Investigation of Ternary Complex Formations of Polyacrylic Acid with Bovine Serum Albumin in the Presence of Metal Ions by Fluorescence and Dynamic Light Scattering Measurements

In this paper, the complex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) in the presence metal ions at pH = 7 has been examined by using fluorescence and dynamic light scattering measurements. It has been observed that the most stable complexes of polyacrylic acid and bovine serum albumin have occurred in the presence of copper(II) ions. The other ions have the ability to form weak complexes between polyions and bovine serum albumin. To prior characterizing the interaction between bovine serum albumin and polyacrylic acid, the dynamic light scattering technique have been applied to determine the intensity-size distributions of the solutions of bovine serum albumin, polyacrylic acid, and ternary mixtures containing various molar ratios of bovine serum albumin to polyacrylic acid (the molar ratios of bovine serum albumin to polyacrylic acid has been taken equal to 0.5, 1.0, 1.5, 2.0 and 2.5) prepared at different molar ratios of copper(II) ions/acrylic acid unit. When the molar ratio of copper(II) ions to acrylic acid in the ternary mixtures has been lower than and equals to 0.3, two peaks have been observed in the curves of the intensity-size distributions due to contents of free bovine serum albumin and ternary complexes of polyacrylic acid-copper(II)-bovine serum albumin whereas when the molar ratio of copper(II) ions to acrylic acid equals to 0.4, the hydrodynamic diameter has pointed out only one peak. This result indicates that soluble and stable ternary complexes has occurred when the molar ratio of copper(II) ions to acrylic acid has been taken equal to 0.4.     

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

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

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.     

A physical-chemical model for cellular uptake of fatty acids: prediction of intracellular pool sizes

If the uptake of fatty acids by liver is a physical, not a biological, process, then the size and location of the intrahepatic pool of fatty acids can be predicted from uptake rates and thermodynamic data. The purpose of the experiments in this paper was to test the accuracy of this idea. Rat livers were perfused with palmitate bound to albumin, and the total amounts of palmitate removed from the perfusate were measured at 3-s intervals. The intrahepatic pools of palmitate calculated from these data were 13.8 and 23.0 nmol/g of liver at ratios of palmitate/albumin (mol/mol) (afferent side) of 2/1 and 4/1, respectively, in the steady state. The intrahepatic pools of palmitate calculated from the distributions of palmitate between membranes, H2O, albumin, and fatty acid binding protein and the measured first-order rate constants for acyl-CoA ligases in mitochondria and microsomes were 12.1 and 34.6 nmol/g for perfusate ratios of palmitate/albumin of 2/1 and 4/1, in the steady state. Intrahepatic pools of palmitate measured after establishment of a steady-state rate of uptake were 15.0 and 31.8 nmol/g for these ratios of palmitate/albumin of 2/1 and 4/1.     

The hyaluronan–protein complexes at low ionic strength: How the hyaluronidase activity is controlled by the bovine serum albumin

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at low HAase over HA concentration ratio and under low ionic strength conditions. The reason is the ability of long HA chains to form electrostatic and non-catalytic complexes with HAase. For a given HA concentration, low HAase concentrations lead to very low hydrolysis rates because all the HAase molecules are sequestered by HA, whilst high HAase concentrations lead to high hydrolysis rates because the excess of HAase molecules remains free and active. At pH 4, non-catalytic proteins like bovine serum albumin (BSA) are able to compete with HAase to form electrostatic complexes with HA, liberating HAase which recovers its catalytic activity. The general scheme for the BSA-dependency is thus characterised by four domains delimited by three noticeable points corresponding to constant BSA over HA concentration ratios. The existence of HA–protein complexes explains the atypical kinetic behaviour of the HA / HAase system. We also show that HAase recovers the Michaelis–Menten type behaviour when the HA molecule complexed with BSA in a constant complexion state, i.e. with the same BSA over HA ratio, is considered for substrate. When the ternary HA / HAase / BSA system is concerned, the stoichiometries of the HA–HAase and HA–BSA complexes are close to 10 protein molecules per HA molecule for a native HA of 1 MDa molar mass. Finally, we show that the behaviour of the system is similar at pH 5.25, although the efficiency of BSA is less.     

The effect of bovine serum albumin on partial reactions of palmitoyl-CoA chain elongation by rat liver microsomes.

In the absence of albumin, v/s curves for both condensation and overall chain elongation demonstrated that the specific activity for overall chain elongation was 3.7 times that of condensation. When the molar ratio of palmitoyl-CoA to albumin was greater than 2 : 1, the specific activity of chain elongation exceeded that of condensation. At these low albumin concentrations, in the absence of NADPH, the beta-ketostearoyl-coA was converted back to palmitate. This cleavage reaction is inhibited by albumin in a concentration-dependent manner. When the palmitoyl-CoA to albumin molar ratio was less than 2 : 1, the specific activity for condensation exceeded that for overall chain elongation and some beta-ketostearate was shown to accumulate under chain elongation conditions. The specific activity for dehydration of beta-hydroxystearoyl-CoA was maximal when the acyl-CoA to albumin molar ratio was between 10 : 1 and 4 : 1 but the rate of this reaction was not markedly influenced by variations in albumin concentration. The specific activity for the NADPH-dependent reduction of 2-trans-octa-decenoyl-CoA was 18 nmol . min(-1) . mg(-1) in the absence of albumin and increased to a maximum of 112 when the substrate to albumin molar ratio was 2 : 1. At higher albumin concentrations the reductase reaction was inhibited. Conversely, the specific activity for the reverse dehydrase was maximal at low albumin concentrations and the rate of this reaction declined as the albumin concentration increased. Our results demonstrate that albumin not only alleviates a substrate induced inhibition but also regulates the metabolic fate of 2-trans-octadecenoyl-CoA and in this regard may possibly substitute for acyl-CoA binding proteins.     

Trace element uptake in liver cells. 2. Effect of different proteins in the medium on the uptake of copper and zinc by hepatoma cells

Cultured rat hepatoma cells (HTC-cells) were used to study the uptake of copper and zinc from a minimal salt-glucose medium, supplemented with albumin from different species or with ovalbumin. Competitive equilibrium dialysis showed that at low molar ratios of metal/protein (less than 1) the affinity for copper of human and bovine albumin was about equal, but that of dog albumin or ovalbumin was much lower. Only a small difference in affinity for zinc could be detected between human albumin and ovalbumin. Supplementing the medium with the different proteins the rate of copper uptake in the cell at a given molar Cu/protein ratio increased as follows: human albumin congruent to bovine albumin less than dog albumin less than ovalbumin. When the molar Cu/protein ratio was increased, a discontinuity was seen with all three albumin species at a ratio of about 1. In contrast, the zinc uptake mimics that of Cu/ovalbumin, and no discontinuity was observed using different molar Zn/protein ratios. These results indicate that the rate of copper and zinc uptake depends strongly on its affinity for the protein: a low affinity leads to a high uptake. The results suggest further that at physiologic concentrations zinc is taken up by a mechanism different from that for copper.     

Interaction of α-lactalbumin with dimyristoylphosphatidylcholine vesicles. III. Influence of the temperature and of the lipid-to-protein molar ratio on the complex formation

We investigated the interaction between α-lactalbumin and sonicated dimyristoylphosphatidylcholine at pH 4 and different temperatures. (1) At 23°C and lipid-to-protein molar ratios below 170, the interaction results in a disruption of the original vesicles to form smaller complex particles. By the sedimentation velocity method we determined for this particle a molar mass of (1.05 ± 0.16) · 10⁶ g·mol^?1. The lipid-to-protein molar ratio within the complex particle is 70/1, as earlier estimated. It follows that there are approximately 1200 lipid and 17 α-lactalbumin molecules per particle. At molar ratios above 170, α-lactalbumin strongly associates with the vesicles. In this case the vesicle entity remains. The ability of α-lactalbumin to break up the vesicles at this temperature is determined by the number of protein molecules which are required in the complex particle. (2) By means of fluorescence polarization of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene and energy transfer of the tryptophan groups of the protein to 1,3-(1,1′-dipyrenyl)propane located in the hydrocarbon region of the vesicles, it is shown that with increasing temperature above 25°C, complexes of decreasing internal lipid-to-protein molar ratio are formed. However, by electron microscopy we show that the overall size of these complexes remains approximately the same, i.e., bars with dimensions $\text{70 × 220}\text{A}\text{?}$. A temperature-reversible transformation occurs between these complexes, which cannot be isolated by gel chromatography. In contrast, the complex of molar ratio 70/1 remains stable at lower temperatures.     

Effects of several common long chain fatty acids on the properties and lipid composition of the very low density lipoprotein secreted by the perfused rat liver.

1. Livers from normal fed male rats were perfused in vitro with a bloodless medium which contained intially 3% bovine serum albumin and 100 mg% glucose. Albumin alone, or myristate (14 : 0), palmitate (16 : 0), palmitoleate (16 : 1), stearate (18 : 0), oleate (18 : 1), or linoleate (18:2) was infused at a constant rate (496 mumol/4 h), as a complex with albumin, during the experiment. 2. The very low density lipoprotein secreted by the liver after infusion of unsaturated fatty acids (16 : 1, 18 :1, 18 : 2) has a faster rate-zonal mobility in the ultracentrifuge and is, therefore, probably a larger particle with fewer moles of phospholipid and cholesterol relative to triacyglycerol (triacyglycerol/phospholipids/cholesterol = 100/25.1/16.4) than the very low density lipoproteins produced after infusion of saturated (14 : 0, 16 : 0, 18 : 0) fatty acids (triacyglycerol/phospholipids/cholesterol = 100/30.1/19.1). The molar ratio of phosphoipids/cholesterol of the very low density lipoprotein was similar regardless of which fatty acid was infused. The predominant fatty acid of the very low density lipoprotein or hepatic triacyglycerol, in all cases, was the infused acid. 3. We conclude that free fatty acid regulates the quantity and proportions of triacyglycerol, phospholipids, and cholesterol secreted by the liver in the very low density lipoprotein, and therefore, may secondarily influence concentrations of lipids in the very low density lipoprotein and other plasma lipoproteins circulating in vivo.     

Optical properties of chlorophyll <Emphasis Type="Italic">a</Emphasis> and β-Carotene mixtures adsorbed on bovine serum albumin

The fluorescence of β-carotene/chlorophyll a mixtures in complex with bovine serum albumin in water solution was found to exceed that of one-pigment complexes, being maximal at initial Car/Chl molar ratios of 8–4, whereas the amount of pigment adsorbed on BSA was maximal for an equimolar mixture. The fluorescence spectra of Car-BSA and Chl-BSA complexes largely overlapped (maxima at 684 and 690 nm, respectively).     

Role of high-density lipoprotein in transport of circulating bilirubin in rats

The analbuminemic rat strain established by Nagase et al. (Nagase, S., Shimamune, K., and Shumiya, S. (1979) Science 205, 590-591) exhibits hereditary deficiency in albumin biosynthesis. Serum bilirubin concentration is rather lower in homozygous (aa) rats (0.009 +/- 0.002 mg/dl) as compared with heterozygous (Aa) rats (0.047 +/- 0.009 mg/dl) or wild-type Sprague-Dawley (AA) rats (0.034 +/- 0.006 mg/dl) as evidenced by high pressure liquid chromatography analysis of bilirubin. After intravenous administration of various amounts of [heme-3H]hemoglobin in rats, [3H]bilirubin derived from [3H]heme of hemoglobin in vivo is more efficiently excreted into bile in aa rats than in Aa or AA rats. [3H]Bilirubin is exclusively bound with high-density lipoprotein (HDL) in aa rats, and a significant amount of [3H]bilirubin is shown to bind with HDL in Aa or AA rats in vivo. Scatchard plots revealed that [3H]bilirubin is bound with HDL in three binding modes depending on the molar ratio of [3H]bilirubin to HDL: Kd = 0.8 X 10(-7) M (molar ratio, 0.02-0.06), Kd = 1.6 X 10(-6) M (molar ratio, 0.06-0.41), and Kd = 1.2 X 10(-4) M (molar ratio, 0.79-9.02). Even under extreme conditions of excess hemoglobin administration, the molar ratio remains under 0.041; and thus, expected the Kd value would remain around 0.8 X 10(-7) M. Binding of [3H]bilirubin to rat serum albumin revealed two distinct binding modes depending on the molar ratio of [3H]bilirubin to rat serum albumin: Kd = 3.6 X 10(-7) M (molar ratio, 0.03-0.21), and Kd = 5.0 X 10(-6) M (molar ratio, 0.21-2.46). Under physiological conditions in Aa or AA rats, the former mode would be more reliable than the latter. Thus, HDL could bind with approximately 4.5 times higher affinity than rat serum albumin in Aa or AA rats under physiological conditions in vivo.     

Use of fluorescence enhancement technique to study bilirubin–albumin interaction

Bilirubin–albumin solution gave an emission spectrum in the wavelength range 500–600 nm with emission maxima at 528 nm when excited at 487 nm. The magnitude of fluorescence intensity increased on increasing bilirubin/albumin molar ratio. At three different albumin concentrations, namely, 1.0, 2.5 and 10.0 μM, there was an initial linear increase in fluorescence up to a molar ratio 1.0 in all cases beyond which it sloped off or decreased. This fluorescence enhancement was used to calculate the binding parameters of bilirubin–albumin interaction and the value of binding constant was found to be 1.72×10⁷ l/mol similar to the published values obtained with other methods. Different serum albumins, namely, human (HSA), goat (GSA), pig (PSA) and dog serum albumins (DSA) bound bilirubin with almost the same affinity when studied by the technique of fluorescence enhancement. Bilirubin–albumin interaction was also studied at different pH and ionic strengths. There was a decrease in bilirubin–albumin complex formation on either decreasing the pH from 9.0 to 7.0 or increasing the ionic strength from 0.15 to 1.0. These results suggest that the technique of fluorescence enhancement can be used successfully to study the bilirubin–albumin interaction.     

A spectroscopic study of the reaction of NAMI, a novel ruthenium(III)anti-neoplastic complex, with bovine serum albumin.

The reaction of Na[transRuCl4Me2SO(Im)] (NAMI; where Im is imidazole), a novel anti-neoplastic ruthenium(III) complex, with BSA, was studied in detail by various physico-chemical techniques. It is shown that NAMI, following chloride hydrolysis, binds bovine serum albumin tightly; spectrophotometric and atomic absorption data point out that up to five ruthenium ions are bound per albumin molecule when BSA is incubated for 24 h with an eightfold excess of NAMI. CD and electronic absorption results show that the various ruthenium centers bound to albumin exhibit well distinct spectroscopic features. The first ruthenium equivalent produces a characteristic positive CD band at 415 nm whereas the following NAMI equivalents produce less specific and less marked spectral effects. At high NAMI/BSA molar ratios a broad negative CD band develops at 590 nm. Evidence is provided that the bound ruthenium centers remain in the oxidation state +3. By analogy with the case of transferrins it is proposed that the BSA-bound ruthenium ions are ligated to surface histidines of the protein; results from chemical modification experiments with diethylpyrocarbonate seem to favor this view. Spectral patterns similar to those shown by NAMI are observed when BSA is reacted with two strictly related ruthenium(III) complexes Na[transRuCl4(Me2SO)2] and H(Im)[transRuCl4(Im)2] (ICR), implying a similar mechanism of interaction in all cases. It is suggested that the described NAMI-BSA adducts may form in vivo and may be relevant for the biological properties of this complex; alternatively NAMI/BSA adducts may be tested as specific carriers of the ruthenium complex to cancer cells. Implications of these findings for the mechanism of action of NAMI and of related ruthenium(III) complexes are discussed.     

Solubilization of polyaromatic hydrocarbons by recombinant bioemulsifier AlnA

AlnA is the protein responsible for the emulsifying and solubilizing activity of the Acinetobacter radioresistens KA53 bioemulsifier alasan. AlnA was produced in Escherichia coli, purified to homogeneity and then used to measure the enhanced solubility of 12 polyaromatic hydrocarbons (PAHs). The amount of PAH solubilized was directly proportional to AlnA concentration. The ratio of PAH solubilized by 40 μg/ml AlnA compared to that soluble in the aqueous buffer varied greatly, from 4 (fluorene) to 81 (hexylbenzylcyclosilane). Calculations of moles PAH solubilized per mole AlnA yielded values from 4.3 (hexylphenylbenzene) to 55.8 (1,10-phenanthrolene). There was no obvious relationship between the amount of PAH solubilized and its molecular weight or intrinsic solubility. Native gel electrophoresis indicated that AlnA formed hexamers in the presence of PAHs. With molar ratios of fluorene to AlnA of 0.75 or less, only the monomer was observed, whereas at ratios of 7.5 or higher, only the hexamer was detected. At an intermediate molar ratio of 2.6, both monomer and hexamer appeared. The data indicate that PAHs are initially solubilized by binding to the monomeric form of AlnA, and as the amount bound increases above one molecule PAH per AlnA, the protein aggregates to form a specific oligomer of 5–8 monomers which allows for the binding and solubilization of more PAH. Electronic Publication     

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.     

The enzymic degradation of ovalbumin and its glycopeptides

1. Ovalbumin glycopeptides, freed from all amino acids other than aspartic acid and a small proportion of leucine by repeated digestion with Pronase, were hydrolysed by 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (glycoaspartamidase) to the corresponding oligosaccharides. The glycoaspartamidase did not attack ovalbumin itself. 2. Ovalbumin, with mannose/hexosamine ratio 5:4, lost 1.5moles of N-acetylglucosamine and more than 2moles of mannose after incubation with alpha-mannosidase and beta-N-acetylglucosaminidase respectively. 3. In ovalbumin glycopeptides with approximate mannose/hexosamine ratios 5:3 and 5:4, one and two N-acetylglucosamine residues respectively were accessible to the action of beta-N-acetylglucosaminidase. 4. A mixture of alpha-mannosidase and beta-N-acetylglucosaminidase, acting on an ovalbumin glycopeptide with mannose/hexosamine ratio 5:3.7, removed nearly 4moles of mannose and 1.5moles of N-acetylglucosamine. 5. alpha-Mannosidase removed about 1.5moles of mannose from the ovalbumin oligosaccharide with mannose/hexosamine ratio approx. 5:3. The subsequent action of beta-N-acetylglucosaminidase liberated less than 1mole of N-acetylglucosamine and made at least 1mole further of mannose accessible to alpha-mannosidase action. 6. It is concluded that the carbohydrate moiety of ovalbumin is linked through a glycosyl group to asparagine. In a molecule with mannose/hexosamine ratio 5:4, there are two beta-N-acetylglucosamine residues linked together in a terminal position, followed by alpha-mannose. There is also present a side chain containing two alpha-mannose units.     

Electron spin resonance study of the copper(II) complexes of human and dog serum albumins and some peptide analogs

Electron spin resonance spectra of the first Cu(II) complexes of human serum albumin, dog serum albumin, l-aspartyl-l-histidine N-methylamide and glycyl-glycyl-l-histidine N-methylamide have been studied using isotopically pure ⁶⁵Cu in its chloride form. At 77° K, the esr spectra of Cu(II) complex of human serum albumin exhibited only one form of esr signal between pH 6.5 and 11. No intermediate forms were detected. The presence of an equally spaced nine-line superhyperfine structure with spacing ～15 G indicated considerable covalent bonding between Cu(II) and four nitrogen atoms derived from the protein. The esr spectrum form of Cu(II) bound to human serum albumin detected at neutral pH would be consistent with the participation of four nitrogens from the α-NH₂ group, two peptide groups, and the imidazole group of a histidine residue. In contrast, the esr spectra of Cu(II)-dog serum albumin complex showed a transition from a low pH form to a high pH form as the pH was increased to 9.5. These spectral changes were found to be reversible upon lowering the pH. Ligand superhyperfine splittings in the low pH form of the esr signal of Cu(II)-dog albumin were not resolved. The distinct pH dependence of the esr signals observed in human and dog serum albumin complexes could be correlated to their respective optical spectra changes as a function of pH. At room temperature and in the pH range between 6 and 11, the esr spectra of Cu(II) complexes of l-aspartyl-l-alanyl-l-histidine N-methylamide and glycyl-glycyl-l-histidine N-methylamide exhibited a well-resolved nine-line superhyperfine structure indicating metal coordination with four equivalent nitrogen atoms of peptide.     

Interrelationships and metabolic effects of fatty acids in the perfused rat liver at hyperthermic temperatures

Livers of fasted rats were perfused for 70 min at 37 degrees-43 degrees C in the presence or absence of acetate, octanoate or palmitate. Hepatic biosynthetic capacity was assessed by measuring rates of gluconeogenesis, ureogenesis, ketogenesis and O2 consumption. In the presence of each fatty acid, gluconeogenesis, ureogenesis and oxygen consumption were maintained at 37 degrees and 42 degrees C. At 43 degrees, the rate of glucose formation decreased markedly and rates of ureogenesis and oxygen consumption were distinctly lower. As the temperature was increased from 37 degrees to 43 degrees C without fatty acids, i.e. albumin only, there was a progressive decrease in the rate of gluconeogenesis while the ratio of net C3 utilized to glucose formed, increased successively. The values of this ratio in the presence of palmitate or octanoate at 43 degrees were smaller than those for albumin or acetate, but higher than the figure of 2 for complete conversion of C3 units to glucose. Although fatty acid was added in equimolar amounts of C2 units, total ketone formation was influenced significantly by chain length. Hepatic ketogenesis was similar at 37 degrees with albumin, palmitate, or acetate, but was stimulated significantly by octanoate at 37 degrees and 42 degrees C. At 42 degrees, ketone formation increased in the presence of palmitate. At 43 degrees C, ketogenesis with palmitate or octanoate decreased, while that with acetate or albumin was maintained at the same lower rates. The ratio of 3-hydroxybutyrate to acetoacetate in the perfusate was increased with palmitate at the end of perfusion at 37 degrees and 42 degrees C or octanoate at 42 degrees and 43 degrees C. Thus, long (palmitate)- and medium (octanoate)- but not short (acetate)-chain fatty acids enhance not only beta-oxidation, but influence the redox state of hepatic mitochondria with an increase in the state of reduction of the pyridine nucleotides. Such a shift in the redox state would be operable in the perfused liver even at 43 degrees C and may be responsible for improved conversion of lactate to glucose when medium- or long-chain fatty acids are present at hyperthermic temperatures.     

Copper and nickel binding to canine serum albumin. A circular dichroism study

The binding of copper and nickel to canine serum albumin has been studied using circular dichroism. In the 320-700 nm region, only a single positive extremum was observed at about 664-667.5 nm for copper bound to canine serum albumin. The intensity of this extremum was found to increase until a Cu2+/albumin molar ratio of 3 was reached. Further addition of Cu2+ led to a decrease in ellipticity. The absence of any extrema in the 560-570 and 480-510 nm regions showed that histidines were not involved in copper binding to canine albumin. In the case of nickel, initial binding was found to take place at the N-terminal tripeptide. At higher nickel to albumin molar ratios, circular dichroism spectra indicated the presence sulfur containing ligands but showed no evidence for the involvement of histidines. Canine serum albumin was found to bind six or more Cu2+ and Ni2+ ions with affinities that are lower than for human or bovine serum albumin.     

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.