The ability to bind albumin is correlated with nitric oxide sensitivity in Moraxella catarrhalis

Moraxella catarrhalis is sensitive to NO generators e.g. S -nitroso- N -acetylpenicillamine (SNAP) and sodium nitroprusside (SNP), but can spontaneously develop higher SNP tolerance. Using SDS-PAGE of outer membrane proteins and immunoblotting for serum albumin, we found that the wild strain bound more blood-medium-derived albumin than the SNP-resistant variant did. There was a negative correlation between NO tolerance and the presence of serum albumin in the medium. We suggest that M. catarrhalis can change its surface properties to avoid binding albumin and thereby increase its resistance to NO. Growth of Moraxella is affected by iron, and that may have influenced our results. Using chrome azurol S plates as an indicator, we noted that both albumin and SNP have a strong affinity for iron(III).     

Multiple fatty acid binding to albumin in human blood plasma

Binding equilibria of long-chain fatty acids to human serum albumin, in serum or plasma, were studied by a dialysis exchange rate technique. Palmitate was added to citrated plasma in vitro and it was observed that between six and ten palmitate molecules were bound to albumin with nearly equal affinity. Observations in vivo gave similar results in the following series: (a) in two volunteers with increased fatty acid concentrations after fasting, exercise, and a cold shower: (b) in three male volunteers in whom high concentrations of non-esterified fatty acids, up to 4.6 mM, were induced by intravenous administration of a preparation of lecithin/glycocholate mixed micelles, and (c) in 81 patients with diabetes mellitus, type I. The binding pattern of palmitate in serum or plasma is essentially different from that observed with palmitate added to buffered solutions of pure albumin when two molecules are tightly bound and about four additional molecules with lower affinity. The differences may partly be explained by the presence of chloride ions in blood plasma, reducing the affinity for binding of the first two fatty acid molecules, and partly by facilitated binding of several molecules of mixed fatty acids, as found in plasma.     

Binding of cardiolipin/lecithin and monoamine oxidase to lipid-depleted mitochondrial membrane structure.

Lipid-depleted pig liver mitochondrial residues were incubated with different proportions of the acidic phospholipid cardiolipin and the zwitterionic phospholipid lecithin in either separate or mixed liposomes. When cardiolipin and lecithin were present in separate liposomes all of the cardiolipin but no lecithin bound to the residues. When present in the same liposomes, cardiolipin also caused binding of lecithin to the mitochondrial residues. When monoamine oxidase solubilized from pig liver mitochondria by extraction of the phospholipids was included in the incubation, binding of the enzyme to the residues occurred in the presence of cardiolipin. The percentage of enzyme bound followed the same trend as the binding of phospholipids to the mitochondrial residues.     

Binding of spin-labeled local anesthetics to phosphatidylcholine and phosphatidylserine liposomes

A homologous series of spin-labeled local anesthetics, 2-[N-methyl N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-p-alkoxybenzoates were shown to bind to phosphatidylcholine and phosphatidylserine liposomes. Under similar conditions, 70% of the ethoxy homolog (R2C) of these spin-labeled local anesthetics bound to synthetic dipalmitoyl lecithin while 98% bound to phosphatidylserine liposomes. Five percent of R2C's bound signal could be released by 4 mm calcium from phosphatidylserine liposomes, but calcium had no effect on R2C bound to synthetic lecithin. The butoxy (R4C) and hexyloxy (R6C) homologs bound to phosphatidylcholine in the order R6C > R4C. All of R6C and all of R4C were bound to phosphatidylserine liposomes, while only 90% of R6C bound to synthetic dipalmitoyl lecithin. Calcium was incapable of displacing bound R4C or R6C from either phosphatidylcholine or phosphatidylserine liposomes. The results are discussed in light of anesthetic binding by electrostatic and Van der Waal's forces to phospholipids.     

Enantioselective binding of mephobarbital to plasma proteins

The enantioselective protein binding of mephobarbital (MPB) was investigated in human plasma and human serum albumin solutions by equilibrium dialysis. A small but statistically significant difference was observed in the in vitro plasma protein binding of the enantiomers; (S)-MPB was approximately 59% bound and (R)-MPB approximately 67% bound. The binding to albumin [(S)-MPB: approximately 29% bound, and (R)-MPB: approximately 41% bound] was less than to plasma proteins but showed somewhat greater enantioselectivity, suggesting that albumin binding is a major source of the enantioselectivity in plasma. The effects of MPB concentration, of varying enantiomeric concentration ratio, and of phenobarbital on the enantioselective binding of MPB were studied. The effect of age was also investigated by measuring the binding in plasma from 8 young (18-25 yr) and 8 elderly (greater than 60 yr) male subjects who took single doses of MPB. The results were in close agreement with the in vitro binding data, and the binding of both enantiomers was marginally but significantly lower in the young compared with the elderly subjects. These differences in binding were consistent with previously observed pharmacokinetic differences between the two subject groups.     

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

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

The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation

Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.     

Oestriol binding to plasma proteins

Simple diffusion experiments indicated that oestriol was retained by human pregnancy plasma more effectively than by albumin solutions of a corresponding concentration. Oestriol bound (Ka = 6 X 10(6) l/mol at 4 degrees C) to a glycoprotein which had been isolated from plasma by adsorption to Concanavalin A. The free energy of binding at 37 degrees C was -38 kJ/mol. Competition experiments indicated that the oestriol binding glycoprotein had properties expected of sex hormone binding globulin. The distribution of oestriol among the protein fractions of human pregnancy plasma--glycoprotein bound 7.8%, albumin bound 78.6%, unbound 13.6%--suggests that this glycoprotein plays little part in the transport of oestriol.     

Molecular aspects of the interaction between albumin and tannin-treated erythrocytes in the process of hemosensitization. Report 1. Stable and unstable effects. Quantitative parameters]

A study was made of the process of interaction between the tannin-treated sheep erythrocytes and the human serum albumin. Protein binding increased, whereas the loose/stable binding ratio decreased with the rise of the initial protein concentration. The process of stable albumin binding is described by the Langmour equation, this permitting to assess the limiting binding values -- about 6-10(5) molecules per erythrocyte. Albumin molecules were bound by erythrocyte with their greatest surface. At any concentration albumin was incapable of occupying more than 85% of the erythrocyte surface; at 90% binding level it blocked only 51--52% of the tannin-treated erythrocyte. The data obtained were regarded as a methodical basis for the preparation of erythrocytic diagnostic agents with the proteins of albumin type.     

Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study.

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

Distribution and percentages of non-protein bound contraceptive steroids in human serum

It has been shown that albumin bound steroids are taken up by the rat brain in addition to nonprotein bound steroids and it has also been suggested that cortisol binding globulin (CBG) may facilitate progesterone uptake by the rat uterus but not the brain. Recently serum sex-hormone binding globulin (SHBG) has been identified in the cytoplasm of sex steroid target cells. Thus the distribution of synthetic steroids between various protein bound and nonprotein bound components in serum may influence their bioavailability at different target tissues. The authors employed a newly developed technique, centrifugal ultrafiltration-dialysis. The results showed that there are no differences in percentages of nonprotein bound ethinyl estradiol (EE2), and cyproterone acetate (CA) with respect to sex or serum SHBG and CBG binding capacities. However serum percentages of nonprotein bound norethisterone (NET) (p0.05) are significantly lower in women than in men. Also the percentages of nonprotein bound NET and D-norgestrel are both very much lower (p0.001) in serum from pregnant women when compared to nonpregnant women. These differences appear to be inversely related to serum SHBG binding capacity. The percentages of nonprotein bound NET and D-norgestrel in heat treated serum from men and nonpregnant women are identical and largely represent the contribution of albumin binding alone. In addition heat labile binding proteins do not appear to influence the percentages of nonprotein bound EE2 and CA and it can be inferred that EE2 and CA are almost exclusively bound by albumin in native serum; 98.5% of EE2 and 93% of CA are bound to albumin. In contrast the percentages of nonprotein bound NET and D-norgestrel in native serum are inversely related to SHBG binding capacity. This data indicate that the nonprotein bound and albumin bound factors of NET and D-norgestrel may vary by as much as 2-3 fold between women who are known to have subnormal or supranormal levels of serum SHBG binding capacity and it is suggested that measurements of serum SHBG binding capacity may provide a method of assessing the lowest effective dose of these 2 progestins in individual subjects to help reduce side effects associated with their use. Future studies should address the effect of serum steroid concentrations on the actual nonprotein bound serum concentrations and distribution of these progestins.     

Use of amphotericin B as optical probe to study conformational changes and thermodynamic stability in human serum albumin

The binding of polyene antibiotic amphotericin B to serum albumin was studied using absorption, fluorescence, and circular dichroism techniques. A hypochromic effect was observed in the absorption spectrum of amphotericin B in the presence of albumin with maxima at 366 nm, 385 nm, and 408 nm, which correspond to the absorption of the monomeric form of amphotericin B. A modification on the circular dichroism spectrum of amphotericin B in the presence of albumin was observed at bands 329 nm and 351 nm (excitronic interaction), which suggests that only amphotericin B monomer is bound to the protein. Amphotericin B perturbs the specific markers for sites I, II, and fatty acid binding site bound to these sites, suggesting that amphotericin B interacts with a great binding area in albumin. Lysines 199 and 525 in albumin participate in the molecular interaction between amphotericin B and the protein. The absorption spectrum of amphotericin B bound to albumin was sensitive to the chemical and thermal treatment of the protein, to neutral-basic transition of albumin and to conformational changes induced by the binding of other ligands to this protein.     

Binding of the chloroplast coupling factor CF to lipid vesicles

The binding of the chloroplast coupling factor CF to lipid vesicles was analyzed by gel filtration. CF can be bound to vesicles made of chloroplast lipids but not of lecithin. The presence in the vesicle walls of a proteolipid subunit of the hydrophobic component of the coupling factor increases the binding of CF. The apparent binding constant and the maximum protein/lipid ratio are calculated. The Ca2+-ATPase activity of bound CF is markedly lower than that of dissolved CF. It is confirmed that the proteolipid is a N,N'-dicyclohexylcarbodiimide sensitive proton channel. The binding of CF on proteolipid vesicles decreases their proton permeability.     

Effects of a lecithin and catalase containing semen extender and a second dilution with different enhancing buffers on the quality of cold-stored canine spermatozoa

In the present study, a diluent containing 0.8% lecithin (Minitube®, Tiefenbach, G) for the cold storage of canine semen was compared to a Tris-egg yolk extender (TRIS-EY) containing 20% egg yolk. For this purpose, aliquots of 10 mixed ejaculates (main fractions) were either diluted with TRIS-EY or with three lecithin extenders containing 0.8% lecithin with or without catalase and tyrosine. All samples were examined by computer assisted sperm analysis (CASA), chlortetracycline assay (CTC) and flow cytometry, sperm chromatin structure assay (SCSA) and zona pellucida binding assay (ZBA). Samples were then cold stored for 8 d and examinations repeated at days 3 and 8. Measurement in the CASA were repeated daily and prior to measurement, each sample was diluted with each of 4 enhancers with or without acetylcarnitine. The use of an enhancer proved to be essential for all extenders and after 8 d of cooling, progressive motility (P) and viability (V) still averaged > 70% and > 80% with the lecithin extenders containing additives, whereas with TRIS-EY and without additives it was significantly lower (P < 0.05). The percentage of capacitated spermatozoa did not differ between extender groups and there was no significant increase in acrosome reactions (AR) within 3 d. The chromatin status of cells was not changed by cooling within 8 d. The ZBA showed that with additives, significantly more spermatozoa bound to oocytes when a lecithin extender with additives was used (P < 0.05). In conclusion, the 0.8% lecithin extender containing catalase, conserved P and V during 8 d of cold storage better than the TRIS-EY extender, however, only when an enhancer was used; addition of acetylcarnitine to the enhancer did not further improve semen quality. The here introduced lecithin extender / enhancer combination is a useful tool for prolonged storage of cooled semen with excellent longevity and binding ability; addition of tyrosine to the extender did not improve semen quality.     

Interaction of liposomes with a varying lipid composition with hepatocytes in vitro

Interaction of liposomes from egg lecithin, phospholipids and gangliosides of rat liver with rat hepatocyte monolayers was investigated. It was shown that liposomes from phospho- and glycolipids of the liver were bound by rat hepatocytes to a far greater degree than lecithin liposomes. Liver gangliosides increased active endocytosis of liposomes by hepatocytes. Preincubation of hepatocytes with gangliosides reduced the binding of phosphoglycolipid liposomes by those cells.     

Albumin prevents nonspecific transferrin binding and iron uptake by isolated hepatocytes

Bovine serum albumin inhibits binding of transferrin by hepatocytes in suspension by 60-70%. Iron uptake is inhibited by less than 20%. A Scatchard analysis of the transferrin-binding data reveals a biphasic plot in the absence of bovine serum albumin, but a monophasic plot in the presence of bovine serum albumin. Bovine serum albumin inhibits low-affinity binding of transferrin (125000 molecules/cell), but has no effect on high-affinity binding (38000 molecules/cell). In pronase-treated cells, transferrin binding is reduced by 40%, and when bovine serum albumin is added, the binding is reduced by a further 40%. Corresponding figures for iron uptake are 70 and 10%, respectively. The results are strong evidence that the major part of iron uptake by hepatocytes occurs from transferrin bound to the plasma membrane transferrin receptor.     

Binding of physostigmine to rat and human plasma and crystalline serum albumins

The binding of 3H-physostigmine (3H-Ph) to human and rat plasma proteins and crystalline serum albumin was studied by ultrafiltration technique. This study showed that the percentage of 3H-Ph bound to rat plasma slightly decreased from 49% to 41% whereas human plasma showed an increase in binding from 29% to 43% over a 50-fold increase in drug concentration. Human plasma samples which were collected in a bag coated with citrate phosphate dextrose adenine-1 solution bound 50% less 3H-Ph than samples collected with EDTA indicating a drug-drug interaction between 3H-Ph and anticoagulants. No significant change in binding was observed if the samples were frozen prior to use. Scatchard plots for binding of 3H-Ph resulted in a positive slope for human plasma and a negative slope for rat plasma; whereas curvilinear Scatchard plots with negative slopes were obtained for binding to human and rat crystalline serum albumin.     

Determination of binding characteristics as a measure for effective albumin using different methods

Background & aimsTransport functions of albumin are of clinical and pharmacological interest and are determined by albumin's properties like posttranslational modifications or bound ligands. Both are affected in pathological conditions and in therapeutic grade albumin solutions. The term effective albumin concentration was introduced as a measure of functionally intact albumin. Our aim was to evaluate the impact of ligands and modifications with different approaches as a measure of effective albumin.Approach & resultsWe used a spin labelled fatty acid and dansylsarcosine to characterize binding properties of albumin i) prepared from plasma of patients and healthy control donors, ii) measured directly out of plasma, iii) research grade albumin, iv) in vitro modified albumin, and v) therapeutic infusion solutions before and after removal of stabilizers.Bilirubin is the main determinant for binding function in patients' albumin. In in vitro prepared albumin bound fatty acids correlated with impaired binding. Human nonmercaptalbumin1, not human nonmercaptalbumin2, showed reduced binding properties. Binding and transport function of therapeutic albumin was severely impaired and restored by filtration. Glycation of research grade albumin had no effect on the binding of dansylsarcosine and only a minor effect on fatty acid binding.ConclusionsOur results suggest that effective albumin -in terms of binding properties- is primarily determined by bound ligands and only to a minor extent by posttranslational modifications. Characterizing albumin directly from plasma better reflects the physiological situation whereas in the case of therapeutic grade albumin stabilizers should be removed to make its binding properties accessible.     

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

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

Mechanisms involved in the cellular uptake of hematoporphyrin by rat hepatoma cells

To clarify the mechanisms involved in the specific uptake of hematoporphyrin by cancer cells, we investigated the interaction of the heme- and/or hematoporphyrin-hemopexin complexes with rat hepatoma dRLh-84 cells. Hemopexin bound to the cells in a saturable, time- and temperature-dependent manner. The cells exhibited 0.55 nmol of binding sites/mg of protein for the heme-hemopexin complex and 0.38 nmol for the hematoporphyrin-hemopexin complex. The dissociation constants (Kd) for the heme-hemopexin and hematoporphyrin-hemopexin complexes were 0.57 and 0.54 microM, respectively. Specific binding of the labeled hemopexin was inhibited by the unlabeled heme- and hematoporphyrin-hemopexin complexes but was unaffected by albumin or neoglycoprotein. Hematoporphyrin bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that hematoporphyrin bound hemopexin was taken up by dRLh-84 cells, via the hemopexin receptors. When the hematoporphyrin-albumin complex was incubated with the cells, the hematoporphyrin-[125I]albumin complex bound to the cells in a time and temperature-dependent manner. Here the binding was not saturated up to 100 micrograms/ml of albumin. The binding of hematoporphyrin-[125I]albumin was partially inhibited by unlabeled albumin and hemopexin. Hematoporphyrin bound to albumin was taken up by the cells at 37 degrees C. Thus, the albumin-dependent uptake of hematoporphyrin by rat hepatoma dRL-84 cells could be differentiated from the hemopexin-mediated uptake of hematoporphyrin.