P-phenylenediamine Oxidase Activity Research Articles

Effect of Ketoprofen on acute phase protein concentrations in goats undergoing castration.

BackgroundThe objective of this study was to determine the effect of ketoprofen on acute phase protein (APPs) concentrations in goats undergoing castration. A total of 16 clinically healthy, male and 12 months old goats were enrolled and each case received ketoprofen (group I) or control (group II) in a randomized fashion. Goats were sedated with Xylazine-HCl, afterwards ketoprofen (3 mg/kg) was injected via jugular vein in group I, whereas physiological saline solution was administered to group II. Goats were castrated by the Burdizzo method. Hematological parameters were determined with a blood cell counter and plasma fibrinogen (Fb), serum haptoglobin (Hp), serum amyloid A (SAA) and ceruloplasmin (Cp) concentrations were measured Millars technique, ELISA kit or p-phenylenediamine oxidase activity prior to castration and throughout the study on 0 to 96 h.ResultsThere were no differences in pre-treatment serum Cp, SAA and Fb concentrations among the groups. Contrarily, there were significant differences in plasma Hp concentrations on 0 to 96 h onwards post-castration. There were no differences in WBC and PCV between groups. Cp, Fb, and SAA were almost constant or showed slight changes at various stages of the study with no significant differences between groups.ConclusionsThe results revealed that, levels of Cp, Fb and SAA may not be affected by castration such as the confounding parameters similarly to stress. More investigations possessing different surgical or non-surgical castration techniques with larger number of goats and focusing on specific markers for stress are suggested for precise analysis.

Mechanisms for cellular NO oxidation and nitrite formation in lung epithelial cells

Airway lining fluid contains relatively high concentrations of nitrite, and arterial blood levels of nitrite are higher than venous levels, suggesting the lung epithelium may represent an important source of nitrite in vivo. To investigate whether lung epithelial cells possess the ability to convert NO to nitrite by oxidation, and the effect of oxygen reactions on nitrite formation, the NO donor DETA NONOate was incubated with or without A549 cells or primary human bronchial epithelial (HBE) cells for 24 h under normoxic (21% O2) and hypoxic (1% O2) conditions. Nitrite production was significantly increased under all conditions in the presence of A549 or HBE cells, suggesting that both A549 and HBE cells have the capacity to oxidize NO to nitrite even under low-oxygen conditions. The addition of oxyhemoglobin to the A549 cell medium decreased the production of nitrite, consistent with NO scavenging limiting nitrite formation. Heat-denatured A549 cells produced much lower nitrite and nitrate, suggesting an enzymatic activity is required. This NO oxidation activity was highest in membrane-bound proteins with molecular size <100kDa. In addition, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and cyanide inhibited formation of nitrite in A549 cells. It has been shown that ceruloplasmin (Cp) possesses an NO oxidase and nitrite synthase activity in plasma based on NO oxidation to nitrosonium cation. We observed that Cp is expressed intracellularly in lung epithelial A549 cells and secreted into the medium under basal conditions and during cytokine stimulation. However, an analysis of Cp expression level and activity measured via p-phenylenediamine oxidase activity assay revealed very low activity compared with plasma, suggesting that there is insufficient Cp to contribute to detectable NO oxidation to nitrite in A549 cells. Additionally, Cp levels were knocked down using siRNA by more than 75% in A549 cells, with no significant change in either nitrite or cellular S-nitrosothiol formation compared to scrambled siRNA control under basal conditions or cytokine stimulation. These data suggest that lung epithelial cells possess NO oxidase activity, which is enhanced in cell-membrane-associated proteins and not regulated by intracellular or secreted Cp, indicating that alternative NO oxidases determine hypoxic and normoxic nitrite formation from NO in human lung epithelial cells.

Dietary Iron Status Has Little Effect on Expression of Ceruloplasmin but Alters That of Ferritin in Rats

Evidence supports a role for ceruloplasmin (ferroxidase I) in the release of iron to the blood from mammalian cells. However, recent studies with cultured cells have suggested that it has the opposite effect, and that iron deficiency enhances expression of ceruloplasmin. We therefore examined in rats how nutritional iron status would affect expression of ceruloplasmin. Groups of male Sprague-Dawley rats were reared on a low iron, starch-based diet for 6-8 wk; half were supplemented by injection of iron dextran. At killing, hematocrits of deficient rats were half normal. Supplemented rats had normal liver concentrations of ferritin and ferritin iron. No ferritin was detected in the livers of the deficient rats. Northern analysis showed that ferritin L and H mRNAs were present in the deficient livers, but expression was half that of the normal rats. There was also twice as much copper. Levels of circulating ceruloplasmin (measured by rocket immunoelectrophoresis) were not altered by iron deficiency, although p-phenylenediamine oxidase activity and plasma copper were reduced approximately 30%. In repeated studies, no differences in the expression of hepatic ceruloplasmin mRNA were detected. Treatment of rats of both sexes with additional iron (25 mg as iron dextran) 5-14 d before killing increased liver ferritin but did not alter liver ceruloplasmin mRNA expression or levels of circulating ceruloplasmin. We conclude that iron status is not an important factor in the expression of plasma ceruloplasmin made by the liver. However, it does have modest effects on steady-state levels of liver ferritin mRNA.

Direct correlationship between proton translocation and growth yield: An analysis of the respiratory chain of Bacillus stearothermophilus

Thermophilic bacilli contain cytochrome caa 3-type cytochrome c oxidase as the main terminal oxidase in the respiratory chain. A mutant strain, named K-17, lacking cytochrome caa 3 and exhibiting very low N,N,N′,N′-tetramethyl- p-phenylene diamine oxidase activity, was isolated by random mutation from Bacillus stearothermophilus K1041 (Sakamoto, J. et al., FEMS Microbiol. Lett., 143, 151–158, 1996). Comparing this mutant with the parent strain K1041, we obserbed the following differences in energy-yielding properties. (i) K-17 gave an cell yield less than one half of that of the wild type, although the doubling time of K-17 was only a little slower than that of the parent strain. (ii) In cellular respiration, the H +/O ratio of K-17 was 2.9–3.1, while that of the wild type was 6.1–6.5. (iii) A low concentration of cyanide inhibited endogenous respiration of the wild-type cells partly with a concomitant reduction of the H +/O ratio to around 3, while it did not significantly affect the respiration rate and the H +/O ratio of the K-17 cells. (iv) Cytochrome bd-type quinol oxidase seemed to operate in the wild-type cells when a low concentration (below 0.5 mM) of cyanide was added, while this enzyme is the main terminal oxidase in K-17. The K-17 cells also contained cytochrome b(o/a) 3-type cytochrome c-551 oxidase. These results demonstrated that the combination of the enzymes involved in the respiratory chain determines the H +/O ratios of the cell and consequently the growth yield of the bacteria.

Purification and Characterization of Fet3 Protein, a Yeast Homologue of Ceruloplasmin

The FET3 gene product of Saccharomyces cerevisiae is an essential component of the high affinity iron transport system. Based on FET3 sequence homology to the multicopper oxidase family and iron oxidation studies in spheroplasts (De Silva, D. M., Askwith, C. C., Eide, D., and Kaplan, J. (1995) J. Biol. Chem. 270, 1098-1101), it was hypothesized that the Fet3 protein (Fet3p) was a cell surface ferroxidase. To further characterize the protein, we have isolated Fet3p from yeast membranes and purified the protein to apparent homogeneity. Consistent with its localization at the plasma membrane, Fet3p is a glycosylated protein. SDS-polyacrylamide gel electrophoresis analysis showed that the protein was present in two differentially glycosylated forms of approximately 120 and 100 kDa. Purified Fet3p is a copper-containing protein that is able to catalyze the oxidation of a variety of organic compounds in addition to ferrous iron. Azide and metal chelators strongly inhibited enzyme activity. Iron appeared to be the best substrate for the enzyme, and the apparent Km for ferrous oxidation was 2 microM. Interestingly, Fet3p was able to effectively catalyze the incorporation of iron onto apotransferrin. We conclude that Fet3p is a ferro-O2-oxidoreductase in yeast, homologous to the human plasma protein ceruloplasmin.

Consideration of the optimum pH for the analysis of serum p-phenylenediamine oxidase activity in thoroughbred horses.

The optimum pH for the measurement of serum p-phenylenediamine oxidase (Ox) activity was given (pH 6.6), and the relationship between serum ceruloplasmin (Cp) concentration and its Ox activity was established in healthy adult horses. In adult horses, serum antigenic Cp concentrations were measured by the single radial immunodiffusion (SRID) method with the affinity-purified antibody to equine plasma Cp and compared with its Ox activity. Efficient co-relation between Cp concentration and Ox activity in the sera (r = 0.93) and its Ox/Cp ratio were given. These results might contribute to the calculation of antigenic Cp concentration from its Ox activity, the analysis of holo-Cp content in serum, and the research for copper metabolism in thoroughbred horses.

Copper transport in the Nagase analbuminemic rat

The copper binding components of serum and potential importance of albumin to copper transport were investigated in adult Nagase analbuminemic and Sprague-Dawley rats of both sexes. There was a sex difference in total plasma copper concentrations, which were 60 and 130% higher than in the parent strain, in male and female Nagase rats, respectively. The higher levels of plasma copper were accounted for by two- and threefold increases in ceruloplasmin, as measured by p-phenylenediamine oxidase activity and copper by atomic absorption after gel chromatography. Other nonalbumin plasma proteins were also present in higher concentrations. Albumin concentrations were one-four-thousandth that of Sprague-Dawley rats, at 15 micrograms/ml (determined by rocket immunoelectrophoresis and comparative Western blotting). The tissue distribution and rate of uptake of intravenously injected 67 Cu(II) were unaffected by the lack of circulating albumin. 67Cu entered the liver of Nagase rats at least as rapidly as in the parent strain and reemerged in the blood on ceruloplasmin at an accelerated rate. The initial binding of 67Cu(II) to plasma components was primarily to transcuprein compared with albumin in the case of Sprague-Dawley rats. In the Nagase rats, the rest of the 67Cu bound primarily to nonalbumin proteins with about the same size as albumin; in Sprague-Dawley rats, it bound primarily to transcuprein. Limited analyses of tissue copper confirmed previous reports showing no striking differences from the parent strain. We conclude that albumin is not critical to the normal distribution and metabolism of copper and that it may serve more as a reservoir for excess plasma copper than as a specific conduit for the delivery of this element to hepatocytes or other cells.

Abnormal accumulation of apo-ceruloplasmin in a rat model of Wilson's disease

We examined the mechanism of decrease in serum ceruloplasmin (Cp) in LEC rats, an animal model of Wilson's disease. Northern blot analysis showed that the level of Cp mRNA in the liver of LEC rats was similar to that in control LEA rats. Moreover the serum level of Cp in LEC rats, determined by ELISA, was 70% of that in LEA rats, but its p-phenylenediamine oxidase activity was significantly decreased. Serum Cp was detected as two bands by polyacrylamide gel electrophoresis and immunoblot analysis. Cp in the band with higher mobility had ferroxidase activity and was the predominant form in LEA rats, whereas that in the band of lower mobility was inactive and was the major form in LEC rats. Dialysis of serum against sodium cyanate to remove copper from Cp resulted in disappearance of the form of Cp of higher mobility with increase in the slower migrating form. These results demonstrate that the faster and slower migrating forms of Cp are a holo-Cp and apo-Cp, respectively, and that LEC rats accumulate apo-Cp in their serum. A disturbance of copper binding to apo-Cp may be responsible for pathogenesis in LEC rats.

Synthesis and Turnover of Ceruloplasmin in Rats Treated with 17β-Estradiol

The effects of estrogen on synthesis and turnover of ceruloplasmin were studied in adult female Fischer rats. Daily treatment with 140 μg 17β-estradiol resulted in a slow rise of ceruloplasmin concentrations, as measured by p-phenylenediamine oxidase activity, leading to a 70% increase by 7 days and a tripling by Day 14. Ceruloplasmin protein concentrations increased to the same degree, based on yields of the protein obtained during purification. Effects of estrogen on rates of synthesis (incorporation of [ 3H]leucine) were followed, using immunoprecipitation of total ceruloplasmin or isolation of its two major isoforms ( R f s 0.4 and 0.6 in native gel electrophoresis). Synthesis was increased by 7 days and was 2.5 times that of controls by Day 14. Both forms of ceruloplasmin showed the same specific activities and degree of increase in rate of synthesis. Rates of ceruloplasmin turnover were unaffected, based on double labeling with 3H- and 14C-leucine, but were three- to fourfold faster than for total plasma protein. The enzymatically more active 0.6 R f form of ceruloplasmin had a faster turnover rate than the 0.4 R f form. Estrogen treatment doubled ceruloplasmin mRNA levels by 7 days and almost tripled them by Day 14. Most of the ceruloplasmin mRNA was associated with the endoplasmic reticulum-bound polyribosomes. We conclude that estrogen increases the rate of synthesis of two forms of ceruloplasmin by indirectly increasing liver concentrations of its mRNA but has no effect on ceruloplasmin turnover.

Rat ceruloplasmin: Resistance to proteolysis and kinetic comparison with human ceruloplasmin

Rat ceruloplasmin was purified from serum using fast protein liquid chromatography and compared to human ceruloplasmin isolated in the same manner. Rat ceruloplasmin was found to be more resistant to plasmin-mediated proteolysis than was human ceruloplasmin. Although both proteins were cleaved initially to products with apparent molecular weights of 116,000 and 20,000 Da, rat ceruloplasmin was resistant to further proteolysis, whereas the human enzyme was cleaved to smaller fragments. Primary structure differences could account for the different relative stabilities between the two enzymes. Kinetic analysis of rat ceruloplasmin produced a biphasic v vs v s plot with apparent K m 's of 40 and 1.5 μ m for iron. When compared with the human enzyme, rat ceruloplasmin showed about one-fourth the ferroxidase activity and had a much broader pH profile than that of human ceruloplasmin. Rates of p-phenylenediamine oxidation by rat ceruloplasmin were about one-half those obtained with human ceruloplasmin, with maximal p-phenylenediamine oxidase activity at pH 5.0 for both enzymes.

Lipid Peroxide Levels and Antioxidant Enzyme Activities in Blood of Breast Cancer Patients.

Supeoxide dismutase, glutathione peroxidase, glutathione S-transferase, and the p-phenylenediamine oxidase activity of ceruloplasmin, as well as lipid peroxides in terms of malondialdehyde, were determined in the blood of 52 breast cancer patients and 29 healthy women. No differences except for superoxide dismutase were found regarding lipid peroxide level and antioxidant enzyme activities in blood plasma and red blood cells between breast cancer patients and matched control women. Superoxide dismutase activity was statistically significantly lower in breast cancer patients than in the control group (p<0.0002). We also found a statistically significant correlation between the p-phenylenediamine oxidase activity of ceruloplasmin and lipid peroxide concentration in the blood plasma of the patients with breast cancer (r=0.342, p<0.05).

Detection of rat ceruloplasmin receptors using fluorescence microscopy and microdensitometry

Rat ceruloplasmin (rCp) has been labeled with the fluorophores fluorescein and rhodamine by using the isothiocyanate derivatives (FITC, RBITC). High p-phenylenediamine oxidase activity of the resulting conjugates was observed (70–90% of native activity). Polyacrylamide gel electrophoresis showed fluorescein-labeled rCp (FITC-rCp) had an increased mobility, while rhodamine-labeled rCp (RBITC-rCp) showed no increase in mobility when compared to native rCp. RBITC-rCp was tested as a probe for ceruloplasmin receptors on rat erythrocytes using fluorescence microscopy to detect membrane binding. Film negatives of blood smears exposed under identical conditions were analyzed by microdensitometry to give relative optical densities for the amount of RBITC-rCp bound per unit area of the plasma membrane. With this technique, binding of rCp was observed to be saturable, reversible, and specific. Competition of the protein ligands superoxide dismutase, catalase, and unlabeled rCp against RBITC-rCp already bound on erythrocytes showed that only rCp could displace the bound RBITC-rCp with 32% specific binding being observed. Low levels of membrane binding were seen after the erythrocytes had been trypsin treated. Platelet binding was also detected and it was saturable, reversible, and trypsin sensitive. However, only 20% of the bound RBITC-rCp could be displaced by rCp. These studies demonstrate a versatile technique for detection and localization of Cp receptors.

Isolation of a cytochrome aa 3 gene from Bradyrhizobium japonicum

Bradyhizobium japonicum strain LO501 is a Tn5-induced mutant that does not express the terminal oxidase cytochrome aa(3) (cytochrome-c oxidase, EC 1.9.3.1). Two and one-half kilobase pairs of LO501 genomic DNA that flanks the transposon was isolated and used as a hybridization probe to obtain the wild-type gene from a cosmid library. Two subcloned fragments from two of the isolated cosmids were ligated into broad host range vectors, and restriction maps of these fragments were generated. The resultant plasmids, pCA1 and pBL33, each contained DNA homologous to that mutated in strain LO501. The two plasmids were each introduced into strain LO501 by conjugal transfer, and it was found that pCA1, but not pBL33, complemented the oxidase mutant. The transconjugant strain LO501[pCA1] expressed wild-type levels of cytochrome aa(3), as discerned spectrophotometrically, and had restored N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity. Furthermore, the frequency of complementation of LO501 cells that received pCA1 by conjugation was 1.0, demonstrating that pCA1 complemented the mutant in trans. The results show that pCA1 contains the entire wild-type gene that was mutated in strain LO501, and this gene is required for cytochrome aa(3) expression.

A ‘blue’ copper oxidase from Nitrosomonas europaea

A soluble copper-containing protein with p-phenylenediamine oxidase activity was purified from Nitrosomonas europaea by flat-bed isoelectric focusing and chromatography on Sephacryl S-300. The native and subunit molecular weights were 127 500 and 40 100, respectively; the isoelectric point was pH 4.63. The protein had an absorption maximum at 607 nm in the oxidized form with an extinction coefficient of 6.0 cm −1 · mM −1 at pH 7.5. On reduction with dithionite, the absorbance was abolished. The electron paramagnetic resonance spectrum of the protein showed evidence for both Type 1 and Type 2 copper in a 1:1 ratio. The protein catalyzed the aerobic oxidation of p-phenylenediamine, cytochrome c-554, and hydroxylamine oxidoreductase. The enzyme catalyzed the reduction of nitrite with cytochrome c-552 as electron donor. In contrast, p-phenylenediamine, reduced cytochrome c-554, and hydroxylamine oxidoreductase were not active as electron donors for nitrite reduction.

Correlation of lysyl oxidase activation with the p-phenylenediamine oxidase activity (ceruloplasmin) in serum.

A dietary deficiency in copper decreases the lysyl oxidase activity in aortic tissue of growing chickens. The decrease has been correlated with a lowering of serum copper and ceruloplasmin (measured as PPD oxidase activity). Injections of CuSO, restored enzyme function and brought serum copper and ceruloplasmin levels back to normal. How- ever, when the CuSO., was given to chicks that had received two injections of estradiol-17/3, a much stronger increase in lysyl oxidase in response to CuSO, was observed. The enhanced response correlated with an enhanced increase in ceruloplasmin, linking the serum copper protein with the restoration of lysyl oxidase activity. Lysyl oxidase is a copper-dependent en- zyme. It requires copper to function as a catalyst and to maintain a constant level of activity in aortic tissue. The enzyme catalyzes the formation of allysines, the ac- tive aldehyde centers which give rise to crosslinks in collagen and elastin (1). Lysyl oxidase activity fails in animals that are fed diets low in copper, but is restored by re- feeding copper-supplemented diets or in- jecting CuSO, intraperitoneally (2). The close interplay between copper and an as- sociated metalloenzyme is evident. Not so evident, however, is a biochemical under- standing of the mechanism. Very little is known of the process by which metal ions are transported in the blood to the tissues. Even less is known of how metal ions enter cells to engage the binding sites of enzymes. The lysyl oxidase system may provide a model for elucidating such events, at least to the extent of iden- tifying transport components for delivering copper to the metal-free enzyme. Studies in vitro have suggested that lysyl oxidase acti- vation coincides with the binding of copper ions to newly synthesized molecules of apoenzyme (3). Although copper ions may limit the activation of this enzyme in vitro, free metal ions are not likely to be the im- mediate source of the protein-bound metal in the intact animal; a serum protein or a complex of copper with specific amino acids (4) probably performs this function in vivo. The present study examines this

Effect of crosslinking cytochrome c oxidase

Bovine heart cytochrome c oxidase and rat liver mitochondria were crosslinked in the presence and absence of cytochrome c. Biimidate treatment of purified cytochrome oxidase, which results in the crosslinkage of all of the oxidase protomers except subunit I when ⩾ 20% of the free amines are modified, inhibits ascorbate- N,N,N′,N′-tetramethyl- p-phenylene diamine oxidase activity. Intermolecular crosslinking of cytochrome oxidase molecules, which results in the formation of large enzyme aggregates displaying rotational correlation times ⩾ 1 ms, does not affect oxidase activity. Crosslinking of mitochondria covalently binds the cytochrome bc 1 and aa 3 complexes to cytochrome c, and inhibits steady-state oxidase activity. Addition of cytochrome c to purified cytochrome oxidase or to cytochrome c-depleted mitoplasts increases this inhibition slightly. Cytochrome c oligomers act as competitive inhibitors of native cytochrome c; however, crosslinking of cytochrome c to cytochrome c-depleted mitoplasts or purified cytochrome oxidase results in a catalytically inactive complex. These experiments indicate that cytochrome c oxidase subunit interactions are required for activity, and that cytochrome c mobility may be essential for electron transport between cytochrome c reductase and oxidase.

Ceruloplasmin in monkey plasma.

An abnormally greenish tint appeared in the blood plasma of adult rhesus monkeys that had been daily administered an intramuscular injection of 1-methyl-2-(β-naphtyl)-aziridine or chlorpromazine. This greenish component in the monkey plasma could not be extracted with cyclohexane, 1, 2-dichloroethane or n-butyl alcohol, and the greenish tint was scarcely changed by the addition of ethylenediaminetetraacetate. The greenish monkey plasma showed a visible absorption band at 610 nm like the blue fraction separated from it. These absorption bands at 610 nm, both in the greenish monkey plasma and the separated blue fraction, disappeared with the addition of ascorbic acid and reappeared with oxygen, as with human ceruloplasmin (Cp). The electron spin resonance spectrum of the greenish monkey plasma was similar to that of human Cp. Furthermore, the major portion of copper in monkey plasma was in the blue fraction, and the total plasma copper and p-phenylenediamine oxidase activity (PPD activity) increased with deepening of the green tint. These results suggested that the abnormal greenish tint of the monkey plasma is due to the presence of the blue copper protein Cp in the plasma. Based on examination of the relationships between total plasma copper and PPD activity in monkey and rat plasma, monkey Cp activity was compared with those of other mammalian species.

Menkes' Kinky hair syndrome: studies of copper metabolism and long term copper therapy.

A patient with Menkes' kinky hair syndrome was treated with oral CuSO4. Plasma copper, ceruloplasmin, red cell copper, and 24-hr urine copper excretion remained essentially unchanged. Intravenous copper infusion resulted in a rise of serum ceruloplasmin. During administration of a test meal of 64Cu(NO3)2, the patient was given alternately a volume of 0.9% NaCl or an equal volume of plasma intravenously. Radioactivity found in the blood was unchanged after each infusion period. During 427 days of subcutaneous copper, plasma p-phenylenediamine oxidase activity and plasma copper rose toward normal. Subsequent balance studies showed that the patient was in negative copper balance because of large losses in feces. Scanning electron microscopy demonstrated persistent pili torti.

The effect of Escherichia coli endotoxin on plasma histaminase activity in the domestic fowl and the involvement of caeruloplasmin

The injection of four-to nine-week-old fowls with Escherichia coli O111:B4 endotoxin (0-1 and 1-0 mg/kg) produced a two to eight fold rise in the histaminase activity of the plasma 24 h afterwards. In some cases this increase was still detectable after 48 h. This activity was strongly correlated with the p-phenylenediamine oxidase activity of caeruloplasmin. Electrophoretic studies with 7-5 per cent polyacrylamide gels indicated that fowl caeruloplasmin also was histaminase and putrescinase activity and that the release of this protein from the liver by endotoxin is largely responsible for the increase in the activity of the plasma. In untreated fowls this activity was lower than published values for several mammals and does not explain the relative resistance of the fowl to the acute effects of endotoxins and large doses of histamine.

Development of Ceruloplasmin in Pigs during the Neonatal Period

Colostrum-free newborn pigs were raised artificially to investigate the appearance of ceruloplasmin in the serum of piglets. The sensitivity of the p-phenylenediamine oxidase activity assay, commonly used as a measure of ceruloplasmin, has been increased 10-fold. Based on this activity, no evidence was found that this copper protein was present in the serum of baby pigs at birth, although some evidence was obtained that the protein moiety (apoceruloplasmin) was present. Holoceruloplasmin usually became detectable in the serum 10-15 hours after birth. Its concentration increased slowly at first and then at an accelerated rate. The ceruloplasmin in the sera of 2-3-day-old piglets was isolated and compared with that isolated from the serum of adults. Contrary to a previous report, the physical and chemical properties of ceruloplasmin synthesized during the first 3 days of life of the piglet showed no significant difference from that synthesized by the adult.