P-phenylenediamine Oxidase Activity Research Articles

Identification and Purification of a Non-Ceruloplasmin Ferroxidase of Human Serum

A non-ceruloplasmin ferroxidase (ferroxidase-II) protein was isolated from human serum and completely resolved from ceruloplasmin by DEAE-Sephadex A-50 chromatography. This protein was isolated and purified approximately 500-fold by utilization of the Cohn IV-1 fraction, DEAE-Sephadex A-50 chromatography, and gel filtration on Agarose A-15m, followed by gel filtration on Agarose A-50m. Ferroxidase-II differed from ceruloplasmin in many respects. Three of the most important differences were that ferroxidase-II (a) was yellow rather than blue as ceruloplasmin, (b) it was not inhibited by azide, and (c) it exhibited no p-phenylenediamine oxidase activity. Ferroxidase-II also differed significantly from the known plasma amine oxidases. The ferroxidase activity of ferroxidase-II was not lost by dialysis or ultrafiltration, but was inactivated by heat treatment and was proportional to the protein concentration at all stages of purification. Oxygen consumption with ferroxidase-II was observed simultaneously with iron oxidation. The ferroxidase-II activity in Wilson's disease serum represented a much larger percentage of the total ferroxidase activity than in normal serum. Although ferroxidase-II was decreased in Wilson's disease serum, it was reduced to a lesser extent than the ceruloplasmin ferroxidase activity. Thus ferroxidase-II may account for the lack of correlation of ferroxidase activity with p-phenylenediamine oxidase activity of Wilson's disease serum and may be responsible for the maintenance of near normal iron metabolism despite the low levels of ceruloplasmin.

Measurement of Human Serum Ceruloplasmin by Its p-Phenylenediamine Oxidase Activity

Abstract Optimum reaction conditions were evaluated for assay of serum ceruloplasmin by measurement of its p-phenylenediamine oxidase activity. The pH optima of p-phenylenediamine oxidase activities of human and rat ceruloplasmins were 5.45 and 5.2, respectively. The p-phenylenediamine oxidase activity of rat ceruloplasmin was markedly inhibited by phosphate (0.1 mol/liter), that of human ceruloplasmin only slightly. These reaction conditions were judged to be optimum for the ceruloplasmin assay: (a) substrate: p-phenylenediamine dihydrochloride, 8.9 mmol/liter; (b) buffer: acetate, 0.1 mol/liter; (c) chloride concentration: 21 mmol/liter; (d) serum dilution: 31-fold; (e) incubation: 37°C for 30 min; (f) spectrophotometry: 530 nm (vs. a "serum blank" containing NaN3). Using these reaction conditions, we devised an improved technique for measuring serum ceruloplasmin. The coefficients of variation of replicate analyses by this technique were 1.25% (for "within-run" precision) and 2.8% (for "day-to-day" precision). The mean concentration of ceruloplasmin in sera from 29 healthy men was 0.315 g/liter (SD = ± 0.049, range = 0.233 to 0.402).

The oxidase systems of Ascaris-muscle mitochondria

1. The oxidase systems of Ascaris-muscle mitochondria were investigated by polarographic and spectroscopic techniques. 2. Spectrophotometric studies at −196° revealed that Ascaris-muscle mitochondria contained low levels of substrate-reducible a-, b- and c-type cytochromes. Cytochrome a 3, present in very low concentrations, was shown to be functional by photochemical action spectrum. 3. Ascaris-muscle mitochondria oxidized (in decreasing order of respiratory activity) succinate, malate, NADH and α-glycerophosphate with H 2O 2 as end-product. 4. The antimycin A-insensitive ascorbate-tetramethyl- p-phenylenediamine (TMPD) oxidase activity was inhibited 33% by 1.0 mM CN −, 58% by CO and 100% by CO plus 1.0 mM CN −. CO inhibited 50% of succinate oxidation. 5. Fluorescence spectrum confirms and further identifies the two flavin components involved with malate oxidation in Ascaris-muscle mitochondria.

The Possible Significance of the Ferrous Oxidase Activity of Ceruloplasmin in Normal Human Serum

The oxidation of Fe(II) by serum was studied at pH 7.35 and at various oxygen concentrations which approach the physiological conditions of human serum. The nonenzymic oxidation of Fe(II) was estimated to be insufficient to account for a rate of Fe(III)-transferrin formation necessary to provide an adequate iron supply for hemoglobin and other biosyntheses if Fe(II) is a relevant source of serum iron. The results suggested that an appreciable catalytic activity was involved in Fe(II) oxidation in serum. The ferroxidase activity of various normal human sera correlates precisely with the p-phenylenediamine oxidase activity of these sera. This catalytic activity is inhibited by azide and cyanide, and is low in sera with reduced ceruloplasmin levels. Fe(II) oxidation is also associated with the ceruloplasmin fraction on diethylaminoethyl cellulose chromatography of serum. The oxidation of Fe(II) by ceruloplasmin is zero order with respect to oxygen (10 to 200 µm), whereas the nonenzymic oxidation is first order. A biological role for the ceruloplasmin of serum in promoting the rate of iron saturation of transferrin and in stimulating iron utilization and the designation of the enzyme as serum ferroxidase is proposed.

Uptake and biliary excretion of Cu64 in rabbits in relation to blood ceruloplasmin.

It has been postulated that one of the important biological functions of the alpha-2 globulin, ceruloplasmin, is the regulation of the biliary excretion of inorganic copper(1). However, there is little experimental evidence to support this theory(2,3). With the availability of an experimental technique for modifying the blood ceruloplasmin levels(4), it became feasible to reexamine the problem as to possible correlations between serum ceruloplasmin, liver uptake, and biliary excretion of Cu64 in the rabbit. Three groups containing 9 to 12 male albino rabbits (12-13 weeks old) were employed. One group was maintained for 10–15 days on a commercial dry ration containing molybdate-sulfate, a diet known to reduce ceruloplasmin levels in the rabbit and greatly elevate total blood copper(4,5). A second group, which served as controls, was maintained on the commercial ration without added salts. A third group was maintained on a ration mixed with inorganic copper, 0.005% copper as Cu2SO4 for 4 weeks in order to elevate serum ceruloplasmin levels (5,6). Ceruloplasmin was measured as p-phenylenediamine oxidase activity in each rabbit serum 24 hours before administration of the isotope(7). Animals were given an intravenous injection of 75 microcuries of high activity Cu64 acetate (6 μg copper) via the marginal ear vein and sacrificed 2, 25, and 50 hours later. Levels of Cu64 activity were measured in 0.1–0.5 ml of bile immediately withdrawn from the gall bladder. Suitable samples of liver and cross sections of cortex and medulla of the kidney were hydrolyzed with sulfuric, perchloric and nitric acids(8). Each sample was returned to a standard volume and Cu64 activity determined. The values obtained were corrected for radioactive decay and expressed as percent of the dose administered (Table I). Radioactive copper was determined in a well-type, thallium-activated sodium iodide crystal scintillation counter. In those rabbits maintained on a high molybdate-sulfate diet there resulted a marked diminution of serum ceruloplasmin levels, uptake of Cu64 by liver, and excretion of copper at 2, 25, and 50 hours (Table I). Cu64 uptake by liver in this group was reduced to 35.7% of the control level 2 hours after injection. Along with a 20% reduction in serum ceruloplasmin, the biliary excretion of Cu64 was virtually zero. This sharp suppression in liver uptake and biliary copper excretion in 2, 25, and 50 hours occurred despite an elevation in the non-ceruloplasmin copper pool(4,5). On the other hand, a significant increase in the concentration of Cu64 in the kidney was noted in this group as compared to the other 2 groups.

Vertebrate distribution of serum ceruloplasmin and sialic acid and the effects of pregnancy

1. 1. Serum ceruloplasmin, as p-phenylenediamine oxidase activity, was assayed in 115 species representing thirty-eight orders from seven vertebrate classes. There was no demonstrable activity in sera from sixteen species representing ten orders from six classes. 2. 2. Five species—the cyclostomes and ranid frogs—were anomalous in that the oxidase activity was stimulated by azide. 3. 3. Comparisons of gravid and non-gravid females demonstrated an increased concentration of ceruloplasmin activity only in certain of the mammalian species. 4. 4. Parallel measurements of total serum proteins and sialic acid provided evidence for striking species differences in the concentration and distribution of the glycoproteins, normally and in the gravid state.

Correlation between serum copper, ceruloplasmin activity and C-reactive protein

The correlation between serum copper, ceruloplasmin activity, and C-reactive protein (CRP) in adults was investigated, with the following results: (1) There was a very strong positive linear correlation between total copper content of serum and p-phenylenediamine (PPD) oxidase (ceruloplasmin) activity when measured by the techniques described herein. (2) Although hypercupremia and CRP generally occurred together, there were sufficient exceptions to indicate that these two serum components are not necessarily interdependent. (3) Further work is needed to establish what other factors are involved with the regulation of serum copper levels.