Rolf A. Løvstad

Copper catalyzed oxidation of ascorbate (vitamin C). Inhibitory effect of catalase, superoxide dismutase, serum proteins (ceruloplasmin, albumin, apotransferrin) and amino acids

The inhibitory effect of catalase and superoxide dismutase on copper catalyzed oxidation of ascorbate is probably due to a binding of copper ions. Scavengers of hydroxyl ions and singlet oxygen had no effect on the ascorbate oxidation rate. Copper binding serum proteins reduced the oxidation rate; the order of effectiveness being: Ceruloplasmin greater than human albumin = bovine albumin greater than apotransferrin. The excellent protection obtained with catalase and ceruloplasmin is possibly due to a strong affinity for cuprous ions generated during the reaction. Cupric ion binding amino acids (His, Thr, Glu, Gln, Tyr) had considerably weaker protective effect than the proteins studied. Apparently they do not compete favorably with ascorbate for cupric ions.

Interaction of phenothiazine derivatives with human ceruloplasmin

Abstract Tranquillizing drugs of the phenothiazine class (promazine, chlorpromazine, triflupromazine and levomepromazine) were oxidized to free radicals by human ceruloplasmin. The blue colour of the enzyme, due to protein-bound cupric atoms, was reduced by addition of phenothiazine derivatives. In the presence of reducing agents (NADH, NADPH, reduced glutathione and ascorbate) the rate of the ceruloplasmincatalyzed oxidation of phenothiazine derivatives was markedly increased. NADH was oxidized during the process, suggesting that the activating effect is due to a reduction of phenothiazine derivative radicals, which rapidly react with several reducing agents. Straight lines were obtained when the reciprocal rate of NADH oxidation was plotted against the reciprocal phenothiazine derivative concentration (1/ V vs 1/[ S ]). The V max -values for the four substrates investigated do not vary significantly. At lower substrate concentrations, however, the rate of triflupromazine oxidation was considerably slower than the rates obtained with the other substrates. Phenothiazine derivatives activate the enzymic oxidation of dopamine and dopa, probably by acting as a cycling intermediate between ceruloplasmin and catecholamine.

The protective action of ceruloplasmin on copper ion stimulated lysis of rat erythrocytes

1. Human ceruloplasmin effectively protects washed rat erythrocytes against copper ion stimulated lysis. 2. Experiments suggest that the protective action is not associated with (a) the oxidase activity of ceruloplasmin, (b) the proteins superoxide radical scavenging ability, (c) its ability to weakly bind cupric ions (nonspecific binding) or (d) ceruloplasmin bound sialic acid.

The protective action of ceruloplasmin on Fe2+ stimulated lysis of rat erythrocytes

Abstract 1. 1. Ceruloplasmin, a copper containing serum protein, was found to effectively protect washed rat erythrocytes against Fe 2+ stimulated lysis. 2. 2. It is proposed that Ceruloplasmin, through its Fe 2+ oxidase activity, prevents the formation of superoxide radicals, known to cause hemolysis.

Interaction of dimethyl-p-phenylenediamine with ceruloplasmin

The stoichiometric interaction of dimethyl-p-phenylenediamine (DPD) with ceruloplasmin-Cu2+ has been investigated by spectrophotometric and electron paramagnetic resonance technique. Intermediates in the reaction sequence, DPD → (DPD)+ → (DPD)2+ → further oxidation products, were characterized. Cerulo-plasmin-Cu2+ was rapidly reduced by DPD as well as by the monoradical ion (DPD)+ but not by (DPD)2+. The two oxidation products (DPD)+ and (DPD)2+ were reversibly reduced by NADH2. The stoichiometry of the reactions confirmed that DPD is oxidized by one electron transfer to ceruloplasmin-Cu2+ in two subsequent steps. Calculations of some of the rate constants were done. The “association constant” k1 for DPD with ceruloplasmin was 750m−1 sec−1, and k′1 for (DPD)+ 800m−1 sec−1 at 10 °. The rate constant for the reoxidation of ceruloplasmin-Cu+ was 0.037 sec−1. When DPD was oxidized by ceruloplasmin, the formation of a relatively stable protein-bound radical was indicated. Kinetic studies of the oxidation of DPD by ceruloplasmin in catalytical amounts were done. The results suggested two different types of active sites or oxidizing units in ceruloplasmin.

Kinetic studies on the ceruloplasmin-catalyzed oxidation of phenothiazine derivatives

Abstract The ceruloplasmin-catalyzed oxidation of several phenothiazine derivatives has been investigated. The results suggest that the activity decreases with increasing Hammett sigma value (para) for the substituent in the 2-position of the substrates, which have three carbon atoms between the nitrogen atom of the ring and the nitrogen atom in the side chain in the 10-position. The V max -values for substrates with identical substituents in the 2-position and a three-carbon side chain in the 10-position do not vary much. However, at lower substrate concentrations compounds with a piperazinyl-propyl side chain are more rapidly oxidized than those with an aliphatic side chain, suggesting that the piperazinyl-propyl side chain enhances the enzyme affinity for the substrate molecule. Compounds with only two carbon atoms in the side chain in the 10-position (promethazine and diethazine) are more slowly oxidized by ceruloplasmin than analogs with a three-carbon side chain (promazine and alimemazine). Phenothiazine derivatives activate the ceruloplasmin-catalyzed oxidation ofcatecholamines. The substrates which are most rapidly oxidized by the enzyme also activate the oxidation of dopamine most effectively.

Interaction of serum albumin with the Fe(III)-citrate complex

1. Fe(III)-citrate forms a red coloured complex with bovine serum albumin (lambda max = 500 nm). 2. Fe(III)-albumin complexes also appear, when Fe(III)-citrate is mixed with albumin, suggesting that albumin competes with citrate for ferric ions.

Fatty acid induced hemolysis. protective action of ceruloplasmin, albumins, thiols and vitamin C

The hemolytic effect of saturated fatty acids increased rapidly, when the number of carbon atoms in the chain exceeded 12. At low fatty acid concentrations (less than 60 microM) the hemolytic effect decreased with increasing number of double bonds in the carbon chain (cis-form fatty acids). A more complex pattern was observed at higher fatty acid concentrations. Trans-unsaturated fatty acids were more hemolytic than cis-analogs. Ceruloplasmin, a serum protein with no fatty acid binding capacity, reduced the hemolytic effect of fatty acids; possibly by interacting with the cell membrane. Reducing compounds (thiols, vitamin C) also protected against fatty acid induced hemolysis.

Activating effect of chlorpromazine on the peroxidase-catalysed oxidation of catecholamines

Abstract 1. Chlorpromazine was found to have an activating effect on the horseradish peroxidase-catalysed oxidation of catecholamines (noradrenaline, dopamine and dopa) to aminochrome, an intermediate in the melanin synthesis. 2. Chlorpromazine, a good electron donor, is enzymatically oxidized by peroxidase to a radical, the rate of chlorpromazine oxidation being much higher than the rate of catecholamine oxidation. 3. The present study suggests that chlorpromazine enhances the catecholamine oxidation by functioning as a cycling (redox) intermediate between peroxidase and catecholamine molecule.

Effects of Oxytetracycline on Solubility and Synthesis of Collagen in Young Rats

In a recent study we demonstrated that the antibiotic oxytetracycline reduces both growth and mechanical strength of bone and skin in young rats. The present study deals with the effects of 14 days of oxytetracycline medication on salt solubility of collagen (cross-linking) and conversion of 14C-proline to 14C-hydroxyproline in collagen (synthesis). At the end of the medication period, significantly higher solubility of collagen was found in the femurs and skin of rats receiving oxytetracycline than in controls. No effect of the antibiotic on the rate of collagen synthesis could be demonstrated. These findings may indicate that oxytetracycline in young rats causes reduced mechanical strength of bone and skin by interfering with the cross-linking of collagen.