Copper catalyzed oxidation of ascorbate (vitamin C). Inhibitory effect of catalase, superoxide dismutase, serum proteins (ceruloplasmin, albumin, apotransferrin) and amino acids

The inhibitory effect of catalase and superoxide dismutase on copper catalyzed oxidation of ascorbate is probably due to a binding of copper ions. Scavengers of hydroxyl ions and singlet oxygen had no effect on the ascorbate oxidation rate. Copper binding serum proteins reduced the oxidation rate; the order of effectiveness being: Ceruloplasmin greater than human albumin = bovine albumin greater than apotransferrin. The excellent protection obtained with catalase and ceruloplasmin is possibly due to a strong affinity for cuprous ions generated during the reaction. Cupric ion binding amino acids (His, Thr, Glu, Gln, Tyr) had considerably weaker protective effect than the proteins studied. Apparently they do not compete favorably with ascorbate for cupric ions.     

Catalysis of the disproportionation of superoxide by metalloporphyrins.

The efficiencies of several metalloporphyrin complexes at catalyzing the disproportionation of superoxide have been determined at pH 10 in both carbonate and borate buffer systems. Catalytic rate constants were obtained for the iron(III) and cobalt(III) derivatives of tetrakis(4-N-methylpyridyl) porphine, for tetraphenylporphinesulfonatoferrate(III) and for hemin. In addition, the effects of added bovine serum albumin and imidazole were studied. The order of catalytic efficiency is FeTMpyP greater than FeTMpyP(Im)2 greater than FeTPPS(Im)2 approximately FeTPPS approximately FeTPPS.BSA approximately Fe(EDTA) greater than or approximately CoTMpyP greater than hemin(Im)2 greater than or approximately hemin.     

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.     

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.     

Inhibition of human lymphocyte ferrochelatase activity by hemin

Ferrochelatase activity in human lymphocytes was found to be 50% inhibited by 10.5 microM hemin under maximal velocity conditions. The inhibition was not prevented by dithiothreitol or glutathione, suggesting that the hemin was not interacting with the sulphydryl groups of ferrochelatase. Human serum albumin, but not bovine serum albumin was able to prevent the inhibition consistent with the known formation of the tightly bound methemalbumin complex with human albumin. Kinetic studies performed under initial velocity conditions with hemin concentrations ranging from 2 to 8 microM revealed the inhibition to be non-competitive with respect to the metal substrate (zinc) and competitive with respect to the porphyrin substrate (mesoporphyrin). The kinetic analysis indicated that hemin binds to both the enzyme and enzyme-metal complex at a site normally occupied by the porphyrin substrate, and a second molecule of hemin could bind to the enzyme-metal complex but with a much lower affinity than the first molecule. We conclude that the product inhibition of ferrochelatase by hemin should be considered as a possible site of regulation of heme biosynthesis.     

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.     

The effects of heme-binding proteins on the peroxidative and catalatic activities of hemin.

The plasma proteins hemopexin (Hx) and albumin (Alb) are known to bind heme with high and medium affinity, respectively. To study how this binding modifies heme catalytic reactivity, the effects of Hx, human serum Alb (HSA), and bovine serum Alb (BSA) on the peroxidase- and catalaselike activities of hemin were investigated. These hemin activities were found to be inhibited by 50 to 60% with either HSA or BSA, and by 80 to 90% with Hx. The heme complexes with Hx or Alb (1:1 = protein:heme) therefore had a much lower reactivity toward H2O2 and Cum-OOH than the nonprotein heme. A kinetic analysis suggested that binding to Hx or Alb inhibited the primary activation of heme by H2O2, the step common for both peroxidase- and catalaselike activities of hemin. It is thought that by complexing heme, the Hx and Alb can prevent the toxic effects of extracellular heme in blood plasma.     

Interaction of serum proteins with CYP isoforms in human liver microsomes: inhibitory effects of human and bovine albumin,alpha-globulins,alpha-1-acid glycoproteins and gamma-globulins on CYP2C19 and CYP2D6

The effects of serum proteins on the in vitro hydroxylation pathways of mephenytoin (CYP2C19) and debrisoquine (CYP2D6) were studied to enhance the predictability of in vivo drug metabolism from in vitro assays. Both CYP substrates are known to be weakly bound to albumin and the applicability of the free drug hypothesis was further appraised. Since bovine serum albumin (BSA) is used widely in in vitro assays, a comparison between human and bovine proteins was made. Four major serum proteins were studied: albumin, alpha1-acid glycoprotein (AGP), alpha- and gamma-globulins. Human serum albumin (HSA) inhibited both CYP activities about 20% more than BSA. The addition of human alpha-globulins, but not the bovine protein, resulted in marked reduction of 86% and 41% in CYP2C19 and CYP2D6 activities, respectively. This reduction of activity was strikingly greater than the fraction bound (14 and 22%, respectively). The inhibition was of the competitive type and the Ki values of human alpha-globulins on CYP2C19 and CYP2D6 were found to be 0.45% (4.5 mg/ml) and 3.5% (35 mg/ml), respectively. The effect of both human and bovine gamma-globulins on CYP isoforms was negligible. The Ki values of human and bovine AGP for CYP2C19 were 1.84% (420 microM) and 0.93% (210 microM), respectively. For HSA, human alpha-globulins and human and bovine AGP, the strongly decreased CYP activities in vitro cannot be explained by the free drug hypothesis. A direct interaction of these serum proteins with CYP enzymes is postulated. Differential effects of bovine and human serum proteins and CYP specific inhibition were observed.     

Immobilized hemin affinity chromatography as a probe for proteins having potentiality to bind with heme

After Sepharose 4B polymer beads were activated by using epichlorohydrin, hemin was binded with them to prepare an immobilized hemin affinity chromatography column. The coupling rate of this column was very high, more than 0.25mg hemin could be fixed by 1g of wet Sepharose 4B beads. The column equilibrated with deionized water and eluated with pH 3.0 NaAc-HAc buffer was applied to capture the proteins in human serum, earthworm body and Bacillus subtilis cells. Three polypeptides in human serum were captured, one of which was verified as serum albumin after comparison to the control. At least one polypeptide in earthworm body, two in Bacillus subtilis cells displayed the powerful binding specificity to hemin. Our experiments demonstrated that the immobilized hemin affinity chromatography was available as a probe for some proteins having potentiality to bind with heme.     

Regulation of rabbit acute phase protein biosynthesis by monokines.

We defined the acute phase behaviour of a number of rabbit plasma proteins in studies (in vivo) and studied the effects of monokine preparations on their synthesis by rabbit primary hepatocyte cultures. Following turpentine injection, increased serum levels of C-reactive protein, serum amyloid A protein, haptoglobin, ceruloplasmin, and decreased concentrations of albumin were observed. In contrast to what is observed in man, concentrations of alpha 2-macroglobulin and transferrin were increased. Co-culture of primary hepatocyte cultures with lipopolysaccharide-activated human peripheral blood monocytes or incubation with conditioned medium prepared from lipopolysaccharide-activated human or rabbit monocytes resulted in dose-dependent induction of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and depression of albumin synthesis, while C-reactive protein synthesis and mRNA levels remained unchanged. A variety of interleukin-1 preparations induced dose-dependent increases in the synthesis and secretion of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and decreased albumin synthesis. Human recombinant tumour necrosis factor (cachectin) induced a dose-dependent increase in synthesis of haptoglobin and ceruloplasmin. In general, human interleukin-1 was more potent than mouse interleukin-1 and tumour necrosis factor. None of the monokines we studied had an effect on C-reactive protein synthesis or mRNA levels. These data confirm that C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin display acute phase behaviour in the rabbit, and demonstrate that, in contrast to their behaviour in man, alpha 2M and transferrin are positive acute phase proteins in this species. While both interleukin-1 and tumour necrosis factor regulate biosynthesis of a number of these acute phase proteins in rabbit primary hepatocyte cultures, neither of these monokines induced C-reactive protein synthesis. Comparison of these findings with those in human hepatoma cell lines, in which interleukin-1 does not induce serum amyloid A synthesis, suggests that the effect of interleukin-1 on serum amyloid A synthesis may be indirect.     

Metals in biochemistry : P.M. Harrison and R.J. Hoare,Chapman & Hall,New York, 1980, 80 pp. $5.95 (paper)

An ultrafiltration technique was used to study stripping by glycine of the first copper and zinc ion equivalents bound by bovine, dog, and rat serum albumins at pH 7.5. Affinity of dog serum albumin for copper was poorer than for the other albumins, consistent with the absence in the former albumin of the copper binding site present at the amino terminus of the latter albumins. Affinities of all three proteins for zinc were similar, suggesting that the albumin amino terminus is not the primary zinc ion binding site.     

Spectrophotometric and fluorometric study of albumins and hemin interactions with aromatic antioxidants

Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants (Ka) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4). Ka values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes with o-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media; Ka values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically; Ka values were of order of approximately 10(-5) M-1 and decreased with the ligand hydrophobicity.     

Immune response in dog. 3. Antibody formation in dogs and rabbits to human serum proteins and keyhole limpet haemocyanin.

Antibody formation in dogs and rabbits to human serum proteins and keyhole limpet haemocyanin (KLH) following primary, secondary and multiple stimulation was analysed. Primary injection of human serum stimulates the formation of precipitin antibodies to beta2 lipoprotein in dogs, beta2 lipoprotein and beta globulin in rabbits. After a secondary dose the rabbits formed precipitins to a whole range of human serum proteins, while the dogs to beta2 lipoprotein, albumin, beta globulin. When the primary dose of antigen was divided over a period of 8 weeks, the dog produced precipitins to beta2 lipoprotein while the rabbit to a wide range of serum proteins. Secondary stimulation of these animals did not increase the number of precipitins formed. Quantitative analysis of the antibody produced show that the best response was with beta2 lipoprotein followed by albumin and beta globulin. As the immunogenicity of the antigen was greater the differences between the two species were narrow. These differences were less pronounced following the primary injection than after the secondary and multiple stimulations. The primary response to KLH (which represents mainly IgM) is better in the dog than in the rabbit, while the secondary response (IgG) was better in the rabbit. The poorer IgG response in the dog compared to the rabbit, observed in all the experiments, is discussed.     

Study of interaction between catechin and native and modified bovine serum albumin by physico-chemical methods

The interaction of native and modified bovine serum albumin (BSA) with catechin, a flavanoid having vitamin P activity, has been studied using equilibrium dialysis, pH-metric, viscosity and spectrophotometric methods. The order of reactivity of catechin binding to proteins was found to be: esterified BSA greater than BSA greater than formylated BSA greater than acetylated BSA with log K values of 3.778, 3.879, 3.748 and 3.813 and free energy change equal to -5.11, -5.16, -5.07 and -5.15 kcal/mole, respectively.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

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.     

Elimination of Homologous And Heterologous (Bovine) Serum Albumin from the Blood Circulation in the Dog)

The elimination of intravenously administered I131-labelled bovine serum albumin has been compared to the elimination rate of relabelled homologous serum albumin in normal and bled dogs, which had lost considerable blood volumes. The investigation shows that during the first four to five days after the administration the elimination is similar of heterologous and homologous serum albumin. This proves that bovine serum albumin can be regarded to be an equivalent plasma expander to homologous serum albumin in the dog. Elimination of homologous as well as heterologous serum albumin follows a simple exponential curve during four to five days after administration. The intravascular half-lives for homologous serum albumin were 6.4 ±1.5 days and 6.4 ± 0.6 days respectively in control and bled dogs. Corresponding values for heterologous (bovine) serum albumin were 5.0 ± 0.3 and 4.8 ± 0.4 days respectively. The quote for cencentrations of homologous and heterologous serum albumin in different tissues was found to be relatively constant approximately 1.4. An exception was the stomach wall in bled dogs which had a quote of 1.1 only.     

Microcalorimetric study of human serum

It was established that albumin of donor blood serum denatures in two temperature ranges. It is shown that the first stage of denaturation with Td = 61.5 degrees C is dominant and corresponds to melting of regions not bound to fatty acids. The second stage with Td = 80 degrees C corresponds to melting of regions bound to fatty acids. Serum denaturation heat is equal to 20.2 J/g dry protein. A change in denaturation heat capacity is 0.21 J/(g.K). Analysis of thermal parameters of deconvolution peaks showed that albumin of donor blood serum is in a fatless state and its multiple binding centers are essentially free as compared with freshly isolated albumin and may play an important role in binding of ligands in vivo. The thermal parameters of denaturation of some important human blood serum proteins including gamma-globulins, transferrin ceruloplasmin and protease inhibitors were also determined.     

Erythrocyte oxidative damage by myeloperoxidase. The protective action of serum proteins

Influence of main serum proteins (albumin, immunoglobulin G) and proteins-antioxidants (ceruloplasmin, transferrin, superoxide dismutase) on the oxidative damage of erythrocytes by myeloperoxidase and hypochlorite was investigated. The proteins were determined to act as protectors and decrease the degree of hemoglobin oxidation, ceruloplasmin and albumin possessing the highest antioxidant activity.     

Study of the effect of hydration on the dynamics of various globular proteins by Rayleigh scattering of M?ssbauer radiation

Hydration relationships of the elastic scattering fraction of M?ssbauer radiation were studied for human serum albumin (HSA), pancreatic trypsin inhibitor and lysozyme within hydration degrees 0 less than or equal to h less than or equal to 0.75 g/g (at T = 295 degrees K) and temperatures 100K less than or equal to T less than or equal to 320 K (for HSA only at h = 0.03; 0.25; 0.41; 0.65). It is shown that the increase of both hydration degree above h greater than 0.1 and temperature above T greater than 200K leads to the appearance of intramolecular mobility in these proteins.