Interaction of p-phenylenediamine with the iron-bleomycin complex.

The iron-bleomycin complex has been shown to catalyze the oxidation of p-phenylenediamine to a stable, purple coloured oxidation product, characterized by an absorption maximum around 520 nm. Molecular oxygen is used for reoxidizing Fe(II)-bleomycin after reduction by p-phenylenediamine. An apparent Michaelis constant of 5.2 mM and a catalytic constant of 17.2 min-1 were obtained from kinetic studies. ATP, ADP and orthophosphate inhibited the catalytic oxidation of p-phenylenediamine, while AMP was without effect. It is proposed that p-phenylenediamine may be used as 'substrate' in kinetic studies involving the 'oxidase' activity of iron-bleomycin.

Stimulatory effect of ascorbate on iron transfer from bleomycin to apotransferrin.

The chemotherapeutic agent, bleomycin, forms a 1:1 complex with both Fe(III) and Fe(II). The rate of ferric ion transfer from bleomycin to apotransferrin is rather slow. However, when ascorbate was added to Fe(III)-bleomycin prior to exposure to apotransferrin, the transfer rate was markedly increased. Ascorbate readily reduces Fe(III)-bleomycin to Fe(II)-bleomycin. A second order rate constant of 2.4 mM-1 min-1 was estimated for this reaction. Fe(II)-bleomycin immediately combines with O2, generating the so-called 'activated bleomycin' complex. The data suggest that a reduced form of iron-bleomycin more readily donates its iron ion to apotransferrin. Reoxidation of ferrous ions, and Fe(III)-transferrin formation occur rapidly.

A study on ascorbate inhibition of ceruloplasmin ferroxidase activity.

The ferroxidase activity of ceruloplasmin is often determined according to the method of Johnson et al. (1967), using apotransferrin for trapping ferric ions generated by the enzyme; spectrophotometrically monitoring the Fe(3+)-transferrin formation at pH 6.0. Reports have shown that ascorbate inhibits this reaction, and it is hypothesized that the effect could be of physiological significance in individuals with a high ascorbate to ceruloplasmin ratio in plasma (e.g. premature babies). The present study shows that the inhibitory effect of ascorbate rapidly decreases with increasing pH. At pH 7.4 no significant effect was observed, the result suggesting that ascorbate is not a physiological inhibitor of ceruloplasmin. Furthermore, experiments demonstrate that at acidic pH the inhibitory effect of ascorbate on the rate of Fe(3+)-transferrin formation is not primarily due to an interaction with ceruloplasmin, but to a reduction of enzymically generated ferric ions before they are bound to apotransferrin.

On the mechanism of citrate inhibition of ceruloplasmin ferroxidase activity.

Ceruloplasmin is a plasma protein, which oxidizes ferrous ions in a catalytic manner. It is considered to function as a ferroxidase in vivo. Citrate was found to inhibit the reaction. The ceruloplasmin catalyzed oxidation of p-phenylenediamines, however, was not affected by citrate. The inhibitory effect is proposed to be due to formation of Fe(2+)-citrate, which does not react with ceruloplasmin. The stability constant for the Fe(2+)-citrate complex estimated from the present inhibition study is in good agreement with previously published data.

A kinetic study of the coupled iron-ceruloplasmin catalyzed oxidation of ascorbate in the presence of albumin.

Ascorbate is catalytically oxidized by a couple iron-ceruloplasmin system, the iron ions functioning as a red/ox cycling intermediate between ceruloplasmin and ascorbate. Serum albumin, an iron binding compound, was found to stimulate the ascorbate oxidation rate. It is proposed that ferrous ions react more rapidly with ceruloplasmin when they are bound to albumin. A Km value of 39 microM was estimated for Fe(2+)-albumin. Citrate and urate inhibit the iron-ceruloplasmin-dependent ascorbate oxidation by chelating ferric ions. In the presence of albumin only citrate reduced the oxidation rate, the observation suggesting the following order of iron binding ability: citrate > albumin > urate. Physiological aspects of the results have been discussed.

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.

Iron transfer from urate-Fe(III) to citrate and ATP.

1. Urate, citrate and ATP, which form stable complexes with ferric ions, are proposed to function as low mol. wt iron binding agents in humans. 2. Citrate and ATP were found to readily take up iron from the urate-Fe(III) complex; the study suggests that citrate and ATP may be physiologically more important iron binding agents than urate.

Exchange of copper between serum albumin and bleomycin.

1. The present in vitro study shows that bleomycin is able to take up copper from albumin, which functions as a copper transport protein in plasma. 2. Calculations, based on plasma concentrations of Cu(II)-albumin and bleomycin, suggest that only a small fraction of bleomycin (less than 3%) may directly accept copper from Cu(II)-albumin.

The reaction of ferric- and ferrous salts with bleomycin.

1. A comparative study shows that ferrous ions give a much better yield of Fe(III)-bleomycin than ferric ions, when iron salt is added to bleomycin in a buffer solution (pH 7.2). 2. The amount of Fe(III)-bleomycin formed after addition of ferric ions was markedly increased in the presence of ferric ion binding compounds (BSA, citrate) or reducing agents (ascorbate, cysteine).

Interaction of iron-bleomycin with catecholamines.

1. Catecholamines were found to reduce Fe(III)-bleomycin to the ferrous state. Aminochrome, an oxidation product of catecholamine, rapidly appears in the reaction solution. 2. The purple colour of Fe(III)-catecholamine is also detected in the reaction solution, suggesting that iron is transferred from bleomycin to catecholamine. 3. Gel filtration studies confirm that catecholamines are able to take up iron from the iron-drug complex.

Interaction of citrate with Fe(III)-bleomycin.

1. Citrate binds to Fe(III)-bleomycin, removing the ferric ion from the iron-drug complex; a reaction that may be of physiological significance. 2. Low concentrations of citrate markedly enhance the rate of iron transfer from Fe(III)-bleomycin to apotransferrin; an iron binding plasma protein.

Stimulatory effect of phosphate, ATP and ADP on iron transfer from Fe(III)-bleomycin to apotransferrin.

1. The rate of ferric ion transfer from Fe(III)-bleomycin to apotransferrin was increased in the presence of orthophosphate, ATP and ADP, while AMP was without effect. 2. Ortho phosphate activation probably involves formation of a Fe(III)-bleomycin-phosphate complex. The optical absorption of Fe(III)-bleomycin at 450 nm is enhanced in the presence of phosphate. 3. ATP and ADP remove the ferric ion from the iron-drug complex; thus making the ferric ion readily available for uptake by apotransferrin. 4. Low concentrations of ATP, ADP and AMP, also enhance the 450 nm absorption of the iron-drug complex. Higher ATP and ADP concentrations reduce both the 450 and 384 nm absorption of Fe(III)-bleomycin.

Interaction of iron-adriamycin complexes with ceruloplasmin and apotransferrin.

1. The present kinetic study suggests that the Fe(II)-adriamycin complex acts as substrate for ceruloplasmin, which oxidizes the complex to the ferric form (Km = 21.7 microM). 2. Apotransferrin readily removes iron from Fe(III)-adriamycin. 3. However, adriamycin, at low concentration, is able to take up some iron from a 20% iron-saturated transferrin solution; a reaction which may take place in vivo.

Interaction of neocuproine, 1,10-phenanthroline and 2,2'-dipyridyl with human ceruloplasmin.

1. Neocuproine binding to ceruloplasmin markedly increases the chlorpromazine-ceruloplasmin-catalyzed oxidation of NADH. 2. 1,10-Phenanthroline and 2,2'-dipyridyl inhibit neocuproine activation in a competitive manner. 3. The order of enzyme chelator complex stability was: phenanthroline greater than dipyridyl greater than neocuproine.

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 promazine with human ceruloplasmin.

Promazine is enzymically oxidized by ceruloplasmin without reduction of the 610 nm absorption band of the enzyme. Fluoride inhibited the reaction in a non-competitive manner. The ceruloplasmin oxidase activity is markedly enhanced when promazine is added in the presence of NADH; possibly through a change in enzyme conformation.

Hemin-induced lysis of rat erythrocytes. Protective action of ceruloplasmin and different serum albumins.

Hemin-induced lysis of rat erythrocytes is markedly reduced by ceruloplasmin (human) and serum albumins from different species, the order of effectiveness beings: bovine albumin approximately equal to ceruloplasmin greater than human albumin approximately equal to dog albumin greater than apotransferrin (human). Although the proteins studied had hemin binding capacity, the best protective agents, ceruloplasmin and bovine albumin, did bind hemin less strongly than human and dog albumin. The results suggest the existence of another protective mechanism, possibly involving an interaction between erythrocyte membranes and serum proteins.

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.

Catecholamine stimulation of copper dependent haemolysis: protective action of superoxide dismutase, catalase, hydroxyl radical scavengers and serum proteins (ceruloplasmin, albumin and apotransferrin).

Copper induced lysis of washed rat erythrocytes was stimulated by catecholamines, the order of effectiveness being: adrenaline greater than noradrenaline approximately equal to dopamine greater than L-DOPA. The degree of effectiveness is related to the rate of copper ion dependent oxidation of catecholamine, adrenaline being more rapidly oxidized than the other catecholamines investigated. Superoxide dismutase, catalase and different hydroxyl radical scavengers (mannitol, tris, formate and ethanol) markedly reduced the haemolytic effect of copper and catecholamine, suggesting the possibility that superoxide radicals and hydrogen peroxide, formed in the reaction system, cooperate in producing hydroxyl radicals, which are directly involved in the haemolytic action. The plasma proteins, ceruloplasmin, albumin and apotransferrin, also reduced the copper-catecholamine induced haemolysis. The protective action is probably not related to the copper binding ability of these proteins.

Studies on cysteine-induced hemolysis.

Two mechanisms of cysteine-induced lysis of washed rat erythrocytes appear to exist; one operating at low, the other at somewhat higher cysteine concentration. DETAPAC (20 microM) and EDTA (0.1 mM) markedly reduced the effect of the latter mechanism. Copper and iron ions, ascorbate, dehydroascorbate and glucose protected the erythrocytes against cysteine-induced hemolysis, while superoxide dismutase, catalase, hydroxyl radical scavengers and antioxidants had no effect. The hemolytic action of cysteine seems to be associated with the stability of the cysteine sulfhydryl group.