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.     

Species-dependent stereoselective drug binding to albumin: a circular dichroism study

Drug binding to albumins from different mammalian species was investigated to disclose evidence of species-dependent stereoselectivity in drug-binding processes and affinities. This aspect is important for evaluating the reliability of extrapolating distribution data among species. The circular dichroism (CD) signal induced by drug binding to the albumins [human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), and dog serum albumin (DSA)] were measured and analyzed. The binding of selected drugs and metabolites to HSA significantly differed from the binding to the other albumins in terms of affinity and conformation of the bound ligands. In particular, phenylbutazone, a marker of site one on HSA, showed a higher affinity for binding to BSA with respect to RSA, HSA, and DSA, respectively. In the case of diazepam, a marker of site two on HSA, the affinity decreased in order from HSA to DSA, RSA, and BSA. The induced CD spectra were similar in terms of energy and band signs, suggesting almost the same conformation for the bound drug to the different albumins. Stereoselectivity was high for the binding of ketoprofen to HSA and RSA. A different sign was observed for the CD spectra induced by the drug to the two albumins because of the prevalence of a different conformation of the bound drug. Interestingly, the same induced CD spectra were obtained using either the racemic form or the (S)-enantiomer. Finally, significant differences were observed in the affinity of bilirubin, being highest for BSA, then decreasing for RSA, HSA, and DSA. A more complex conformational equilibrium was observed for bound bilirubin.     

Immobilized metal ion affinity chromatography of serum albumins.

The interaction of several serum albumins with chelated (iminodiacetate, IDA) and immobilized (agarose-IDA) metal ions, Co2+, Ni2+, Cu2+ and Zn2+, was studied. There was no retention of human, bovine, porcine, murine and avian albumins on IDA-Zn(II) and IDA-Co(II) columns. However, all albumins studied, i.e., those of: man, cow, pig, dog, rabbit, rat, mouse, chicken and pigeon were retained on IDA-Cu(II) columns, and all except dog albumin were retained also on IDA-Ni(II). The recognition of albumins by chelated and immobilized transition metals seems to be related to an affinity for the imidazole side chains. It is postulated that one to three imidazoles is involved in this interaction, under the employed experimental conditions (pH 7.0; 1 M sodium chloride). There is no evidence for any significant contribution of tryptophan or cysteine (Cys 34) residues to the chromatographic event. The retention of defatted albumin and albumin oligomers (human), on IDA-Cu(II) columns was not significantly different from that of non-defatted albumin or albumin monomer, respectively.     

Trinitrophenylation of the bilirubin binding site of human serum albumin.

Human serum albumin has been modified with 2,4,6-trinitrobenzenesulphonic acid and picryl chloride in low ratios of reagents/albumin. The derivatives have been investigated by spectrophotometry and by thin layer chromatography of the hydrolysates in order to assess the specificity of the reagents. The same reaction conditions were used to modify albumin previously complexed with bilirubin in the ratio of 1:1. The affinity of bilirubin to the modified albumins was estimated by an improved perozidase method. It is concluded that TNBS and picryl chloride react almost quantity with epsilon-amino groups of lysine on the albumin molecule. The results also suggest that at least on TNBS reactive amino group and at least one picryl chloride reactive amino group are located in or near the high-affinity bilirubin binding site.     

Hemin-induced lysis of rat erythrocytes. Protective action of ceruloplasmin and different serum albumins

Hemin-induced lysis of rat erythrocytes is markedly reduced by ceruloplasmin (human) and serum albumins from different species, the order of effectiveness beings: bovine albumin approximately equal to ceruloplasmin greater than human albumin approximately equal to dog albumin greater than apotransferrin (human). Although the proteins studied had hemin binding capacity, the best protective agents, ceruloplasmin and bovine albumin, did bind hemin less strongly than human and dog albumin. The results suggest the existence of another protective mechanism, possibly involving an interaction between erythrocyte membranes and serum proteins.     

Phosphate ester serum albumin affinity tags greatly improve peptide half-life in vivo

A series of phosphate ester based small molecules designed to bind tightly to serum albumin were applied to the amino terminus of an anticoagulant peptide in an effort to increase its protein binding in vivo. The tagged peptides exhibited high affinity for both rabbit and human serum albumin when passed through liquid chromatographic columns with serum albumins incorporated into the stationary phase. The peptides were then administered intravenously to rabbits and found to have a greater than 50-fold increase in plasma half life. The highest affinity peptides showed a reduction in bioactivity consistent with their sequestration away from their protein target in the presence of 0.1% rabbit serum albumin.     

Spectral and kinetic studies on the binding of trithiomolybdate to bovine and canine serum albumin in vitro: the interaction with copper

Spectral studies showed that copper and trithiomolybdate participated in a three-way interaction with bovine and canine serum albumin. The interaction with the proteins was affected by increased pH and ionic strength. Kinetic studies of binding equilibria indicated that [³⁵S] trithiomolybdate bound to both albumins at a single site. The affinity of the site, but not the capacity of the protein, was increased by copper. It was concluded that the site was distinct from the N-terminal copper (and nickel) binding site, which is present on BSA but absent from CSA. Whether or not the N-terminal site has a role in copper transport is discussed. Reversible thiomolybdate-copper-protein interactions of this type may play a fundamental role in the pathogenesis of Mo-induced syndromes, since as the normal binding patterns are perturbed the interprotein equilibria are altered and the copper distribution patterns are modified.     

Quenching of the emission of tryptophan, tyrosine, and serum albumins by cupric ion

The effect of cupric ion on the emission of tryptophan, tyrosine, and serum albumins is studied by emission spectroscopy and lifetime measurements. It is found that whenever cupric ion is bound to tryptophan or tyrosine, their emissions are quenched completely. The quenching may be due to an electron transfer mechanism. The fluorescence of complexes of cupric ions with serum albumins is partially quenched; this is because energy is transferred from tryptophan to the complexed cupric ions by a dipolar energy transfer mechanism. It is deduced from the present study that the tryptophan in the human serum albumin molecule is between 11 and 16 Å from the nearest eupric ion binding sites (assumed to be at the surface of the protein) and that one of the tryptophan in the bovine serum albumin molecule is very close to the cupric ion binding sites and the other is near the center of the bovine serum albumin molecule. It is also found that the deuterium solvent effect on serum albumin fluorescence is very small, and that the quenching of bovine serum albumin fluorescence at the N-F transition is the result of quenching of the fluorescence of both tryptophans. The phosphorescence lifetime apparatus, capable of measuring decay times of signals with intensities changing over a few orders of magnitude, and the ratio spectrofluorometer, both of which were constructed in this laboratory, are also described.     

Albumin affinity tags increase peptide half-life in vivo

Small organic molecules that bind tightly to serum albumin were applied to the amino terminus of an anticoagulant peptide in an effort to increase its protein binding in vivo. The tagged peptides were evaluated for their ability to be retained on liquid chromatographic columns with serum albumins incorporated into the stationary phase. Those which demonstrated significant affinity were administered intravenously to rabbits and found to have significantly increased plasma half-lives. Novel affinity tags were identified by appending a focused library of compounds to a model tetrapeptide and evaluating the resulting compounds' ability to bind to the serum albumin columns. The most promising were synthesized as the full length peptides and again evaluated in vivo. They were found to have still longer half-lives than the first generation compounds.     

Structural studies on metal-serum albumin. IV. The interaction of Zn(II), Cd(II) and Hg(II) with HSA and BSA.

There have been no detailed and reliable studies on the environment and configuration of Zn(II), Cd(II) and Hg(II) in the metal centers of human serum albumin and bovine serum albumin to date. In this paper the authentic evidence for the involvement of the cystinyl sulfur atoms in the ligation to the zinc group ions has been obtained from the X-ray photoelectron spectra. The belief that each of the zinc group ions possesses several similar binding sites in human- and bovine serum albumin and is bound to the deprotonated thiol group (-RS-) of the cysteinyl residues to form tetrahedral and linear metal centers has been further confirmed by the treatment of ligand to metal charge transfer data with Jorgensen's method. According to these results, we have inferred that these binding sites may be located at the seventeen disulfide bridges, most likely at the seven pairs of adjacent disulfide bridges between positions 75 and 567, in the serum albumins.     

Effect of molecular parameters on the binding of phenoxyacetic acid derivatives to albumins

The interaction of 12 phenoxyacetic acid derivatives with human and serum albumin as well as with egg albumin was studied by charge-transfer reversed-phase (RP) thin-layer chromatography (TLC) and the relative strength of interaction was calculated. Each phenoxyacetic acid derivative interacted with human and bovine serum albumins whereas no interaction was observed with egg albumin. Stepwise regression analysis proved that the lipophilicity of the derivatives exert a significant impact on their capacity to bind to serum albumins. This result supports the hypothesis that the binding of phenoxyacetic acid derivatives to albumins may involve hydrophobic forces occurring between the corresponding apolar substructures of these derivatives and the amino acid side chains.     

A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent,pinostrobin

The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, K_a in the range of 1.49 – 6.12 × 10⁴ M⁻¹, with 1:1 binding stoichiometry. Based on the PS–albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics.     

Comparison of copper binding components in dog serum with those in other species.

The copper content of dog serum and its distribution to copper binding proteins was compared with that of rat and mouse. Total serum Cu concentrations of dogs and mice were one third those of the rat. Plasma ceruloplasmin, determined by azide-inhibitable oxidase activity with two substrates, was 8-fold less in the dog and 9- to 20-fold less in the mouse than in the rat, and, in both dogs and mice, there was 70-75% less ceruloplasmin Cu, determined by atomic absorption after gel filtration. In the dog, the largest proportion of total and exchangeable serum Cu was with the transcuprein fraction. Only one third as much Cu was with albumin in the dog (and mouse) versus the rat, and this was released much more readily through dialysis. In dogs and mice, the exchangeable (nonceruloplasmin) serum copper pool was half the size of that in rats and humans. Especially in the mouse (but also in rats and dogs), a small proportion of the exchangeable pool appeared bound to ferroxidase II. We conclude that the dog may rely more on transcuprein and low molecular weight complexes and less on albumin and ceruloplasmin for transport of copper to cells.     

The Role of Histidine Residues in the Non-Enzyme Covalent Attachment of Glucose and Ascorbic Acid to Protein

Copper ions have been suggested to play a role in the non-covalent glycosylation (glycation) of proteins via transition metal-catalysed oxidations. We have further investigated “autoxidative glycosylation” by comparison of the behaviour of dog and bovine serum albumin with respect to the oxidative reactions of glucose and ascorbate. The proteins possess similar numbers of total amino residues available for glucose attachment but dog serum albumin contains fewer histidine groups and also lacks a high affinity copper-binding site. We find that the higher copper-binding capacity of bovine serum albumin is reflected in a lower rate of ascorbate oxidation as well as less protein oxidative damage than is the case for dog serum albumin. We also observe that modification of bovine serum albumin histidine groups by diethylpyrocarbonate enhances ascorbate-mediated protein fluorophore formation.     

Association of Serum Levels of Iron,Copper, and Zinc,and Inflammatory Markers with Bacteriological Sputum Conversion During Tuberculosis Treatment

Iron, copper, and zinc are key micronutrients that play an important role in the immune response to Mycobacterium tuberculosis. The present study aimed to evaluate the association between serum levels of those micronutrients, inflammatory markers, and the smear and culture conversion of M. tuberculosis during 60 days of tuberculosis treatment. Seventy-five male patients with pulmonary tuberculosis (mean age, 40.0?±?10.7 years) were evaluated at baseline and again at 30 and 60 days of tuberculosis treatment. Serum levels of iron, copper, zinc, albumin, globulin, C-reactive protein, and hemoglobin, and smear and cultures for M. tuberculosis in sputum samples were analyzed. Compared to healthy subjects, at baseline, patients with PTB had lower serum iron levels, higher copper levels and copper/zinc ratio, and similar zinc levels. During the tuberculosis treatment, no significant changes in the serum levels of iron, zinc, and copper/zinc were observed. Lower serum copper levels were associated with bacteriological conversion in tuberculosis treatment (tuberculosis-negative) at 30 days but not at 60 days (tuberculosis-positive). C-reactive protein levels and the C-reactive protein/albumin ratio were lower in tuberculosis-negative patients than in tuberculosis-positive patients at 30 and 60 days after treatment. Albumin and hemoglobin levels and the albumin/globulin ratio in patients with pulmonary tuberculosis increased during the study period, regardless of the bacteriological results. High serum globulin levels did not change among pulmonary tuberculosis patients during the study. Serum copper levels and the C-reactive protein/albumin ratio may be important parameters to evaluate the persistence of non-conversion after 60 days of tuberculosis treatment, and they may serve as predictors for relapse after successful treatment.     

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.     

Species-dependency in chiral-drug recognition of serum albumin studied by chromatographic methods

Stereoselective binding of benzodiazepine and coumarin drugs to serum albumin from human and six mammalian species were studied by chiral chromatographic techniques. The applied methods were affinity chromatography on the albumins immobilized on Sepharose 4B, high-performance liquid chromatography (HPLC) separation on columns based on human serum albumin (HSA) and bovine serum albumin (BSA), and chiral HPLC analysis of ultrafiltrates of solutions containing the racemic drug and the native protein. Substantial differences in preferred configurations and conformations were detected among the species. The binding stereoselectivity of the 2,3-benzodiazepine drug, tofisopam, in human, is opposite to that in all other species. In the binding of 1,4-benzodiazepines, dog albumin is very similar to HSA. Highly preferred binding of (S)-phenprocoumon was found with dog albumin.     

Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol.

Methoxypolyethylene glycols of 1900 and 5000 daltons have been attached covalently to bovine serum albumin using cyanuric chloride as the coupling agent. When sufficient polymer is attached, the modified bovine serum albumin appears to lose its immunogenicity in the rabbit and, on intramuscular or intravenous injection, elicits antibodies neither to itself nor to native bovine serum albumin. It does not react with antibodies raised against native bovine serum albumin. Bovine serum albumin to which methoxypolyethylene glycol has been attached exhibits a blood circulating life in the rabbit rather similar to native bovine serum albumin, except that it is not removed from circulation by the eventual development of antibodies. Modified bovine serum albumins which had been iodinated with 125I, or prepared with [14C]cyanuric chloride, were injected intravenously in rabbits. Both labels appeared almost quantitatively in the urine after 30 days. The modified bovine serum albumins showed substantial changes in properties, such as solubility, electrophoretic mobility in acrylamide gel, ion exchange chromatography, and sedimentation, as compared with the unmodified protein.     

Investigations of interaction mechanism and conformational variation of serum albumin affected by artemisinin and dihydroartemisinin

In this work, binding interactions of artemisinin (ART) and dihydroartemisinin (DHA) with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated thoroughly to illustrate the conformational variation of serum albumin. Experimental results indicated that ART and DHA bound strongly with the site I of serum albumins via hydrogen bond (H-bond) and van der Waals force and subsequently statically quenched the intrinsic fluorescence of serum albumins through concentration-dependent manner. The quenching abilities of two drugs on the intrinsic fluorescence of HSA were much higher than the quenching abilities of two drugs on the intrinsic fluorescence of BSA. Both ART and DHA, especially DHA, caused the conformational variation of serum albumins and reduced the α-helix structure content of serum albumins. DHA with hydrophilic hydroxyl group bound with HSA more strongly, suggesting the important roles of the chemical polarity and the hydrophilicity during the binding interactions of two drugs with serum albumins. These results reveal the molecular understanding of binding interactions between ART derivatives and serum albumins, providing vital information for the future application of ART derivatives in biological and clinical areas.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.