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During oxydase reaction spectral characteristics of ceruloplasmin at absorption of copper ions and protein part of the molecule are shown to change. It has been ascertained, that when irradiating ceruloplasmin by UV-light the functioning of intramolecular electron transport chain is broken, the degree of positive cooperativity (a Hill's constant) on substrate decreases. It is supposed, that these changes are caused by disturbance of interdomain interactions in a protein molecule.

Purpose: Ceruloplasmin (CP), an important serum antioxidant, was previously found to reduce the incidence of ventricular fibrillation (VF) induced by ischemia and reperfusion in isolated rat hearts. The present study investigated whether CP sterilized by gamma-irradiation maintains its antiarrhythmic capacity and in vitro antioxidant properties., Materials and Methods: Isolated rat hearts submitted to regional ischemia (15 min), were reperfused (10 min) with native CP or with CP irradiated at various doses (1-3 kGy) in the absence or presence of tyrosine (Tyr)., Results: All untreated hearts showed VF at reperfusion, which were all irreversible ventricular fibrillation (IVF). No IVF were found in hearts treated with native CP or gamma-irradiated CP. Cardioprotection afforded by irradiated CP (with or without Tyr) was slightly higher than that obtained with native CP. No VF at all (100% prevention) was found in hearts treated with CP irradiated alone or in the presence of tyrosine at 3 kGy. Tyrosine and irradiated tyrosine had no cardiotoxic or protective effects on reperfusion-induced arrhythmias. The Oxygen Radical Absorbing Capacity (ORAC), measured in vitro with beta-phycoerythrin (beta-PE) fluorescent indicator, was slightly higher for gamma-irradiated CP in the presence of Tyr., Conclusions: Ceruloplasmin sterilized by gamma-irradiation maintains antioxidant and antiarrhythmic effects in the post-ischemia reperfused isolated rat heart.

The present paper describes a protective role of L-tyrosine against aggregation of caeruloplasmin and haemoglobin therapeutic proteins during their sterilization by gamma-irradiation in the aqueous phase. Irradiation of proteins, known to induce their degradation in the presence of oxygen, generates aggregation under oxygen-free conditions. It was found that L-tyrosine present during irradiation in deoxygenated media prevents protein aggregation even at high doses (10 kGy), as asserted by SDS/PAGE and high-performance size-exclusion chromatography. The protective role of L-tyrosine, allowing the gamma-irradiation treatment of therapeutic proteins in solution without conformational alteration, is probably exerted by scavenging oxygen radicals produced by irradiation-induced water radiolysis. It was also found that haemoglobin had a greater stability than caeruloplasmin under gamma-irradiation treatment.

It was previously shown that protein aggregation induced by gamma-irradiation can be prevented by L-tyrosine, even when irradiation was done in solution at doses as high as 10 kGy. It is now reported that caeruloplasmin irradiated at low to moderate doses appears more resistant to trypsin-mediated proteolysis than native caeruloplasmin. Ceruloplasmin presents an irradiation-dose-dependent decrease of oxidase activity and a slight increase of ferroxidase activity at low irradiation doses (2 kGy), followed by a decrease at high doses (4-8 kGy). In all cases, the catalytic activities are higher when caeruloplasmin is irradiated under tyrosine protection. Irradiated caeruloplasmin in the presence of tyrosine preserves its oxidase and ferroxidase activities. Caeruloplasmin irradiated in solution at a dose of 2-3 kGy and higher, in the presence and absence of L-tyrosine, remained sterile during at least 8 weeks of storage at 4 degrees C, as evaluated by the plate-count-agar method for assessing total viable lactic acid bacteria content after incubation for 48 h at 30 degrees C.

Photoinduced changes of human ceruloplasmin (hCp) under laser and ultraviolet (UV) radiation have been studied. A complex character of hCp spectral modifications points to a different degree of the protein molecule folding. After the influence of UV radiation (240-390 nm) in a dose range 906-4530 J/m2 the decrease of oxidase hCp activity was registered. These changes are thought to be connected with modification of the conformation of hCp active centre or copper reduction that was confirmed by the decrease of optical density at 610 nm. An inactivation constant (kf = 3.8 x 10(-4) 1/s) and a photoinactivation quantum yield (1.4 x 10(-4)) were calculated. Kinetic dependencies of o-phenilen-diamine oxidation by intact and UV-modified glycoprotein do not correspond to Michaelis-Menten equation. UV radiation in a dose range 151-4530 J/m2 induced increasing of ceruloplasmin superoxiddismutase activity. The influence of He-Ne laser radiation (1.3 mW; lambda = 632.8 nm, exposure time from 1 up to 30 min) leads to the raise of the hCp functional activity; superoxiddismutase activity changes more significantly. Possible photophysical and photochemical processes in hCp molecule, leading to photomodification and changes of protein functional activity have been represented on the scheme.

Superoxide dismutase activity in blood red cells of patients with urethral strictures reflects the disease severity and the response to relevant treatment. Antioxidant therapy with dibunol on the first postoperative days, administration of vitamin E before and after the operation and laser biocontrolled chronotherapy for 2 weeks before and after the operation with repetition in 1-2 months are recommended in addition to conventional treatment to reduce probability of complications and recurrences after surgical treatment of urethral strictures.

The concentrations of ceruloplasmin could not be determined by comparing the oxidase activities toward p-phenylenediamine and o-dianisidine in rat whole serum with those of purified ceruloplasmin because of the different specific amine oxidase activities of ceruloplasmin purified from normal and gamma-irradiated rats and the lability caused by freezing and thawing. A competitive enzyme linked immunosorbent assay with antigens immobilized on the solid phase was developed to measure the ceruloplasmin in rat serum. The blocking materials and pH of the coating buffer had an effect on the amount of ceruloplasmin bound to the microtiter plates. The blocking materials nonspecifically reacted with the rabbit anti-rat ceruloplasmin IgG, and consequently the coating sensitivity was decreased and standard curves were not formed completely. Because rabbit anti-rat ceruloplasmin IgG did not show any cross reactivity with rat albumin and hemoglobin, these proteins did not affect the assay at a concentration of 200 micrograms/ml. This assay is useful for measuring the concentration of ceruloplasmin in normal and irradiated rat serum.

1. The mechanism of interaction of CP with O2- radicals in chemical and enzymatic systems of superoxide radical generation as well as in the pulse radiolysis technique was studied. 2. It is found that CP does not exert any kinetic influence on the decomposition of superoxide radical and, unlike SOD, cannot catalyze the reaction of disproportionation of these radicals in systems with chemical and enzymatic generation of O2-. 3. The data obtained confirm the suggestion that CP interacts with precursors of O2- radicals. 4. The irradiation of CP does not change its inhibiting activity in the reaction of the formation of superoxide radicals in systems with enzymatic O2- generation, but decreases its oxidase activity. 5. The results obtained demonstrated that the increase in the radiation dose resulted in the decrease of the inhibiting activity of SOD, whereas the activity of CP did not change.

A study of the peripheral blood indicates that persons constantly working in the zone of rigid radiation control showed a statistically valid reduction of the hemoglobin concentration, appearance of spinous and spheric erythrocyte forms, reduction of the mechanical resistance. Simultaneously 18% of males and 26% of females revealed moderate hypochromic anemia with a normal level of serum iron. The intensity of Fe-transferrin electron paramagnetic resonance was statistically reduced, that may be related to reduction of trivalent iron to bivalent and formation of a pool of free bivalent iron--a potent inductor of lipid peroxidation. Activation of oxidative processes in blood is confirmed by significant fluctuations of the blood ceruloplasmin level.

The study deals with the mechanism of organism's adaptive responses to the effect of radiation in widely ranging dose. Post-irradiation metabolic changes were evaluated in canine blood as well as in murine blood, spleen, bone marrow and liver using the EPR spectroscopy. It was shown that the dynamics of changes in transferrin and ceruloplasmin pools and ribonucleotide reductase activity were phase-dependent with the maxima at the 2nd, 6th and 10-12th days after irradiation. Such dynamics was observed at various irradiation doses applied. The data allow us to suggest that the nonspecific compensatory--adaptive reactions of organisms develop as the response to irradiation. The dose-response function of the reaction intensity was found to be linear. The shape of the dose-response curve indicates that the minimum response of organism depends on the dose linearly up to 3.2 Gy (for dogs) as well as the maximum one. However, in the case of low-dose irradiation (0.25 or 0.5 Gy) there were deviations of maximum responses from the linearity, i.e. the amplification of the amplitude of compensatory adaptive reactions. These effect were shown to be dependent upon initial individual characteristics of animal blood and to be related to the "depressed" or "activated" state of organism prior to irradiation. The ribonucleotide reductase activity was measured in bone marrow and spleen of animals by the EPR method. The nature of non-repairable DNA damage is discussed in view of the inactivation of ribonucleotide reductase.

The change of ceruloplasmin level of blood serum of guinea-pigs was studied on the 12th h after irradiation with gamma-rays from 60Co, using doses of 1-10 Gy, power of the dose of 0.68 Gy/min. The enzyme level was raised by the increase of the irradiation dose as it was reduced by the dose of 10 Gy in comparison with the control animals. This was probably due to inactivation of the molecules of ceruloplasmin by radicals and peroxides, obtained by this hyperlethal dose. Sera proteins showed a change in the increase of the dose: a reduction of albumins and an increase in beta- and gamma-globulins. Alpha-2-globulins, with which ceruloplasmin moved electrophoretically at low doses, showed slowly a slow increase in comparison with the control value, but at high doses-phase globulins. Oxidase activity of ceruloplasmin was determined by 01 todianisidine, but sera proteins-by agarose electrophoresis. The level of s ceruloplasmin in animals, irradiated by LD50 = 4.1 Gy, showed chronological change-an increase at early periods, maximum on the 12th h and a reduction at late periods close to the normal range. The level of serum copper followed the change in the enzyme level by analogy.

The low temperature (77 K) irradiation of oxidized ceruloplasmin and Rhus vernicifera laccase at the 330 nm absorption which arises from type 3 copper leads to the reduction of type 1 copper as demonstrated by bleaching of the 610 nm chromophore and the decrease of the EPR signal associated with this species. Type 2 copper remains unaffected. Concomitant with the type 1 copper reduction, a new EPR signal which is possibly that of a biradical appears. Upon thawing, type 1 copper is reversibly oxidized and the radical signal disappears. Irradiation of oxidized protein at the absorption band of type 1 copper produces no spectral change. An EPR study at room temperature confirms the wave-length specificity and reversibility of the photoreduction of type 1 copper and radical formation. Radical appearance and disappearance at room temperature are extremely slow (tau1/2 approximately 30 min). Optical studies at room temperature show that upon anaerobic irradiation of laccase in the 330 nm absorption band, both type 3 and type 1 chromophores are slowly reduced. Upon return to the dark and in the presence of O2, both type 3 and type 1 centers are reoxidized. Oxidizing equivalents either from O2 or K3Fe(CN)6 are required for the reoxidation reaction. These studies demonstrate that there is a direct energy transfer between type 3 and type 1 copper sites in blue copper oxidases.

Exposure of human caeruloplasmin, an acute phase protein with antioxidant properties, to a mixture of xanthine/hypoxanthine and xanthine oxidase as a source of reactive oxygen intermediates decreased its ferroxidase and ascorbate oxidase activities and its ability to inhibit lipid peroxidation. Immunological reactivity was also altered. Exposure to hydrogen peroxide mimicked these effects. Exposure to low-intensity u.v. irradiation depressed caeruloplasmin's ability to inhibit iron-catalysed hyaluronic acid degradation. The results may explain the mechanism of the observed inactivation of caeruloplasmin within human rheumatoid synovial fluid.

The authors studied changes in serum protein concentrations in rats exposed 10 days to continuous irradiation at progressively diminishing dose rates imitating the radioactive decay of a fissile mixture, up to a total dose of 9 Gy, both during and up to 240 days after discontinuing irradiation. The prealbumin concentration (from the 6th day) and the A1-globulin concentration (from the 1st day) fell to 70% during irradiation. Afterwards, the A1-globulin level fell significantly up to the 28th day and then, from the 60th to the 240th day, rose again. During irradiation the serum haptoglobin concentration rose significantly from the 1st to the 10th day and the ceruloplasmin concentration on the 10th day. An increase in both protein concentrations (to 172% and 190%) was also observed on the 14th postirradiation day, after which the serum haptoglobin, ceruloplasmin and A1M concentration fell abruptly up to the 60th day.

Studied was the change in the oxidase activity of ceruloplasmin (CP) upon the treatment of chickens with gamma rays of 60Co at 1, 2, 3, 5, 7, 10 Gy and dose power of 0.7 per min. The minimum of the oxidase activity of CP at 3 G was found to be 0.014 AE, which, upon raising (higher doses) reached 0.044 AE. Serum copper also rose upon raising the rate of treatment--from 2.09 op to 3.76 microM. Birds that were vaccinated and treated at 4 G showed on the 15th and the 40th day a drop of the oxidase activity of CP from 0.015 AE for the control group to 0.012 AE for the experimental groups. Blood copper rose from 2.55 microM to 3.67 microM.

Changes in the concentration of some serum proteins are determined by quantitative two-dimensional immunoelectrophoresis in rats subjected to continuous irradiation with 400R/day for 11 days. At this dose rate albumin levels were decreased by 36% and IGG levels by 70% in comparison with the control (C2) values. The concentration of haptoglobin, haemopexin and caeruloplasmin was increased by 75-140% on day 10 as compared with the control (C2) values.

The intensity of the ESR signal of methemoglobin, Fe3+-transferrin, and Cu2+-ceruloplasmin in blood and spleen of mice exposed to 6 Gy radiation was shown to undergo considerable changes at early times of acute radiation sickness.

Studied were the changes in the concentration of ceruloplasmin through altering its oxidase activity in the blood serum by gamma treatment, using a 60Co source at the rate of 3 Gy and dose power of 0.68 Gy/min-1. The oxidase activity of the enzyme was determined colorimetrically with an o-dianisidine substrate. The concentration of ceruloplasmin rose from 34 E/l prior to irradiation to 129 E/l following treatment. The analyses were carried out immediately before using gamma rays and at the 30th min, at the 15th and 40th hour, and on the 10th, 17th, and 30th day after that. By the 30th day the ceruloplasmin level was found to drop up to 90 E/l. The serum proteins also changed their values following treatment. Albumin and gamma-globulins dropped from 52.3 to 43.2 per cent and from 20.1 to 13.1 per cent, respectively. Alfa-1 rose from 8.5 to 9.8 per cent, alfa-2 globulins--from 13.0 to 17.7 per cent, and beta-globulins--from 6.0 to 15.1 per cent. Alfa-2 globulins were shown to follow the same pattern of rising as ceruloplasmin and serum copper. As a result of the irradiation disease that developed after treatment a paraclinical picture of an acute inflammatory process set in.