Transport of heptafluorostearate across model membranes. Membrane transport of long-chain fatty acid anions I

Heptafluorostearic acid, an isogeometric derivative of stearic acid, has a pK(a) value of about 0.5. To evaluate the suitability of heptafluorostearate as model compound for anions of long-chain fatty acids in membrane transport, monolayer and liposome studies were performed with lipid mixtures containing phospholipids;-cholesterol-heptafluorostearate or stearate (100:40:20 molar ratios). Transfer of heptafluorostearate and stearate from liposomes to bovine serum albumin (BSA) was followed by measuring the intrinsic fluorescence of BSA. The percentage of heptafluorostearate, equivalent to the amount placed in their outer monolayer, transferred from liposomes (120;-130 nm diameter) to BSA was 55.7 +/- 3.7% within 10 min at 25 degrees C and 55 +/- 2% within 5 min at 37 degrees C. Slow transfer of 22.7 +/- 2.5% of heptafluorostearate at 25 degrees C followed with a half-life of 2.3 +/- 0.4 h and of 20 +/- 4% at 37 degrees C with a half-life of 0.9 +/- 0.1 h until the final equilibrium distributions between BSA and liposomes were reached, 79 +/- 6% to 21 +/- 5% at 25 degrees C and 75 +/- 5% to 25 +/- 4% at 37 degrees C. The pseudounimolecular rate constants for flip-flop of heptafluorostearate equal k(FF,25) = 0.24 +/- 0.05 h(-) and k(FF,37) = 0.6 +/- 0.1 h(-), respectively. By comparison, transfer of stearate required only 3 min to reach equilibrium distribution.The difference between heptafluorostearate and stearate may be explained by a rapid flip-flop movement of the un-ionized fatty acids which exist in different concentrations in accordance with their pK(a) values. Half-life of flip-flop of heptafluorostearate makes it suitable to study mediated membrane transport of long-chain fatty acid anions.     

Fatty acyl coupled catalase

Bovine liver catalase (hydrogen-peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) was derivatized by 9"(10")-[4'-(2-(4,6-dichloro-1,3,5-triazinyl) oxy)butoxy] stearic acid and the fatty acyl-coated enzyme was separated from native catalase and excess reagent by hydroxyapatite chromatography. The derivatization of catalase resulted in coupling the long-chain fatty acyl residues to lysine, histidine and arginine, while other amino acids remained essentially unaffected. The fatty acyl-coated enzyme was water soluble at pH greater than 7.0 but became octanol and ether soluble at pH less than 6.5. The derivatized enzyme retained 50-80% of the catalatic- and peroxidative-specific activities. The free carboxyl function of the coupled long-chain fattyl acyl residues could serve as substrate for ATP-dependent CoA-thioesterification catalyzed by the rat liver microsomal long-chain fatty acyl-CoA synthase.     

Fatty acyl coupled catalase

Bovine liver catalase (hydrogen-peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) was derivatized by 9″(10″)-[4′-{2-(4,6-dichloro-1,3,5-triazinyl)oxy}butoxy]stearic acid and the fatty acyl-coated enzyme was separated from native catalase and excess reagent by hydroxyapatite chromatography. The derivatization of catalase resulted in coupling the long-chain fatty acyl residues to lysine, histidine and arginine, while other amino acids remained essentially unaffected. The fatty acyl-coated enzyme was water soluble at pH > 7.0 but became octanol and ether soluble at pH < 6.5. The derivatized enzyme retained 50–80% of the catalatic- and peroxidative-specific activities. The free carboxyl function of the coupled long-chain fattyl acyl residues could serve as substrate for ATP-dependent CoA-thioesterification catalyzed by the rat liver microsomal long-chain fatty acyl-CoA synthase.     

Biogeochemical features of lipids in endolithic microbial communities in the Ross Desert (McMurdo Dry Valleys), Antarctica.

Endolithic microbial communities inhabiting porous rocks in the cold, dry mountainous regions of Antarctica have been studied extensively as examples of life's adaptations to extreme environments. Here, we examine hydrocarbons and fatty acids occurring in these communities in order to clarify their biogeochemical features with respect to source organisms, microbial activity, fossilization processes and the influence of Gondwanaland sediments. Unusually, long-chain (>C19) n-alkanes and anteiso-alkanes were often the major hydrocarbons in the samples. A suite of n-alkanoic acids (n-C9-n-C32) and long-chain anteiso-alkanoic acids (a-C20-a-C30) were found, along with short-chain iso- and anteiso-alkanoic acids, and n-alkenoic acids. The relationship between long-chain n-alkanoic acids (n-C20-n-C32) and long-chain anteiso-alkanoic acids suggests that these compounds probably originated from the same group of microorganisms, such as bacteria or endolithic lichens, under moderate pH conditions (pH 3-5). Relatively high trans/cis-C16:1 alkenoic acid ratios suggest the presence of unfavorable environmental conditions in the endolithic microbial habitat. Normal-alkenoic/alkanoic acid ratios may be a useful marker for the fossilization of endolithic microbial communities. Thermally matured triterpanes and steranes from fossilized associations on Mount Fleming strongly suggest the presence of Gondwanaland sediments formed during Devonian and Jurassic (400-180 million years ago).     

Absolute configuration of 3-hydroxy acids formed by Stenotrophomonas maltophilia: application of multidimensional gas chromatography and circular dichroism spectroscopy.

The soil bacterium Stenotrophomonas maltophilia was found to transform various long-chain fatty acids selectively into 3-hydroxy fatty acids of shorter chain length. Their chiral evaluation was performed by multidimensional gas chromatography (MDGC) on modified cyclodextrin phase comparing the enantiodistribution of 1,3-diol formed without loss of stereochemical information from a representative microbial product with those of synthetic (3RS)- and (3S)-1,3-diols. Enantiomeric excesses of 84-98% (R) were determined for the microbially produced 3-hydroxy acids. In addition, the CD exciton chirality method was applied to determine their absolute configuration. Derivatization with 9-anthryldiazomethane and 2-naphthoylimidazole led to the required bichromophoric structures. Their CD spectra displayed a positive first Cotton effect around 254 nm and a negative second Cotton effect around 237 nm, which confirmed the (R)-configuration of the bacterial products.     

Ionization and phase behavior of fatty acids in water: application of the Gibbs phase rule

The phase behavior of several medium-chain (10- and 12-carbon) and long-chain (18-carbon) fatty acids in water was examined as a function of the ionization state of the carboxyl group. Equilibrium titration curves were generated above and below fatty acid and acid-soap chain melting temperatures and critical micelle concentrations, and the phases formed were characterized by X-ray diffraction, 13C NMR spectroscopy, and phase-contrast and polarized light microscopy. The resulting titration curves were divided into five regions: (i) at pH values less than 7, a two-phase region containing oil or fatty acid crystals and an aqueous phase; (ii) at pH approximately 7, a three-phase region containing oil, lamellar, and aqueous (or fatty acid crystals, 1:1 acid-soap crystals, and aqueous) phases; (iii) between pH 7 and 9, a two-phase region containing a lamellar fatty acid/soap (or crystalline 1:1 acid-soap) phase in an aqueous phase; (iv) at pH approximately 9, a three-phase region containing lamellar fatty acid-soap (or crystalline 1:1 acid-soap), micellar, and aqueous phases; and (v) at pH values greater than 9, a two-phase region containing micellar and aqueous phases. Interpretation of the results using the Gibbs phase rule indicated that, for oleic acid/potassium oleate, the composition of the lamellar fatty acid/soap phase varied from approximately 1:1 to 1:3 un-ionized to ionized fatty acid species. In addition, constant pH regions observed in titration curves were a result of thermodynamic invariance (zero degrees of freedom) rather than buffering capacity. The results provide insights into the physical states of fatty acids in biological systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and some properties of a thermostable acid esterase from Acidiphilium sp. AIU 409

Extracellular and cell-bound esterases produced by Acidiphilium sp. AIU 409 were homogeneously purified from culture broth and cells, respectively, and some properties were investigated. Both esterases more rapidly hydrolyzed p-nitrophenyl acyl esters containing long-chain fatty acids from C 8:0 to C 18:0 than those containing short-chain fatty acids from C 2:0 to C 6:0. The Km values for p-nitrophenyl long-chain fatty acid esters from C 8:0 to C 18:0 were approximately 1.3-1.5 mM. The enzymes were stable at 50 degrees C for 2 days between pH 3.0 and 6.5, and optimum pH and temperature were 5.0 and 70 degrees C, respectively. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride and SDS. The molecular mass of both enzymes was estimated to be approximately 64 kDa by SDS-PAGE. The 23 amino acid sequence from the NH(2)-terminus was also the same in both enzymes. These results suggest that extracellular esterase might be composed of the same components as cell-bound esterase.     

Effects of hydrogen ion and fatty acid concentrations on the binding of aflatoxin B1 to human albumin

The influence of pH and long-chain fatty acids on the interaction between aflatoxin B1 and human albumin was investigated by fluorescence spectroscopy. Both the binding of aflatoxin B1 to albumin and the fluorescence of albumin-bound aflatoxin are pH-dependent over the pH range of 6-9.5. The data indicates that the carcinogen has a higher affinity for the basic(B) than for the neutral(N) conformation of human albumin. Palmitic, stearic and oleic acids up to a molar ratio of 2 over albumin, increases the binding strength of aflatoxin B1 by means of an allosteric mechanism. Furthermore, the pH-dependence of the aflatoxin-albumin interaction is affected by the presence of oleic acid by narrowing the pH range over which the dependence occurs. At molar ratios of oleic acid to albumin in excess of 4.25 at pH6, 3.1 at pH7.4 and 2.4 at pH9 cause a decrease in aflatoxin B1 fluorescence as a result of reduced binding to albumin.     

Shifts in the Membrane Fatty Acid Profile of Streptococcus mutans Enhance Survival in Acidic Environments

Acid adaptation of Streptococcus mutans UA159 involves several different mechanisms, including the ability to alter its proportion of long-chain, monounsaturated membrane fatty acids (R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). In the present study, we examined the mechanism and timing of changes in fatty acid ratios and the potential benefit that an increased proportion of long-chained fatty acids has for the organism during growth at low pH. Cells taken from steady-state cultures at intermediate pH values of 6.5, 6, and 5.5 showed incremental changes from the short-chained, saturated membrane fatty acid profile normally seen in pH 7 cultures to the long-chained, monounsaturated fatty acids more typically observed in acidic cultures (pH 5). Our observations showed that the bacterium was capable of effecting the majority of changes in approximately 20 min, far less than one generation time. However, reversion to the distribution of fatty acids seen in cells growing at a pH of 7 required a minimum of 10 generations. Fatty acid composition analysis of cells taken from cultures treated with chloramphenicol suggested that the changes in fatty acid distribution did not require de novo protein synthesis. Cells treated with the fatty acid biosynthesis inhibitor cerulenin were unable to alter their membrane fatty acid profiles and were unable to survive severe acidification. Results presented here indicate that membrane fatty acid redistribution is important for low pH survival and, as such, is a component of the S. mutans acid-adaptation arsenal.     

Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments

Acid adaptation of Streptococcus mutans UA159 involves several different mechanisms, including the ability to alter its proportion of long-chain, monounsaturated membrane fatty acids (R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). In the present study, we examined the mechanism and timing of changes in fatty acid ratios and the potential benefit that an increased proportion of long-chained fatty acids has for the organism during growth at low pH. Cells taken from steady-state cultures at intermediate pH values of 6.5, 6, and 5.5 showed incremental changes from the short-chained, saturated membrane fatty acid profile normally seen in pH 7 cultures to the long-chained, monounsaturated fatty acids more typically observed in acidic cultures (pH 5). Our observations showed that the bacterium was capable of effecting the majority of changes in approximately 20 min, far less than one generation time. However, reversion to the distribution of fatty acids seen in cells growing at a pH of 7 required a minimum of 10 generations. Fatty acid composition analysis of cells taken from cultures treated with chloramphenicol suggested that the changes in fatty acid distribution did not require de novo protein synthesis. Cells treated with the fatty acid biosynthesis inhibitor cerulenin were unable to alter their membrane fatty acid profiles and were unable to survive severe acidification. Results presented here indicate that membrane fatty acid redistribution is important for low pH survival and, as such, is a component of the S. mutans acid-adaptation arsenal.     

Inhibition of type 1 and type 2 5alpha-reductase activity by free fatty acids,active ingredients of Permixon

In different cell systems, the lipido-sterolic extract of Serenoa repens (LSESr, Permixon inhibits both type 1 and type 2 5alpha-reductase activity (5alphaR1 and 5alphaR2). LSESr is mainly constituted of fatty acids (90+/-5%) essentially as free fatty acids (80%). Among these free fatty acids, the main components are oleic and lauric acids which represent 65% and linoleic and myristic acids 15%.To evaluate the inhibitory effect of the different components of LSESr on 5alphaR1 or 5alphaR2 activity, the corresponding type 1 and type 2 human genes have been cloned and expressed in the baculovirus-directed insect cell expression system Sf9. The cells were incubated at pH 5.5 (5alphaR2) and pH 7.4 (5alphaR1) with 1 or 3nM testosterone in presence or absence of various concentrations of LSESr or of its different components. Dihydrotestosterone formation was measured with an automatic system combining HPLC and an on-line radiodetector.The inhibition of 5alphaR1 and 5alphaR2 activity was only observed with free fatty acids: esterified fatty acids, alcohols as well as sterols assayed were inactive. A specificity of the fatty acids in 5alphaR1 or 5alphaR2 inhibition has been found. Long unsaturated chains (oleic and linolenic) were active (IC(50)=4+/-2 and 13+/-3 microg/ml, respectively) on 5alphaR1 but to a much lesser extent (IC(50)>100 and 35+/-21 microg/ml, respectively) on 5alphaR2. Palmitic and stearic acids were inactive on the two isoforms. Lauric acid was active on 5alphaR1 (IC(50)=17+/-3 microg/ml) and 5alphaR2 (IC(50)=19+/-9 microg/ml). The inhibitory activity of myristic acid was evaluated on 5alphaR2 only and found active on this isoform (IC(50)=4+/-2 microg/ml).The dual inhibitory activity of LSESr on 5alpha-reductase type 1 and type 2 can be attributed to its high content in free fatty acids.     

Fatty acid transduction of nitric oxide signaling: multiple nitrated unsaturated fatty acid derivatives exist in human blood and urine and serve as endogenous peroxisome proliferator-activated receptor ligands

Mass spectrometric analysis of human plasma and urine revealed abundant nitrated derivatives of all principal unsaturated fatty acids. Nitrated palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids were detected in concert with their nitrohydroxy derivatives. Two nitroalkene derivatives of the most prevalent fatty acid, oleic acid, were synthesized (9- and 10-nitro-9-cis-octadecenoic acid; OA-NO2), structurally characterized and determined to be identical to OA-NO2 found in plasma, red cells, and urine of healthy humans. These regioisomers of OA-NO2 were quantified in clinical samples using 13C isotope dilution. Plasma free and esterified OA-NO2 concentrations were 619 +/- 52 and 302 +/- 369 nm, respectively, and packed red blood cell free and esterified OA-NO2 was 59 +/- 11 and 155 +/- 65 nm. The OA-NO2 concentration of blood is approximately 50% greater than that of nitrated linoleic acid, with the combined free and esterified blood levels of these two fatty acid derivatives exceeding 1 microm. OA-NO2 is a potent ligand for peroxisome proliferator activated receptors at physiological concentrations. CV-1 cells co-transfected with the luciferase gene under peroxisome proliferator-activated receptor (PPAR) response element regulation, in concert with PPARgamma, PPARalpha, or PPARdelta expression plasmids, showed dose-dependent activation of all PPARs by OA-NO2. PPARgamma showed the greatest response, with significant activation at 100 nm, while PPARalpha and PPARdelta were activated at approximately 300 nm OA-NO2. OA-NO2 also induced PPAR gamma-dependent adipogenesis and deoxyglucose uptake in 3T3-L1 preadipocytes at a potency exceeding nitrolinoleic acid and rivaling synthetic thiazo-lidinediones. These data reveal that nitrated fatty acids comprise a class of nitric oxide-derived, receptor-dependent, cell signaling mediators that act within physiological concentration ranges.     

Apparent monomer activity of saturated fatty acids im micellar bile salt solutions measured by a polyethylene partitioning system

Partitioning of saturated fatty acids between discs of polyethylene film and aqueous buffer has been characterized and subsequently used to measure monomer activities of fatty acids in micellar solutions of bile salt. Partitioning of fatty acids between polyethylene and buffer achieved equilibrium in about 24-48 hr. Partition coefficients for fatty acids 10:0 and 16:0 were essentially independent of concentration, as expected for true partitioning. Experiments with various pH buffers showed that only the protonated form of fatty acids 12:0 and 16:0 participated in partitioning, and the midpoints of the partition coefficients vs. pH curves were 4.5-5.0 and 6.5-7.0, respectively. Experimentally determined partition coefficients at pH 7.4 ranged from 2.03 +/- 0.09 for 9:0 to 56,100 +/- 13,850 for 17:0. The addition of each methylene group increased the partition coefficient by a factor of about 3.75, corresponding to an incremental free energy change for each methylene group of -3433 J.mole(-1) (-820 cal.mole(-1)). Monomer activities of solutions of 14:0 and 16:0 dissolved in 20 mM taurodeoxycholate were linearly dependent on the total fatty acid concentration. 1 mM 14:0 and 16:0 in 20 mM taurodeoxycholate had monomer activities of 1.3 x 10(-5) M and 5.6 x 10(-7) M, respectively. Solutions prepared with a constant concentration ratio of fatty acid to taurodeoxycholate had essentially constant monomer activities between 8 and 20 mM taurodeoxycholate. These studies support the hypothesis that fatty acid interaction with bile acid micelles is similar to a phase distribution system, so that some measurable monomer activity of fatty acid exists in equilibrium with the mass of fatty acid contained in the micelle.     

Linker mutagenesis of a bacterial fatty acid transport protein. Identification of domains with functional importance

The product of the fadL gene (FadL) of Escherichia coli is a multifunctional integral outer-membrane protein required for the specific binding and transport of exogenous long-chain fatty acids [C12-C18]. FadL also serves as a receptor for the bacteriophage T2. In order to define regions of functional importance within FadL, the fadL gene has been mutagenized by the insertion of single-stranded hexameric linkers into the unique SalI restriction site that lies towards the 3' end of the gene and into four HpaII restriction sites distributed throughout the coding region. The five insertion mutants were classified into three groups based on their specific growth rates (alpha) in minimal media containing the long-chain fatty acid oleate (C18:1) as a sole carbon and energy source: Oleslow, alpha = 0.035-0.045; Ole +/-, alpha = 0.020-0.035; and Ole-, alpha less than or equal to 0.005 (wild-type, alpha = 0.07-0.10). The hexameric insertion at the SalI site (fadL allele termed S1; insertion after amino acid 410) conferred an Oleslow phenotype and resulted in a reduction of long-chain fatty acid transport (36% the wild-type level). This insertion mutant, however, bound oleic acid at wild-type levels and was fully functional as a receptor for the bacteriophage T2. The modified FadL-S1 protein did not have the heat-modifiable property characteristic of wild-type FadL. Insertions in the four HpaII sites (fadL alleles termed H1, H2, H3, and H5; after amino acids 41, 81, 238, and 389, respectively) resulted in all three classes of mutants. The fadL insertion mutant H5 was defective for long-chain fatty acid transport but bound oleic acid at significant levels. Together with the S1 allele, these data suggest that the carboxyl terminus of FadL is crucial for long-chain fatty acid transport. The insertion mutants H1 and H2 were defective for both oleic acid binding and transport suggesting that the amino terminus of FadL is important for long-chain fatty acid binding and transport. The fadL linker mutant H3 was defective in oleic acid binding yet had significant levels of oleic acid transport. These studies delineated for the first time different regions of the fadL gene that encode domains of FadL implicated in the binding and transport of long-chain fatty acids.     

Diet preferences of warblers for specific fatty acids in relation to nutritional requirements and digestive capabilities

During energy-demanding periods of the annual cycle such as migration or during cold days in winter, birds store fat comprised mostly of 16- or 18-carbon unsaturated fatty acids. In such situations, birds may feed selectively on foods with specific fatty acids that enable efficient fat deposition. We offered wild-caught yellow-rumped warblers Dendroica coronata paired choices between semi-synthetic diets that differed only in their fatty acid composition. Warblers strongly preferred diets containing long-chain (18:1; carbon atoms:double bonds) unsaturated, unesterified fatty acids to diets containing long-chain saturated, unesterified fatty acids (18:0) and they preferred diets containing mono-unsaturated fats (18:1) to diets containing poly-unsaturated fats (18:2). The preference for diets containing long-chain unsaturated fatty acids to diets containing long-chain saturated fatty acids was consistent in birds tested one week after capture at 21°C, one month after capture when cold-acclimated (1°C), and six weeks after capture at 21°C. Birds acclimated to a diet with 50% of the fat comprised of unesterified stearic acid (18:0) lost mass and reduced their food intake when we reduced ambient temperature from 21°C to 11°C over three days. We conclude that especially in energy-demanding situations there are limits to the yellow-rumped warblers' ability to assimilate some long-chain saturated fatty acids and that this digestive constraint can explain in part why yellow-rumped warblers prefer diets containing long-chain unsaturated fatty acids to diets containing long-chain saturated fatty acids.     

Phospholipid and triacylglycerol fatty acid content and pattern in the cardiac endothelium of rats depleted in long-chain polyunsaturated omega 3 fatty acids

Rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3-depleted rats) display several features of the metabolic syndrome including hypertension and cardiac hypertrophy. This coincides with alteration of the cardiac muscle phospholipid and triacylglycerol fatty acid content and/or pattern. In the present study, the latter variables were measured in the cardiac endothelium of normal and omega3-depleted rats. Samples derived from four rats each were obtained from 16 female normal fed rats and three groups of 36-40 female fed omega3-depleted rats each aged 8-9, 15-16 and 22-23 weeks. At comparable mean age, the ratio between the square root of the total fatty acid content of phospholipids and cubic root of the total fatty acid content of triacylglycerols was lower in omega3-depleted rats than in control animals. The total fatty acid content of triacylglycerols was inversely related to their relative content in C20:4omega6. Other differences between omega3-depleted rats and control animals consisted in a lower content of long-chain polyunsaturated omega3 fatty acids in both phospholipids and triacylglycerols, higher content of long-chain polyunsaturated omega6 fatty acids in phospholipids, higher activity of delta9-desaturase (C16:0/C16:1omega7 and C18:0/C18:1omega9 ratios) and elongase [(C16:0 + C16:1omega7)/(C18:0 + C18:1omega9) and C20:4omega6/C22:4omega6 ratios], but impaired generation of C22:6omega3 from C22:5omega3 in the former rats. These findings support the view that cardiovascular perturbations previously documented in the omega3-depleted rats may involve impaired heart endothelial function.     

Growth characteristics and polyene sensitivity of a fatty acid auxotroph of Candida albicans.

A fatty acid auxotroph of Candida albicans 6406, designated A' 44 and originally isolated as an oleic acid requiring strain, has been shown to be a delta9 desaturase mutant. Although lacking this step in fatty acid biosynthesis, it appears to retain the ability to desaturate monounsaturated fatty acids. The polyene sensitivity of the organism grown on different fatty acid supplements varied between 0-08 +/- 0-02 and 1-20 +/- 0-30 microgram amphotericin B methyl ester ml-1 for exponentially growing cells. In spite of this variation, the sterol composition remained fairly constant, the major differences lying in fatty acid composition. Stationary-phase cells were more resistant to amphotericin B methyl ester, although again this change was not associated with changes in sterol content. The organism was most resistant when grown in the presence of oleic or linoleic acid. Protoplasts derived from resistant organisms grown on these two fatty acids were also resistant, indicating that the structure of the cell wall was less important than that of the plasma membrane in determining polyene sensitivity under these conditions.     

Bovine serum albumin-(7-hydroxycoumarin-4-acetic acid) complex: applications to the fluorometric measurement of fatty acid concentrations

A covalent complex between bovine serum albumin and 7-hydroxycoumarin-4-acetic acid (BSA-HCA) shows a strong fluorescence band at lambdamax = 450 nm upon excitation at 375 nm. Quenching of the fluorescence emission accompanies the association of fatty acids (FA) to BSA-HCA and the application of the complex as a spectrofluorometric probe for measurement of fatty acid concentrations in aqueous solution is examined. Binding constants for various long-chain fatty acids (Kd = 14-460 nM) and calibration curves characterizing the probe have been determined. Standardized assay conditions allow for accurate measurements in the concentration range of 10 nM to 5 microM. BSA-HCA provides a stable and sensitive fluorescence-based FA probe with potential biochemical applications.     

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.