Antioxidative properties and degradation of serum proteins by active oxygen forms (O2-., OCl-) generated by stimulated neutrophils

The ability of major serum proteins (albumin, immunoglobulin G) and free radical scavenger proteins (ceruloplasmin, superoxide dismutase, transferrin) to interact with O2-. and OCl- was studied. The interaction between serum proteins and OCl- was shown to be nonspecific and cause protein degradation. During SDS polyacrylamide gel electrophoresis ceruloplasmin and transferrin were degraded in the highest degree. Protein damage was also recorded by fluorescence changes. It is suggested that the damaging influence of active oxygen species secreted by stimulated neutrophils into the extracellular space can be abolished only by ceruloplasmin.     

Sertoli cells synthesize and secrete a ceruloplasmin-like protein

Sertoli cells synthesize and secrete a ceruloplasmin-like protein (testicular ceruloplasmin) that is immunologically similar to serum ceruloplasmin. Rat serum ceruloplasmin was purified and an antiserum was produced to the purified protein which specifically immunoprecipitated a 130,000 dalton protein from rat serum. This ceruloplasmin antiserum was found to also immunoprecipitate a 130,000 dalton protein synthesized and secreted by Sertoli cells. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was required during the immunoprecipitation procedure to prevent the proteolytic degradation of testicular ceruloplasmin. Immunoprecipitation of proteins secreted by Sertoli cells with an antiserum to rat serum proteins was found to precipitate two proteins, testicular ceruloplasmin and testicular transferrin.     

The oxidant hypochlorite (OCl-), a product of the myeloperoxidase system, degrades articular cartilage proteoglycan aggregate.

The myeloperoxidase-derived oxidant, hypochlorite (OCl-) was shown to be able to degrade proteoglycan aggregate prepared from bovine articular cartilage. Exposure of proteoglycan aggregate to OCl- concentrations less than 10(-4) M resulted in a decrease in the size of the constituent proteoglycan monomers, which were unable to reaggregate with hyaluronate due to the loss of the hyaluronic acid binding region as indicated by immunoblotting using the monoclonal 1-C-6 antibody. Analysis of the [35S]-labeled core proteins by SDS/polyacrylamide electrophoresis and fluorography indicated a decrease in the size of the core protein. These data suggest that concentrations of OCl- below 10(-3) M results in the cleavage of the proteoglycan core protein in or near the hyaluronic acid binding region. The physiological consequences of these data are discussed. Exposure to higher concentrations (greater than 10(-3)) of OCl- caused more extensive degradation of the core protein; however, there was no evidence to suggest that OCl- cleaves glycosaminoglycan (GAG) chains.     

Glycosyl phosphatidylinositol-anchored ceruloplasmin is expressed by rat Sertoli cells and is concentrated in detergent-insoluble membrane fractions.

The copper-binding protein, ceruloplasmin, is both a serum component and a secretory product of Sertoli cells. Studies on serum ceruloplasmin have demonstrated it to be a ferroxidase that is essential for iron transport throughout the body. We report here that a glycosyl phosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed by Sertoli cells. Sertoli cell GPI-anchored proteins were selectively released by phosphatidylinositol-specific phospholipase C and were analyzed by Western blotting. A 135-kDa band was identified as ceruloplasmin by multiple antibody recognition and by amino acid sequence analysis. The presence of the GPI anchor on ceruloplasmin was confirmed by Triton X-114 phase partitioning experiments and by recognition with an antibody to the GPI anchor. GPI-anchored ceruloplasmin was enriched in detergent-insoluble glycolipid-enriched membrane microdomains (DIGs) of Sertoli cells. This is the first report of GPI-anchored ceruloplasmin in Sertoli cells and the first study of GPI-anchored ceruloplasmin in DIGs. We suggest that GPI-anchored ceruloplasmin may be the dominant form expressed by Sertoli cells and that Sertoli cell DIGs may play a role in iron metabolism within the seminiferous tubule.     

Interaction of hypochlorite with oxyhemoglobin leads to liberation of iron in a catalytically active form]

Interaction of hemoglobin with hypochlorite (OCI-) induces changes in hemoglobin absorption spectra resulting in Soret band decrease and shift similar to those observed under the action of hydrogen peroxide (H2O2). Hemoglobin decomposition is accompanied by free iron release, as estimated by coloured iron-phenanthroline complex formation. The released iron is catalytically active: the incubation of hemoglobin with H2O2, OCl- or activated neutrophils increases the intensity of H2O2-dependent chemiluminescence of hemoglobin. In both reactions OCl- was more efficient than H2O2. These results show that hemoglobin can serve as a source of catalytically active ("free") iron in the reaction with OCl- and with H2O2.     

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.     

Serum protein degradation by hypochlorite

The structural integrity of serum proteins: albumin, immunoglobulin G, transferrin, ceruloplasmin and superoxide dismutase, and the functional activity of the latter two enzymes after their interaction with hypochlorite were studied. It was shown that the interaction between the proteins and hypochlorite resulted in protein injury and degradation of their native structure. In the case of ceruloplasmin and transferrin, a practically complete protein "dissipation" occurred, the albumin and superoxide dismutase structures being injured in a lesser degree. The inactivation of ceruloplasmin was slower than that of superoxide dismutase. The protein degradation by hypochlorite seems to be the main factor restricting the ability of the proteins to act as antiinflammatory drugs.     

The effect of albumin,ceruloplasmin, and other serum constitutents on Fe(II) oxidation

This study has analyzed the role of several serum constituents, that have been proposed to effect the following reactionin situ: {fx1-1} {fx1-2} These reactions were monitored by measuring the rate of Fe(II) oxidation in the presence of apo-transferrin (reaction A) and Fe(III)-transferrin formation (reaction B) at 465 nm. Reactions A and B were found to be kinetically equivalent. The results show that, singly or in combination, bicarbonate, orthophosphate, citrate, apo-transferrin, and/or albumin have less than one-tenth of the ability to enhance the oxidation of Fe(II) compared to the serum enzyme, ceruloplasmin. It was also found that the rate of Fe(II) oxidation by serum Fe-ligands was influenced by the efficiency of oxygen utilization. Whereas ceruloplasmin produces a 4∶1 ratio of Fe(II) oxidized to oxygen utilized, the non-enzymic components yield a 2∶1 or 3.09∶1 ratio. These data support the role of ceruloplasmin as an antioxidant that prevents the formation of the intermediate active oxygen species O ₂ ⁻ · and H₂O ₂ ^· through the Fe(II) auto-oxidation reaction. A hitherto unrecognized factor in the control of nonenzymic oxidation of Fe(II) was serum albumin. This protein, at >25 μM, was found to sharply dampen the rate of Fe(II) oxidation in the presence of a physiological concentration of bicarbonate, citrate, and transferrin Albumin did not appear to affect the ceruloplasmin catalyzed oxidation of Fe(II) at pH 7.0. The addition of ceruloplasmin effected up to a 44 × increase in the rate of Fe(II) oxidation and Fe(III)-transferrin formation even in the presence of 0.60 mM albumin.     

Hypochlorous acid and human blood low density lipoproteins modified by hypochlorous acid increase erythrocyte adhesion to endothelial cells

The ability of hypochlorous acid (HOCl) (anion form - hypochlorite, OCl-) and HOCl/OCl- -modified human blood low density lipoproteins (HOCl-LDLs) to stimulate erythrocyte adhesion to endothelial cell monolayers was studied. LDLs were modified by incubating at different HOCl/OC- concentrations. This led to a damage of proteins and lipids. We found (1) a more than 20-fold decrease of LDL fluorescence intensity (extinction at 285 nm, emission at 340 nm), (2) accumulation of secondary (TBA-reactive substances) and final (Schiff bases) products of lipid peroxidation, and (3) increase in the electrophoretic mobility of LDLs. Preincubation of endothelial cells (ECs) with HOCI/OCl- (up to 50 microM) enhanced erythrocyte adhesion to the EC monolayer. Preincubation of ECs with HOCl-LDLs (up to 250 microM of HOCI//OCl- during LDL modification) (1) caused an increase in the cholesterol/phospholipid molar ratio in EC and (2) enhanced adhesion of erythrocytes to endothelium. Application of HOCl/OCl- at concentrations above 50 microM or treatment of LDLs with 500 microM HOCl resulted in the cytotoxic effect on ECs and led to a decrease in the molar cholesterol/phospholipid ratio in ECs and adhesion of erythrocytes to endothelium. The results suggest that HOCl/OCl- at physiological concentrations stimulates the adhesion of blood cells to the endothelium and cholesterol accumulation in the vessel wall ECs either directly or due to LDL modification. Both effects could be important in the development of many vascular diseases.     

Interaction of tert-butyl hydroperoxide with hypochlorite induces peroxyl radicals. A chemiluminescence study

The interaction of hypochlorite (HOCl/OCl-) with tert-butyl hydroperoxide ((CH3)3COOH) was investigated by chemiluminescence. It was shown that the addition of HOCl/OCl- to (CH3)3COOH induces a fast chemiluminescent flash. The intensity of this flash increases with the increase in both HOCl/OCl- and (CH3)3COOH concentration. The chemiluminescence is quenched in a concentration-dependent manner in the presence of free radical spin traps N-tert-butyl nitrone and alpha-(4-pyridyl-1-oxyl)-N-tert-butyl nitrone. This fact proves that free radicals take part in the interaction of HOCl/OCl- and (CH3)3COOH. Hypochlorite yielded a very similar chemiluminescence spectrum in its reaction with (CH3)3COOH as Ce4+. It differed considerably from the spectrum in the system H2O2 and HOCl/OCl-. It is well known that the interaction of Ce4+ and (CH3)3COOH produces peroxyl radicals. These results confirm the hyothesis that the interaction of HOCl/OCl- and (CH3)3COOH is mediated by peroxyl radicals. Thus, organic hydroperoxides always present in unsaturated lipids can induce lipid peroxidation processes in the reaction with HOCl/OCl-.     

Glycemic control reverses increases in urinary excretions of immunoglobulin G and ceruloplasmin in type 2 diabetic patients with normoalbuminuria.

To examine whether urinary excretions of plasma proteins with molecular radii of 45-55 A and different isoelectric points such as IgG (pI = 7.4) and ceruloplasmin (pI = 4.4) increase selectively in normoalbuminuric type 2 diabetic patients, urinary albumin excretion rate (AER), renal clearances of IgG, ceruloplasmin and alpha2-macroglobulin, and creatinine clearance (Ccr) were studied in timed overnight urine samples of 36 diabetic outpatients and 16 control subjects. Furthermore, to examine effect of glycemic control on these urinary protein excretions, the same analysis was performed before and after glycemic control in 17 diabetic inpatients admitted for glycemic control. Renal clearances of IgG and ceruloplasmin were significantly higher in diabetic outpatients than in the control group, whereas AER and renal clearance of alpha2-macroglobulin did not differ. Glycemic control caused significant decreases in renal clearances of IgG and ceruloplasmin, accompanied with tendency for Ccr to decrease (p = 0.055). The present results, together with our previous finding of selectively increased urinary excretions of 45-55 A sized plasma proteins in parallel with enhanced glomerular filtration rate after acute protein loading, led us to conclude that enhanced intraglomerular hydraulic pressure may cause increases in clearances of IgG and ceruloplasmin, and that this change can be reversed by strict glycemic control in normoalbuminuric diabetic patients.     

Chlorination of N-acetyltyrosine with HOCl, chloramines, and myeloperoxidase-hydrogen peroxide-chloride system.

N-acetyl-L-tyrosine (N-acTyr), with the alpha amine residue blocked by acetylation, can mimic the reactivity of exposed tyrosyl residues incorporated into polypeptides. In this study chlorination of N-acTyr residue at positions 3 and 5 in reactions with NaOCl, chloramines and the myeloperoxidase (MPO)-H2O2-Cl- chlorinating system were invesigated. The reaction of N-acTyr with HOCl/OCl- depends on the reactant concentration ratio employed. At the OCl-/N-acTyr (molar) ratio 1:4 and pH 5.0 the chlorination reaction yield is about 96% and 3-chlorotyrosine is the predominant reaction product. At the OCl-/N-acTyr molar ratio 1:1.1 both 3-chlorotyrosine and 3,5-dichlorotyrosine are formed. The yield of tyrosine chlorination depends also on pH, amounting to 100% at pH 5.5, 91% at pH 4.5 and 66% at pH 3.0. Replacing HOCl/OCl- by leucine/chloramine or alanine/chloramine in the reaction system, at pH 4.5 and 7.4, produces trace amount of 3-chlorotyrosine with the reaction yield of about 2% only. Employing the MPO-H2O2-Cl- chlorinating system at pH 5.4, production of a small amount of N-acTyr 3-chloroderivative was observed, but the reaction yield was low due to the rapid inactivation of MPO in the reaction system. The study results indicate that direct chlorination of tyrosyl residues which are not incorporated into the polypeptide structure occurs with excess HOCl/OCl- in acidic media. Due to the inability of the myeloperoxidase-H2O2-Cl- system to produce high enough HOCl concentrations, the MPO-mediated tyrosyl residue chlorination is not effective. Semistable amino-acid chloramines also appeared not effective as chlorine donors in direct tyrosyl chlorination.     

Horse Plasma Ceruloplasmin Molecular Weight and Subunit Analysis

Ceruloplasmin is a blue copper-containing serum glycoprotein with oxidase activity. It as been proposed that the physiological function of ceruloplasmin involves the oxidation of ferrous iron and its incorporation into apotransferrin (1). There are several reports demonstrating that ceruloplasmin is made up of multiple chains (2-3-4-5-6-7). Ryden (8) has questioned the multichain structure of ceruloplasmin from human, pig, horse and rabbit sera, arguing that the dissociation observed by previous workers could be attributed to cleavage of labile bands in the protein by enzymatic contaminants present in commercial preparations of the protein. By introducing aminocaproic acid, a general protease inhibitor, at the beginning of the enzyme preparation, Ryden proposed a single-chain structure for ceruloplasmin. On the contrary the results presented by Freeman and Daniel (9) showed that human ceruloplasmin is a multichain protein.

In this paper we report a new purification method for horse ceruloplasmin which furnishes a homogeneous protein preparation in high yield and with good reproducibility.

This procedure allowed to determine with greater accuracy the molecular mass of the protein, of 120000 dal-tons by gel cromatography and 115000 daltons by SDS gel electrophoresis. The protein is composed of one unit only and contains 6 copper atoms. Horse cerulopla-smin is a glycoprotein containing about 20% carbohydrate by weight.     

Two-dimensional electrophoresis of horse serum proteins: genetic polymorphism of ceruloplasmin and two other serum proteins

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum proteins revealed genetic polymorphism of ceruloplasmin (Cp) and two unidentified serum proteins tentatively designated serum protein 1 (SP1) and serum protein 2 (SP2). Family data were consistent with the hypothesis that the observed Cp and SP1 phenotypes were each controlled by two co-dominant, autosornal alleles. The three common SP2 phenotypes were shown to be controlled by two codominant, autosomal alleles. Population data and limited family data indicated the occurrence of two additional SP2 alleles. Altogether more than 600 horses representing 13 different breeds were typed for Cp, SP1 and SP2, and allele frequency estimates were calculated. SP2 was highly polymorphic in all breeds studied whereas SP1 and Cp showed quite low degrees of polymorphism. SP1 polymorphism was observed in seven breeds while Cp polymorphism was observed only in the Icelandic toelter horse breed.     

A comparison of two procedures used for complexing Fe(III) with human apotransferrin: I. Physicochemical properties of the Fe(III) . transferrin products

Samples of human Fe.transferrin (Fe.HTr) were prepared from a single batch of apotransferrin (apo.HTr) by either the Fe(III)-citrate or the Fe(II)-ceruloplasmin (ferroxidase) method. By using 55Fe, 55Fe.HTr prepared by the citrate method and 55Fe.HTr prepared by the ceruloplasmin method contained 2.2-2.3 and 2.0 Fe/mol, respectively. For both 55Fe.HTr preparations, the isotope was shown to be associated with the protein from the measurement of absorbance at 465 nm and dialysis studies. However, passage of the 55Fe.HTr (ceruloplasmin) reaction mixture through DEAE-cellulose caused 55-60% of 55Fe to be lost from the protein, although no decrease in absorbance at 465 nm was observed. Ion-exchange chromatography of 55Fe.HTr (citrate) did not induce loss of 55Fe. Absorbance measurements showed significant differences between the two Fe.HTr preparations with respect to the ratios A212/A278 and A463/A278. Using an excitation wavelength of 275 nm, the fluorescence intensity ratios relative to apo.HTr were 0.275 and 0.309 for Fe.HTr (citrate) and Fe.HTr (ceruloplasmin), respectively. Electron spin resonance (ESR) measurements confirmed that Fe.HTr (citrate) and Fe.HTr (ceruloplasmin) were saturated with Fe. Hyperfine coupling constants and other features of the resonance profile revealed distinct differences between the two Fe.HTr preparations. Dialysis against H2O caused Fe.HTr (citrate), but not Fe.HTr (ceruloplasmin), to lose absorbance at 465 nm. The ESR profile of Fe.HTr (citrate), after dialysis against H2O, was reduced to multiple splittings and a lack of resolution of the central hyperfine structure. Addition of Na2CO3 restored the absorbance (465 nm) and the ESR pattern of Fe.HTr (citrate). In contrast, these properties of Fe.HTr (ceruloplasmin) were little affected by dialysis against H2O. However, the addition of trisodium citrate to Fe.HTr (ceruloplasmin) caused a reduction in absorbance at 465 nm and a change in ESR profile to resemble that of Fe.HTr (citrate) after dialysis in H2O; these changes, caused by citrate binding to Fe.HTr (ceruloplasmin), were restored to normal by the addition of Na2CO3. The data indicate that different protein conformations result from complexing Fe(III) with apo.HTr by these two different procedures. The two Fe.HTr products may differ, conceivably, in their abilities to transfer Fe to cells.     

Reactions of hypochlorite with catalase

OCl-/HOCl imposed a rapid inactivation of catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6), some of which was slowly reversible upon subsequent exposure to H2O2. Ethanol accelerated this restoration of activity by H2O2. OCl- caused biphasic changes in the visible absorption spectrum of catalase, which were partially reversed by dithionite. A scheme of reactions involving axial ligation of one or two OCl- to heme iron, followed by heterolytic or homolytic cleavages of the O-Cl bond, is proposed to account for the behavior of the system.     

Binding and uptake of copper from ceruloplasmin

Specific binding of [67Cu]ceruloplasmin to plasma membrane containing preparations from rat tissues was shown in the presence of an excess of nonradioactive Cu(II) or ceruloplasmin. With Cu(II) there was positive cooperativity and an apparent KD of 10(-7) M. The effects of both "cold" ligands was partly additive. No "specific" binding was shown with Zn(II), unrelated proteins and after boiling the membranes. Total and specific binding of [67Cu]ceruloplasmin were 2-7 fold greater for heart and brain than for liver preparations, per g tissue or per mg protein, +/- correction for yield of 5'-nucleotidase. Cu(II) also inhibited uptake of [67Cu] from ceruloplasmin by CHO cells, but monensin did not, suggesting uptake of ceruloplasmin Cu occurs at the cell surface.     

Fragmentation of human ceruloplasmin induced by hydrogen peroxide

We investigated the fragmentation of human ceruloplasmin induced by H2O2 to study its oxidative damage. When ceruloplasmin was incubated with H2O2, the frequency of the protein fragmentation increased in a proportion to the concentration of H2O2. It also increased in a time-dependent manner and was accompanied by gradual loss of the oxidase activity. Hydroxyl radical scavengers such as azide and mannitol inhibited the fragmentation of ceruloplasmin. The deoxyribose assay showed that hydroxyl radicals were generated in the reaction of ceruloplasmin with H2O2. Incubation of ceruloplasmin with H2O2 resulted in a time-dependent release of copper ions. The released copper ion may participate in a Fenton-like reaction to produce hydroxyl radical, which enhanced the fragmentation. The protection of the fragmentation by copper chelators such as diethylenetriaminepentaacetic acid and bathocuproine indicates a role for copper ion in the reaction. These results suggest that the fragmentation of ceruloplasmin induced by H2O2 is due to hydroxyl radicals formed by a copper-dependent Fenton-like reaction.     

Protection by carnosine-related dipeptides against hydrogen peroxide-mediated ceruloplasmin modification

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. Previous studies showed that these compounds have an antioxidant function. We investigated the protective effects of carnosine and related compounds on the modification of human ceruloplasmin that is induced by H2O2. Carnosine, homocarnosine, and anserine significantly inhibited the fragmentation and inactivation of ceruloplasmin that is induced by H2O2. All three compounds also inhibited the release of copper ion from protein, and the formation of hydroxyl radicals in the ceruloplasmin/H2O2 system. These compounds inhibited the fragmentation of human serum albumin that is induced by the copper-catalyzed oxidation system, as well as by the iron-catalyzed oxidation system. These results suggest that carnosine, homocarnosine, and anserine might protect ceruloplasmin against H2O2-mediated oxidative damage through a combination of copper chelation and free radical scavenging.     

Presence of reduced type 1 copper in ceruloplasmin as revealed by reaction with hydrogen peroxide

The reaction of hydrogen peroxide with ox or sheep ceruloplasmin leads to approximately 10% increase of the optical absorption band at 610 nm and of the Type 1 EPR signal. No inactivation or denaturation of the protein is apparent up to 15 H2O2 molar excess. Oxygen is able to restore about 50% of the Type 1 copper absorption in ascorbate-reduced ceruloplasmin, while the other half is recovered after addition of H2O2. It appears that H2O2 undergoes a specific redox reaction with ceruloplasmin, which reveals a fraction of the total copper to be present in the native protein as reduced copper. This fraction is apparently Type 1 copper, while Type 2 is not affected by H2O2.