Purification of undegraded ceruloplasmin from outdated human plasma

A method for the rapid isolation of homogeneous undegraded ceruloplasmin from outdated human plasma is reported. The procedure consists of a precipitation step with polyethylene glycol 4000, batchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin was purified 1740-fold and the yield from outdated plasma was 67%. The purified ceruloplasmin was found to be homogeneous on anionic polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, isoelectric focusing, and low-speed equilibrium centrifugation. The isoelectric point as determined by isoelectric focusing was 4.4. The purified enzyme was sensitive to storage; when a sample was resubmitted to PAGE after 4 months of storage at 4 degrees C, two bands were obtained and the fast-moving band showed no oxidase activity. The molecular weight estimated by gel electrophoresis and sedimentation equilibrium centrifugation was 130,000.     

Simultaneous phenotyping of pig plasma α-protease inhibitors (PI1, PO1A, PO1B, PI2) and four other proteins (PO2, TF, CP, HPX) by a simple method of 2D horizontal electrophoresis

A simple and rapid method of 2D agarose gel (pH 5.4)-horizontal polyacrylamide gel (pH 9.0) electrophoresis was developed for the simultaneous phenotyping of pig plasma alpha-protease inhibitors (protease inhibitor-1 and -2; postalbumin-1A and -1B), postalbumin-2, transferrin, ceruloplasmin and haemopexin. These eight plasma proteins were clearly visible on gels stained with Coomassie Brilliant Blue G250. The 2D patterns and mobilities of several variants of alpha-protease inhibitors were described. By using two agarose gels and 10 polyacrylamide gels, 120 samples were easily analysed in a day. Since alpha-protease inhibitors show extensive polymorphism and as the gene for postalbumin-2 is closely linked to the halothane sensitivity locus Hal, this method is a useful tool for conducting parentage control and for predicting Hal genotypes of individual pigs.     

Proteomics analysis of phosphotyrosyl-proteins in human lumbar cerebrospinal fluid

Cerebrospinal fluid (CSF) is a secretion product of several different central nervous system (CNS) structures, including the choroid plexus in the ventricles. Pathological CNS processes are reflected in the protein composition of CSF. To elucidate the molecular events that occur in the homeostatic and pathological processes of the CNS, the high-throughput characterization of differentially expressed proteins, and of post-translationally modified proteins, is needed for proteomics studies of CSF. Among the post-translational modifications of proteins, phosphorylation is the most common and important mechanism for the reversible regulation of protein function. In this study, CSF phosphotyrosyl (p-Tyr)-proteins were detected with antibodies and were analyzed with proteomics methods. Three different combination methods--1D gel electrophoresis and Western blotting, immunoprecipitation and 2D gel electrophoresis, and 2D gel electrophoresis and Western blotting--were used to detect p-Tyr-proteins in human lumbar CSF samples. Six protein spots, representing four proteins on a 2D Western blot, were identified as p-Tyr-proteins with the 2D gel electrophoresis and Western blotting method. Those four p-Tyr-proteins are kallikrein-6 precursor, complement C4 gamma-chain, gelsolin, and ceruloplasmin precursor. Additionally, four other nonphosphorylated CSF proteins--beta-2-glycoprotein I precursor, fibulin-1 precursor, EGF-containing fibulin-like extracellur matrix protein 1 precursor, and angiotensinogen precursor--were characterized for the first time.     

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.     

Transport of silver in virgin and lactating rats and relation to copper

Virgin and lactating Sprague Dawley rats were used to determine whether the pathways of silver transport to tissues and milk resemble those for copper. Rats were injected i.p. with small amounts of ¹¹⁰AgNO₃. Blood and tissues were examined at various times thereafter for total radioactivity and for incorporation into copper binding proteins in plasma and milk. As with ⁶⁷Cu, much of the ¹¹⁰Ag was rapidly incorporated into the liver. Skeletal muscle, spleen, mammary gland, ovaries, uterus and adrenals also were significant initial accumulation sites, with or without lactation. Lactation enhanced uptake by the mammary gland, and radioactivity rapidly entered the milk and milk ceruloplasmin. In the plasma, most of the ¹¹⁰Ag bound to a single component of apparent molecular weight 800 k throughout the 52 h period examined. A small proportion was also incorporated into plasma ceruloplasmin, as determined by immunoprecipitation and native gel electrophoresis. There was little or no association of ¹¹⁰Ag with albumin or transcuprein. The binding of ¹¹⁰Ag to the 800 kDa protein was tight. Off rates during pH 7 dialysis were <2.5%/day even in the presence of 100 M histidine or Cu(II), but were accelarated by mercaptoethanol. Subunits of 145 and 45 kDa in virtually pure peak fractions were those of ₁-macroglobulin. We conclude that silver resembles copper in aspects of its tissue distribution, response to lactation, and incorporation into ceruloplasmin. However its main plasma carrier appears to be ₁-macroglobulin, a different macroglobulin than that involved in copper transport.     

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.     

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. There are several reports demonstrating that ceruloplasmin is made up of multiple chains. Ryden 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 epsilon-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 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 120,000 daltons by gel chromatography and 115,000 daltons by SDS gel electrophoresis. The protein is composed of one unit only and contains 6 copper atoms. Horse ceruloplasmin is a glycoprotein containing about 20% carbohydrate by weight.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Ceruloplasmin Is a Potential Biomarker for aGvHD following Allogeneic Hematopoietic Stem Cell Transplantation

Acute graft-versus-host-disease (aGvHD) is the major cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recently, diagnostic biomarkers for aGvHD have been shown to play important roles in evaluating disease status and mortality risk after allo-HSCT. To identify plasma biomarkers for aGvHD with high sensitivity and specificity, a quantitative proteomic approach using 8-plex isobaric tags for relative and absolute quantitation (8-plex iTRAQ) was employed to screen differentially expressed proteins in peripheral blood before and after the onset of aGvHD. Four target proteins, ceruloplasmin (CP), myeloperoxidase (MPO), complement factor H (CFH), and alpha-1-acid glycoprotein (AGP), were chosen for preliminary validation with enzyme linked immunosorbent assay (ELISA) in 20 paired samples at both the time of diagnosis of aGvHD and the time of complete response. The most promising candidate, ceruloplasmin, was further validated at fixed time points after allo-HSCT and during aGvHD. The plasma ceruloplasmin levels were significantly increased during the period of aGvHD onset and were markedly decreased as aGvHD resolved. The plasma ceruloplasmin levels at different time points post-transplant in the aGvHD (+) group were significantly higher than those in the aGvHD (−) group (p<0.001). The elevation of ceruloplasmin level in patients with active aGvHD was independent of infection status. Patients whose ceruloplasmin levels were elevated above 670 μg/ml at 7, 14 and 21 days after allo-HSCT had a remarkably increased probability of subsequently developing aGvHD. In conclusion, our results suggest that plasma ceruloplasmin is a potential plasma biomarker of aGvHD, and it also has prognostic value for risk-adapted prophylaxis during the consecutive time points monitored in the first month after allo-HSCT.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.     

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.     

Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin.

1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.     

Plasma ceruloplasmin Evidence for its presence in and uptake by heart and other organs of the rat

Evidence for the presence of the plasma protein, ceruloplasmin, in heart and other tissues of the rat was sought using various techniques. With p-phenylenediamine, ceruloplasmin-like oxidase activity was detected in heart postmitochondrial and 100 000 × g supernatants in amounts far exceeding those that could be accounted for by residual blood. Much lower levels were detected in kidney, brain and liver. Oxidase activity of heart purified on DEAE-cellulose in the same way as rat plasma ceruloplasmin and behaved identically also in disc gel electrophoresis. The presence of ceruloplasmin in heart extracts was confirmed immunologically by Ouchterlony diffusion, using rabbit antibody raised against pure rat ceruloplasmin. When pure [³H]leucin-labeled ceruloplasmin was infused intravenously into a copper-deficient rat, radioactivity was concentrated in the heart and brain within 2 h; radioactive counts per g attained 11 and 3 times those of plasma in the two organs, respectively. A lesser concentration occurred in the liver. The results suggest that circulating ceruloplasmin (made by the liver) finds its way into the cells of some organs, especially the heart, a phenomenon which may be related to the function of ceruloplasmin to provide copper to the cytochrome oxidase of various tissues.     

Copper regulation of ceruloplasmin in copper-deficient rats.

In copper-deficient rats, oral intubation of copper increases the rate of ceruloplasmin synthesis without affecting general synthesis of plasma or liver proteins. It also restores the enzyme from half to full activity. Copper given by injection at doses commonly employed has additional nonspecific effects on protein synthesis and in some strains of rats produces severe hemolysis. In contrast to deficient rats, in normal rats copper does not elevate plasma ceruloplasmin unless hemolysis also occurs. Thus, at least in deficiency, copper availability controls the rate of synthesis, acitvation, and plasma concentration of ceruloplasmin.     

Purification of riboflavin-binding proteins from bovine plasma and discovery of a pregnancy-specific riboflavin-binding protein.

Riboflavin-binding proteins have been purified from bovine plasma using flavinyl agarose beads. At least three major protein bands, migrating in regions assigned to the beta- and gamma-globulins of plasma, are observed by cellulose acetate electrophoresis. These proteins coelute from a calibrated Sephadex G-100 column in the volume corresponding to a molecular weight of approximately 150,000; a small amount of another riboflavin-binding protein (molecular weight approximately 37,000) is also present. Polyacrylamide gel electrophoresis of the proteins, with detection by autoradiography of those having tightly bound [2-14C]riboflavin, reveals one protein band which is present only in preparations from pregnant cows. This protein has been purified to apparent homogeneity by storing the mixture of riboflavin-binding proteins at 8 degrees C for 3 weeks, which precipitates the other, less stable proteins. Hence, bovine plasma, like that of the laying hen, contains a number of riboflavin-binding proteins, one of which correlates with pregnancy.     

Mitochondrial involvement in genetically determined transition metal toxicity II. Copper toxicity

Copper, like iron, is an essential transition metal ion in which its redox reactivity, whilst essential for the activity of mitochondrial enzymes, can also be a source of harmful reactive oxygen species if not chelated to biomolecules. Therefore, both metals are sequestered by protein chaperones and moved across membranes by protein transporters with the excess held in storage proteins for future use. In the case of copper, the storage proteins in the mitochondria are a distinct ceruloplasmin and metallothionein (MT). If the cell accumulates too much copper or copper is needed by other cells, then copper can be chaperoned to the trans-Golgi secretory compartment where it is transported into the Golgi by ATP-dependent pumps ATP7A/B. In liver, the copper is then incorporated into ceruloplasmin in vesicles that travel to the plasma membrane and release ceruloplasmin into the plasma. This paper reviews the genetic basis for diseases associated with copper deficit or excess, particularly those attributed to defective ATP7A/B transporters, with special emphasis on pathologies related to a loss of mitochondrial function.     

Molecular characterization of human and bovine ceruloplasmin using MALDI-TOF mass spectrometry.

Using SDS-PAGE and MALDI-TOF mass spectrometry, we investigated the difference in the molecular structure between human and bovine ceruloplasmin. In both cases, we found that the protein is present in two majors forms of different molecular mass. The difference between human and bovine ceruloplasmin was more obvious when characterized by MALDI-TOF than with the SDS-PAGE analysis. Furthermore, we established that the N-glycoside content of both enzymes is dissimilar and that the N-glycosyl moieties are distributed in a distinctive fashion in two glycoproteins. Finally, it appeared that both proteins exhibited different cleavage patterns after treatment with trypsin. This study indicates that human and bovine ceruloplasmin differ not only in sugar composition but also in primary structure.     

Highly purified ceruloplasmin messenger RNA from rat liver

Summary Highly purified ceruloplasmin mRNA was isolated from rat liver polyribosomes. The molecular weight of ceruloplasmin mRNA is in a range from 1.05 to 1.25 · 10⁶ daltons which is large enough to code for a putative precursor of ceruloplasmin (∼700 amino acids). Ceruloplasmin mRNA contains 3′-terminal poly(A) the length of which varies from 38 to 165 nucleotides. The 5′-end of ceruloplasmin mRNA is blocked with confronting m⁷G residue which is a component of cap I (m⁷G^5′ppp^5′X^mpAp). The addition of ceruloplasmin mRNA to wheat-germ cell-free system programmed the synthesis of a product that was largely precipitated by anti-ceruloplasmin immunoglobulins. The translation product was homogeneous in polyacrylamide gel-sodium dodecylsulfate electrophoresis. Cell-free translation of ceruloplasmin mRNA was sensitive to inhibition by cap analogue.     

An electron spin resonance (ESR) study on the mechanism of ascorbyl radical production by metal-binding proteins

The mechanism of ascorbate oxidation by metal-binding proteins (ceruloplasmin, albumin and transferrin) was investigated in vitro and in isolated plasma by the measurement of the ascorbyl free radicals (AFR) by electron spin resonance (ESR). In plasma of 13 healthy volunteers, a spontaneous and variable pro-duction of AFR was detected, which was increased by a 10 M ascorbate overloading; however, this increase was not correlated to the intensity of the spontaneous AFR signal. The addition of Cu and ceruloplasmin to plasma increased the ESR signal, while the addition of transferrin decreased the signal intensity in a dose-dependent manner. In vitro, we demonstrated that ascorbate was oxidized by human serum albumin and by ceruloplasmin, and that this oxidase-like activity was lost by trypsin or heat treatment of these proteins. These two proteins positively interacted in the oxidation of ascorbate, since addition of crude albumin to a solution of ascorbate and ceruloplasmin increased the intensity of ESR signal in a dose-dependent manner. The treatment of albumin by a metal chelator (DDTC) abolished these positive inter-actions. The respective roles of copper and iron in ascorbate oxidation were studied and showed a dose-dependent effect of these ions on ascorbate oxidation. The role of iron was confirmed by the inhibiting effect of metal-free transferrin on iron-dependent ascorbate oxidation. Concerted actions between iron carrying albumin and copper carrying ceruloplasmin appear responsible for the production of AFR in vitro and in vivo. © Rapid Science 1998     

Isolation and characterization of copper-binding sites of human ceruloplasmin

A tryptic cleavage procedure was used to obtain stable copper-containing peptide regions of human ceruloplasmin. Tryptic digestion-derived copper peptides were fractionated by gel filtration chromatography, yielding two fractions, one with an apparent molecular weight of 11000 and the other 1000. The high molecular weight fraction (11K fraction) was found to contain 50% of the copper atoms of the ceruloplasmin molecule and behaved as a single copper-containing component by gel filtration chromatography and by isoelectric focusing. The low molecular weight fraction (1K fraction) was found to be a mixture of three or four copper peptides by isoelectric focusing. Acrylamide gel electrophoresis studies, amino acid composition analysis and terminal amino acid determinations showed the 11K fraction to be a complex composed of at least three peptides arising from different regions of the ceruloplasmin molecule. Two of the peptides of the 11K complex appear to be derived from the 19K fragment of the ceruloplasmin molecule (18); one peptide in the complex appears to correspond to the aspartic acid-rich portion, residues 7-30, and the other to the histidine-rich portion, residues 103-157. Most preparations of ceruloplasmin are reported to consist of three non-covalently linked fragments that have molecular weights of 67K, 50K and 19K. Dwulet and Putnam (20) proposed a model for the sequence structure of ceruloplasmin where the molecule exhibits a three-fold repeat pattern of two alternating structures, here termed X and Y.(ABSTRACT TRUNCATED AT 250 WORDS)