysis by rat pancreatic lipase of esters of C2-C18 fatty acids with C1-C18 primary n-alcohols

The rate at which rat pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) hydrolyzes the esters of primary n-alcohols containing from 1 to 18 carbon atoms with fatty acids containing from 2 to 18 carbon atoms was determined. The speed of hydrolysis was influenced, apparently independently, by both the acyl and the alkyl chains. With respect to the fatty acid moiety, the esters of dodecanoic acid were usually split at the most rapid rate. Esters of butyric acid were the next most susceptible. In the case of the alcohol moiety, esters of heptyl alcohol were hydrolyzed most rapidly. On the basis of the pattern of the relative rates of hydrolysis, it is proposed that the influence of the alcohol component is a result of its orienting the ester molecule at the oil/water interface. The fatty acid effect is attributed to enzyme-substrate specificity.     

Transfer of stearic acids from albumin to polymer-grafted lipid containing membranes probed by spin-label electron spin resonance

Human serum albumin (HSA) has been spin-labelled with stearic acids having the nitroxide moiety attached to the hydrocarbon chain either at the 5th or at the 16th carbon atom (n-SASL, n = 5 and 16, respectively) with respect to the carboxyl groups. Its interaction with sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) mixed with submicellar content of poly(ethylene glycol:2000)-grafted dipalmitoyl phosphatidylethanolamine (PEG:2000-DPPE) has been studied by conventional electron spin resonance (ESR) spectroscopy. In the absence of bilayer membranes, the ESR spectra of nitroxide stearic acids non-covalently bound to HSA are single component powder patterns, indicative of spin labels undergoing temperature dependent anisotropic motion in the slow motional regime on the conventional ESR timescale. The adsorption of HSA to DPPC bilayers results in two component ESR spectra. Indeed, superimposed to an anisotropic protein-signal appears a more isotropic signal due to the labels in the lipid environment. This accounts for the transfer of fatty acids from the protein to DPPC bilayers. Two spectral components with different rotational mobility are also singled out in the spectra of n-SASL bound to HSA when DPPC/PEG:2000-DPPE mixtures are present in the dispersion medium. The fraction, f(L)(16-SASL), of spin labels transferred from the protein to lipid/polymer-lipid lamellar membranes has been quantified performing spectral subtraction. It is found that f(L)(16-SASL) decreases on increasing the content of the polymer-lipid mixed with DPPC. It is strongly reduced in the low-density mushroom regime and levels off in the high-density brush regime of the polymer-lipid content as a result of the steric stabilization exerted by the PEG-lipids. Moreover, the fraction of transferred fatty acids from HSA to SSL is dependent on the physical state of the lipid bilayers. It progressively increases with increasing the temperature from the gel to the liquid-crystalline lamellar phases of the mixed lipid/polymer-lipid membranes, although such a dependence is much weaker in the brush regime.     

Peptide hydrophobicity and partitioning in poly(ethylene glycol)/magnesium sulfate aqueous two-phase systems.

Several amino acids and peptides were partitioned in poly(ethylene glycol) (PEG)/magnesium sulfate (MgSO4) aqueous two-phase systems. The partition coefficients measured for amino acids and peptides were proportional to the difference in PEG concentration between the phases. The partitioning data were used to calculate the relative hydrophobicities of individual amino acids, which were then used to estimate the hydrophobicities of peptides. The partition coefficients of several dipeptides were predicted from these estimated hydrophobicities. A series of peptide fragments that compose the pentapeptide leucine enkephalin was also partitioned in the PEG/MgSO4 system. Again, the partitioning depended upon the hydrophobicities of the individual exposed amino acids.     

Generation of selective microenvironment at the cell periphery through bulk-phase hydrophilic polymers

In order to understand the previously demonstrated effect of poly(ethylene glycol) on the stimulation of lymphocyte responses to syngeneic tumor cells (Ben-Sasson, S.A. and Henkart, P.A. (1977) J. Immunol. 119, 227–231), we have investigated the effects of addition of poly(ethylene glycol) to the medium in a number of cellular systems. The binding of trimeric IgG to tumor-lymphocyte Fc receptors was greatly enhanced by poly(ethylene glycol); a substantial increase in binding of trimeric IgG to non-Fc-receptor-bearing tumor cells was also observed. Similarly, the binding of labeled bovine serum albumin to lymphocyte surfaces was increased by poly(ethylene glycol), implying that nonspecific binding of proteins to cells was generally enhanced. The dose-response curve of concanavalin A mitogenesis was shifted to the right, as would be expected from a local increase in concanavalin A concentration. Antibody binding to erythrocytes as detected by complement lysis was similarly increased. It was found that in aqueous two-phase mixtures created by poly(ethylene glycol) and dextran, erythrocytes partition into the dextran phase through exclusion into dextran-rich microdroplets. It is proposed that addition of poly(ethylene glycol) to cell culture media creates a similar separate phase around the cell surface in which the local concentration of proteins is greater than that in the bulk medium. This concept explains many of the diverse effects of addition of poly(ethylene glycol) to the medium. It also can partially explain the requirement for serum to observe the poly(ethylene glycol) effect on the lymphocyte response to syngeneic tumor cells.     

Permeability characteristics of the adipocyte cell membrane and partitioning characteristics of the adipocyte triglyceride core.

The unidirectional rates of passive permeation of a homologous series of saturated fatty acids and bile acids into rat epididymal adipocytes were measured to determine the permeability characteristics of this mammalian cell membrane. For fatty acids containing 5 to 12 carbon atoms the logarithm of the permeability coefficient was a linear function of the number of carbons in the fatty acid chain: fatty acids with less than five carbon atoms showed anomalously high permeabilities. Using the data for the fatty acids with 5 to 12 carbon atoms, the incremental free energy of transfer (delta delta F w leads to l) of the -CH2 moiety from the aqueous environment into the fat cell was calculated to equal -547 cal mole-1. The delta delta F w leads to l of the -OH moiety calculated from data using bile acids as the probe molecules was +1,225 cal mole-1. After rupturing the fat cells by freeze-thawing, partition ratios also were measured between bubber and the lipid phase of the adipocyte core using both the fatty acid series and a series of terminal diols as probe molecules. Using these partition ratios delta delta F w leads to l for the -CH2 and -OH substituent groups was calculated to equal -830 and +2,070 cal mole-1, respectively. On the basis of these studies, two conclusions were drawn. First, like many epithelial surfaces and the erythrocyte membrane, the fat cell membrane exhibits anomalously high permeabilities to small molecular weight, polar compounds. Since this behavior in the adipocyte, as in the erythrocyte, cannot be attributed to structures such as tight junctions, it must be explained on the basis of some physico-chemical feature of the cell membrane itself. Secondly, the values of the delta delta F w leads to l indicate that the adipocyte membrane is less polar than the intestinal and gallbladder membranes but more polar than the membranes of Nitella and the erythrocyte.     

Fatty Acid and Aliphatic Hydrocarbon Composition of Sarcina lutea Grown in Three Different Media

Sarcina lutea was grown in Trypticase Soy Broth, Nutrient Broth, and a chemically defined medium. Gas chromatographic analysis of lipid components demonstrated that the composition of the medium had an effect on the relative per cent composition of the aliphatic hydrocarbons and fatty acids present in the cells. The branched olefinic hydrocarbons from the organisms grown in Trypticase Soy Broth showed no predominance or only a slight predominance of odd-numbered carbon chains, whereas the hydrocarbons from cells grown in the other two media showed an obvious predominance of odd-numbered carbon chains. The monocarboxylic fatty acid content and distribution showed only minor differences, with all normal saturated fatty acids present in relatively small quantities for cells grown in Nutrient Broth and in a chemically defined medium.     

A role for hormone-sensitive lipase in the selective mobilization of adipose tissue fatty acids.

The mobilization of fatty acids from rat and human fat cells is selective according to molecular structure, and notably carbon atom chain length. This study aimed at examining whether the release of individual fatty acids from triacylglycerols (TAG) by hormone-sensitive lipase (HSL) plays a role in the selectivity of fatty acid mobilization. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 18 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation degree from 0 to 3 double bond(s), was measured by comparing the composition of non-esterified fatty acids (NEFA) to that of the original TAG. The relative hydrolysis (% in NEFA/% in TAG) differed between fatty acids, being about 5-fold and 3-fold higher for the most (18:1n-7) than for the least (24:0) readily released fatty acid by recombinant rat and human HSL, respectively. Relationships were found between the chain length of fatty acids and their relative hydrolysis. Among 12-24 carbon atom saturated fatty acids, the relative hydrolysis markedly decreased (by about 5- and 3-times for recombinant rat and human HSL, respectively) with increasing chain length. We conclude that fatty acids are selectively released from TAG by HSL according to carbon atom chain length. These data provide insight on the mechanism by which fatty acids are selectively mobilized from fat cells.