Labile conformation of type 2 Cu2+ centres in human ceruloplasmin.

1. The investigation of human ceruloplasmin by spectral methods (EPR and spectrophotometry) demonstrated that type 2 Cu2(+)-containing centres occur not in one, but in two stable forms, differing in EPR and optical spectra. The differential optical spectra of these forms were recorded and the differences in molar absorption coefficients determined. 2. By the EPR method, it was shown that both forms of these centres exist in the blood serum of control donors, as well as in the serum of patients. The relative content of these forms depends on the organism physiological state or on the presence of some pathological condition. 3. The ferroxidase activity of ceruloplasmin against hemoglobin was proved spectrophotometrically. The involvement of other serum proteins in this process cannot be ruled out. The conformational state of ceruloplasmin molecules plays an essential role in its oxidase activity.     

Electron paramagnetic resonance study of storage effects on ceruloplasmin in human serum compared with purified ceruloplasmin in aqueous solution

The EPR signal amplitude of human serum ceruloplasmin shows significant changes as a function of time and temperature during storage. The same behavior occurs with aqueous solutions of purified ceruloplasmin. From the observation that the spectral lines of the EPR signal of ceruloplasmin from unmanipulated serum are identical to those coming from purified ceruloplasmin, we conclude that only type I Cu2+ of ceruloplasmin are involved in the signal changes. A temperature-dependent electron shift toward type I Cu2+ paramagnetic centers, occurring via the type II and type III Cu2+ species of the protein, is believed responsible for the process. The possible origin of the reducing electrons is discussed. A procedure to obtain reproducibility of recording of EPR spectra of ceruloplasmin in physiological fluids is proposed.     

Reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate

The reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate was studied at pH 5.5. The analysis of optical and EPR spectra at 9 GHz showed that ceruloplasmin contains five paramagnetic copper ions, two of which, X and Y, not involved in enzymatic activity, are chelated by diethyldithiocarbamate; the complex thus formed is easily removed by high-speed centrifugation. However, the enzyme depleted of these two X and Y copper ions is able to compete with the Cu(II)-diethyldithiocarbamate complex, as time elapses, recovering both Cu(II) atoms. In addition diethyldithiocarbamate acts as a reducing agent for the two type-I copper atoms when added in large excess to the enzyme or the anion treated enzyme.     

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.     

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and partial characterization of goose ceruloplasmin

The preparation and properties of ceruloplasmin from goose blood plasma are described. Ammonium sulfate was used to precipitate the crude protein followed by adsorption and elution from DEAE-Sephadex A-50. Further treatment with an ethanol-chloroform mixture and Sephadex G-200 yielded an intensely blue protein possessing a high degree of chemical purity and biological activity. Goose ceruloplasmin, existing in two forms, appears to be a single polypeptide, apparent Mr121,300, with an A610/A280 ratio of 0.07. Copper represented 0.32%, which corresponded to six atoms of copper per protein molecule. Although the amount of EPR-detectable copper was the same as in mammalian ceruloplasmins there were some differences in EPR parameters, mainly in A parallel. Goose ceruloplasmin's amino acid composition, although similar in many residues to human ceruloplasmin, was lower in tyrosine, cystine/cysteine, and acidic amino acids. Valine was found as the N-terminal amino acid. Hexose, hexosamine, sialic acid, and fucose accounted for 6.65% of the weight. Goose protein contained only half the sialic acid of human ceruloplasmin. Two values for Km using either p-phenylenediamine (0.64 and 0.053 mM) or o-dianisidine (0.76 and 0.15 mM) were evaluated from Lineweaver-Burk plots. EPR studies on reactions with water radiolysis products at cryogenic temperatures allowed us to discover that goose ceruloplasmin, like human and bovine ceruloplasmins, possesses superoxide dismutase activity.     

Chicken ceruloplasmin. Evidence in support of a trinuclear cluster involving type 2 and 3 copper centers

Ceruloplasmin was isolated to purity from chicken plasma by a single-step chromatography on amino-ethyl-derivatized Sepharose. Molecular mass, as estimated by nonreducing sodium dodecyl sulfate-electrophoresis, was approximately 140 kDa, slightly higher than that found for ceruloplasmins from other sources. Specific activity as p-phenylenediamine oxidase was five times higher than that reported for mammalian ceruloplasmins. The copper content was estimated to be 5.01 +/- 0.35 atoms per protein molecule, 50% of which was EPR-detectable. The EPR spectrum was completely devoid of any signal typical of the type 2 copper as seen in the other blue multicopper oxidases and in ceruloplasmin from mammalian species. Anaerobic reduction of chicken ceruloplasmin resulted in the disappearance of the 330 nm optical band typical of type 3 copper, which was followed by the appearance of an EPR signal typical of type 2 copper. Subsequently, the type 1 copper and finally the newly formed type 2 copper were reduced. The original optical and EPR spectra were recovered within few minutes upon exposure of reduced ceruloplasmin to air. It is concluded that in oxidized chicken ceruloplasmin type 2 copper interacts with the diamagnetic pair responsible for the 330 nm absorption in such a way as to become EPR-undetectable and that the interaction is relieved by reduction of the pair. Whether this interaction is intrinsically weaker in other blue oxidases and ceruloplasmins studied or is lost with standard preparation procedures remains to be established.     

Immunoenzyme determination of the total level of ceruloplasmin in the serum from patients with hepatocerebral dystrophy]

An immunoenzymatic method for ceruloplasmin analysis (IEA) based on the use of horseradish peroxidase-labelled monospecific antibodies as markers has been developed. IEA can be used for direct measurements of ceruloplasmin in blood serum, as can be evidenced from the coincidence of calibration plots obtained after the use of potassium-phosphate buffer and ceruloplasmin-free sera. The procedure allows the determination of the total content of ceruloplasmin present in the blood sera of patients with hepatocerebral dystrophies both in the active and inactive forms. The minimum ceruloplasmin concentration detectable by this method is 5 x 10(-9) g/ml. The method was used to determine ceruloplasmin levels in the blood of patients with various grades of hepatocerebral dystrophy. Analysis of blood sera from 6 patients revealed that the ceruloplasmin concentrations determined by IEA were very close, whereas the oxidase activities of this protein differed more than 7-fold. The amount of enzymatically active ceruloplasmin as determined from the oxidase activity made up to 10-68% of the total ceruloplasmin content in the sera, depending of the severity of the pathology.     

Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy.

The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.     

Spectral characteristics of the mechanism of oxidase activity of ceruloplasmin

The absorbance and EPR spectra of type 1 and 2 copper-binding centres which are present in ceruloplasmin (Cp) molecule were shown to disappear upon the reduction of the enzyme by ascorbate under anaerobic conditions. The fluorescence band attributed to type 3 Cu was altered concomitantly. The electron-accepting nitroxyl radical added to reduced Cp restored the absorbance, EPR and fluorescence spectra of the oxidase. Only type 1 and 3 copper ions, as judged by spectral changes, can be reduced by ascorbate and then reoxidized by the nitroxyl radical in the azide-treated Cp. The spectral properties of Cp provided by copper ions of different types change simultaneously and concordantly upon oxidation/reduction. This seems to be caused by cooperative interaction of these ions involved in the electron transfer from the donating substrate to the accepting molecule of the nitroxyl radical (in model studies of oxidase reaction) or oxygen (under natural conditions). The copper ions in the active centre of Cp constitute an intramolecular electron transport chain, which may, at least in vitro, function without one of its links.     

EPR study of serum ceruloplasmin and iron transferrin in myocardial infarction.

Electron paramagnetic resonance (EPR) analysis of frozen serum from myocardial infarction patients has been conducted. Signal at g=4.3 was found definitively attributable to iron(III)-transferrin complex. Imcrease of serum ceruloplasmin as compared to normal was confirmed, with a concomitant decrease of iron-transferrin content. A mechanism for such correlated variation is hypothesized.     

Effect of ischemic preconditioning on free radical centers of the isolated rat heart during ischemia and early reperfusion

The effect of ischemic preconditioning on the free-radical state of isolated rat myocardium fixed by rapid freezing at the 25th min of normothermic total ischemia and the 3rd min of reperfusion was studied by the EPR method. It was shown that EPR spectra registered at -40 degrees C consist of two free-radical signals: of the semireduced forms of ubiquinone and flavine coynzymes. It was found that during ischemia and at the beginning of reperfusion, the preconditioning results in a narrowing of the spectra (as compared with control) due to an increase in the narrow ubisemiquinone EPR signal portion, and a decrease in the total concentration of free-radical centers: by 16% in the case of ischemia, and 23% in the case of reperfusion. It was concluded that in both cases the changes were due to a decrease in the concentration of myocardial flavosemiquinones as a result of ischemic preconditioning. We registered the microvawe power saturation curves for these two stages, which corresponded to control and ischemic preconditioning. In the case of ischemia these dependences had similar shapes; however, in the case of reperfusion they differ from each other due to changes in the relative intensities of the EPR signals from ubisemiquinone and flavosemiquinones in the integral myocardial free-radical spectra.     

Immunochemical and enzymatic study of ceruloplasmin in rheumatoid arthritis

Forty adult patients (30 women and 10 men) with rheumatoid arthritis (RA), treated with nonsteroidal anti-inflammatory drugs, were studied. Serum levels of immunoreactive ceruloplasmin, oxidase activity of the ceruloplasmin and total copper, as well as the specific oxidase activity (enzyme activity per unit of mass) and the copper/immunoreactive ceruloplasmin relationship were significantly higher in the group of patients than in the healthy control group (p < 0.001). However, no significant difference was found for the concentration of non-ceruloplasmin copper between both groups. A statistically significant negative correlation was obtained for the concentration of serum thiobarbituric acid-reacting substances with the immunoreactive ceruloplasmin and its oxidase activity in the group of patients (p < 0.005). These results suggest that in RA increases of serum copper are produced at the expense of the fraction linked to the ceruloplasmin, diminishing the proportion of apoceruloplasmin and other forms poor in copper. Although the increase in the serum concentration of ceruloplasmin might offer an additional safeguard against oxidative stress. it does not appear to have a beneficial effect upon the activity of the illness as evaluated by means the biological inflammation markers C-reactive protein, erythrocyte sedimentation rate and sialic acid.     

Unusual stability properties of a reptilian ceruloplasmin

Ceruloplasmin from the turtle Caretta caretta was isolated to purity by using the single-step procedure recently developed by us to purify sheep and chicken ceruloplasmin. It has a Mr of ca. 145,000 and a total copper content of 5.1 +/- 0.2 atoms of copper per molecule, 50% of which are detectable by EPR. The spectroscopic features include an absorption maximum at 603 nm in the electronic spectrum and the total absence of any resonance attributable to Type 2 copper in the EPR spectrum. Turtle ceruloplasmin was found to be unusually resistant to aging and proteolysis, when compared to ceruloplasmins isolated from other species. p-Phenyl-endiamine oxidase activity measurements revealed an unusually low catalytic efficiency, while the kinetic parameters of Fe(II) oxidation were consistent with those reported for other species of ceruloplasmin.     

The influence of radiotherapy on ceruloplasmin and transferrin in whole blood of breast cancer patients

Ceruloplasmin and transferrin are proteins which play a potential role in the process of breast cancer development. These molecules contain Cu²⁺ (ceruloplasmin) or Fe³⁺ ions (transferrin) and thus constitute paramagnetic centers, which can be studied using electron paramagnetic resonance method. The aim of the study was to determine how paramagnetic centers in whole blood of breast cancer patients change under the influence of radiation therapy. Samples of whole blood were taken from 17 women with breast cancer treated with radiotherapy. The measurements were carried out at 170 K using X-band electron paramagnetic resonance (EPR) spectrometer Bruker EMX-10. Two distinct EPR lines, derived from high-spin Fe³⁺ in transferrin and Cu²⁺ from ceruloplasmin, were revealed in all frozen samples. The amplitude and integrated intensity of the EPR signal from Cu²⁺ in ceruloplasmin significantly decreased in all patients after the delivery of the radiation fraction. When comparing the integral intensity of the signal from Fe³⁺ in transferrin, three different situations were identified which are patient specific: a significant increase, an insignificant change, or a significant decrease after the irradiation. A decreased level of Cu²⁺ from ceruloplasmin in patients after radiotherapy means a low level of ceruloplasmin in the plasma or an increased content of reduced Cu⁺ ions. Differences in the integrated intensity of the EPR signal from transferrin translate directly into the amount of bound iron. The observed changes could indicate how well the organism fights against cancer and how easily it adapts to the situation of biochemical stress.     

Oxidation of reduced cucumber ascorbate oxidase

Reoxidation process of reduced cucumber ascorbate oxidase (1.10.3.3) with dioxygen was investigated in detail through absorption, circular dichroic (CD) and electron paramagnetic resonance (EPR) spectra. One of the three type I coppers and the type II copper were reoxidized more rapidly than other type I coppers. The principal active site of ascorbate oxidase was considered to be comprised of one type I, one type II and a pair of type III coppers similarly to the active sites in laccase and ceruloplasmin. Remaining two type I and a pair of type III coppers were also disclosed to contribute to the oxidation of L-ascorbate.     

EPR study of iron status in human body during intensive physical activity

The iron metabolism was studied in serum blood samples collected from 26 professional sportsmen undergoing intensive physical exercises using EPR combined with hematological and biochemical laboratory tests. Only 23% of EPR spectra (n = 6) were practically normal while in the rest spectra additional abnormal absorption lines were detected. Presumably, the significant portion of new signals may be caused by different cytochromes. Moreover, the anisotropic signals with g ₁ ? 2.02; g ₂ ? 1.94 and g ₃ ? 1.86 registered in some spectra pointed to the sulfur-iron centers. There was nearly linear correlation between the concentration of Fe³⁺ in transferrin (Fe³⁺-Tf) obtained from the EPR spectra and the serum iron concentration measured by absorption photometry both for sportsmen and controls (healthy individuals and patients with different diseases). At equal serum iron concentrations the Fe³⁺-Tf level was higher in sportsmen than that in controls. The Pearson correlation coefficient (r) for Fe³⁺-Tf and serum iron values was equal to 0.89 in sportsmen versus r = 0.97 in controls. Additional new lines in serum EPR spectra of professional sportsmen prove the suitability of EPR assay for scheduled medical exams since routine biochemical and hematological tests are insufficient to discover all abnormalities in iron metabolism under intensive physical exercises.     

Detection of intracellular forms of secretory aspartic proteinase in Candida albicans.

The extracellular proteinase (EPR) of Candida albicans was induced in a medium containing bovine serum albumin as sole nitrogen source. There were two intracellular forms in cells induced to produce EPR, a 43 kDa protein (EPR) and a 45 kDa protein (cross-reacting material of EPR; CRM-EPR); these were detected by immunoblotting using anti-EPR antiserum. The 43 kDa protein (EPR) may be the same as the extracellular form judging by molecular mass, and the 45 kDa protein (CRM-EPR) may be a precursor form of EPR. Many dense granules were observed by electron microscopy near the plasma membrane of the mother cells in EPR-producing cells. Both the 43 and 45 kDa proteins were recovered in a membrane fraction and were solubilized by Triton X-100. When the membrane fraction was further fractionated by sucrose density gradient centrifugation, the 43 and 45 kDa proteins were differentially fractionated. This suggests that they were located in different membrane-bound structures and is consistent with an assumption that the 45 kDa protein is a precursor for EPR.     

Milk ceruloplasmin and its expression by mammary gland and liver in pigs

Concentrations of ceruloplasmin and copper in milk and blood plasma, the nature of milk ceruloplasmin, and the effects of lactation and gestation on these parameters, as well as the expression of ceruloplasmin mRNA by the mammary gland, were examined in pigs. As seen previously in humans, ceruloplasmin and copper concentrations in sow milk were much higher a few days after birth than 1 month later, averaging 26.5 and 6.6 mg ceruloplasmin/L (by immunoassay) and 1.67 and 0.34 mg total Cu/L, on days 3 and 33 postpartum, respectively. Values for ceruloplasmin oxidase activity (measured with p-phenylene diamine) were 7.8 and 1.3 nmol/min/L, respectively. Daily milk ceruloplasmin production went from 61 to 22 mg/day and daily copper output from 38 to 12 mg/day. In contrast, there was little or no variation in serum ceruloplasmin concentration during lactation or gestation, although total plasma copper was high at the end of gestation. Milk ceruloplasmin was of the same apparent size as serum ceruloplasmin, as determined by SDS-PAGE and immunoblotting, and ceruloplasmin mRNAs of liver and mammary gland were indistinguishable by Northern analysis and RT-PCR of the various exons. Expression of total RNA and ceruloplasmin mRNA, as detected in biopsies of mammary gland, increased markedly upon onset of lactation and then declined during the next month in conjunction with a drop in milk ceruloplasmin production. The results indicate that milk ceruloplasmin, while being the same protein as in plasma, is not derived from the plasma but is produced by the mammary gland.     

Interaction of ascorbate oxidase with inorganic anions

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.