Copper Erythrocuprein

Erythrocuprein, which contains about 60 wt % of the erythrocyte copper, hepatocuprein, and cerebrocuprein act as superoxide dismutases. Each contains two atoms of copper per molecule, having mol wt ca 34,000. The superoxide ion, O", and peroxide, two main toxic by-products of... 

H. Markowitz, G.E. Cartwright, and M.M. Wintrobe, Copper metabolism. XXVII. Isolation and properties of an erythrocyte cuproprotein (erythrocuprein). J. Biol. Chem. 234, 40-45 (1959). 

Superoxide dismutases have been isolated from a wide variety of eukaryotes including yeast, wheat germ, garden peas, chicken liver and erythrocytes. These enzymes contain copper and zinc. Copper containing proteins which also display this catalytic activity had been isolated from blood, brain and liver tissues many years previously and were known as erythrocuprein, cerebrocuprein and hepatocuprein. 

In 1969, McCord and Fridovich discovered that a copper-containing protein, erythrocuprein, isolated from red blood cells, catalyzed the dismutation of superoxide O - [17]. This enzyme was renamed superoxide dismutase (SOD). A striking positive correlation is now known to exist between the life-span potential of mammal species and the ratio of SOD activity and specific metabolic rate of their tissues [18]. The ubiquitous vitaminE, glutathione peroxidases and superoxide dismutases provide a primary protective barrier against the toxicity of free radicals and peroxides in mammalian cells. 

It was not until thirty years after the copper-containing protein erythrocuprein was discovered (T. Mann, D. Kei-LiNG, 1939) that its enzymatic nature was proved. At that time, this protein was shown to convert superoxide radicals into molecular oxygen and H2O2 (J.M. McCord, 1969 I. Fridovich, 1969) it was Eridovich who coined the term superoxide dismutase (SOD). This presumably most important of antioxidants accelerates the conversion of superoxide radicals 10,000 times more effectively than spontaneous dismutation. 

Several forms of copper proteins have been reported to exist, the most important of which is ceruloplasmin. In addition, there are known to be specific copper-binding proteins such as erythrocuprein (in red blood cells), cerebrocuprein (in brain cells), and hepatocuprein (in liver cells (42,43,44),... 

It may be questioned whether all these proteins are separate entities. There are many similarities between hemocuprein and erythrocupiein, and also between hepatocuprein, horse liver copper protein, and human hepato-cuprein. Porter suggested that the erythrocuprein of Markowitz et al. (P14), cerebrocuprein I, and human hepatocuprein may be identical proteins, although he recognizes some differences between them. 

A combination of spectroscopic methods (EPR, light absorption and CD) was used in order to obtain information on the copper binding site of bovine hemocuprein (erythrocuprein), which has been considered to be intermediate in its spectroscopic properties between the blue copper proteins and complexes of copper with model peptides (94). 

Our knowledge of the structural properties and enzymic function of a large number of copper proteins has accumulated during the last few decades. The main results have been comprehensively reviewed 1—4) or presented at symposia (5—54). This survey is devoted to erythro-cuprein, one of the most actively studied copper proteins. Erythrocu-prein is sometimes called haemocuprein, hepatocuprein, cerebrocuprein, cytocuprein, or erythro-cupro-zinc protein. Alternatively, the name superoxide dismutase has been suggested as descriptive of its activity the enzyme-catalyzed disproportionation of anionic monovalent superoxide radicals. However, whether or not the enzymic reaction is specific for Oi- still needs to be investigated1). Thus, the name erythrocuprein is used throughout this review. 

During the last seven years intensive studies of both human and bovine erythrocuprein have been performed (65—83). Carrico and Deutsch were able to demonstrate the identity of erythro-, cerebro-, and hepatocuprein using copper proteins from human tissues the amino-acid analysis, immunochemical behaviour, and physicochemical properties were identical in all three. In 1970 they found, in addition to the 2 copper atoms, a second metallic component, namely 2 atoms of zinc (69). 

Usually the apoprotein was prepared by employing excessive dialysis against cyanide, EDTA or 1,10-phenanthroline (69, 70, 71, 72). However, these apoproteins still contained considerable amounts of copper and zinc (5—20% of the original content). We have devised a new method (76, 78) using chelator-equilibrated gel columns. EDTA proved most convenient, as already observed (69, 72). Concentrated erythrocuprein samples were layered on top of a Sephadex-G.-25 column which was previously equilibrated with 10 mM EDTA, pH 3.8. The migration rate of the protein was adjusted in a way such that 8—10 hours elapsed before... 

The presence of copper in erythrocuprein was first demonstrated by Mann and Keilin (55). In 1970 zinc was found in human erythrocuprein (69). The metal ions were measured by different spectrophotometric assay procedures using 2,2-biquinoline, bis cyclohexanone oxalyldihy-drazone, and dithizone as chelating ligands (85, 97—100). Alternatively, atomic absorption spectroscopy (86), neutron activation analyses, and emission spectroscopy (<5< ) were successfully employed. From the neutron activation analyses it became apparent that metals other than zinc and copper were present in amounts less than 0.1 g-atom per 33,000 g of protein. From the different analyses (Table 3) it can be concluded that erythrocuprein contains 2 g-atoms of each of copper and zinc. 

Table 3. Copper and zinc content of erythrocuprein isolated from human and bovine tissues. Data taken from a (64), b (69), c(72), d (74), e (82), f (55), and converted values using a molecular weight of 34,000 from g (66), h (58), i (87)...

Carrico and Deutsch (69) reported a very poor exchange of the metals when using 64Cu or 65Zn. However, a slow transfer of radioactive copper into erythrocuprein can be observed if 64Cu is added to whole blood. Approximately 25% of the radioactivity added was present in erythrocuprein after 12 hours (60). 

It is interesting to note the unusual diminished absorption in the 280 nm region ( 265 = 17,000). This was attributed at the low content of aromatic amino-acid residues, especially typosine and tryptophan. The absorption in the visible region was extremely low. Apart from the broad absorption band at 675 20 nm ( 675=275), a shoulder can be observed at 440 nm (Fig. 7). According to the definition given by Malkin and Malmstrom (3), erythrocuprein is classified as a non-blue copper protein. 

In contrast to a number of other Cu enzymes, the erythrocuprein copper was fully detectable by EPR (3,81, 114). Exposure of the protein to 6 M guanidine hydrochloride caused no measurable changes in the... 

For a long time erythrocuprein was thought to act exclusively as a copper-transporting protein. This was a very attractive conclusion since over 50% of the erythrocyte copper content is present in erythrocuprein (60). However, in the absence of any known function of a metalloprotein, it is always tempting to assign to it the role of storage or transport of the respective metal ions. For example, caeruloplasmin was considered to be the main copper-transporting protein in blood plasma. It subsequently turned out that this copper protein is a key enzyme in iron metabolism, responsible for the oxidation of Fe2+ to the Fe3+ bound in transferrin (130—132). 

Unfortunately, our knowledge of the structure of nearly all copper proteins including erythrocuprein is rather limited. Thus for the time being great caution is necessary in the interpretation of physical and chemical properties. Extensive studies using Cu model complexes of low molecular weight were carried out as an approach to understanding these structural and functional properties (4, 8, 53, 187, 188). 

The reactivity of native erythrocuprein was higher compared to the superoxide dismutase activity shown in Table 12. Of utmost importance was the observation that the model chelates and CUSO4 were virtually inactive compared to the native enzyme. The difference was 4 orders of magnitude which implies a much higher specificity for this enzymic reaction of the cupreins. The powerful reactivity of erythrocuprein is further demonstrated by the fact that the apoprotein displayed a detectable enzymic activity due to traces of copper which were undetectable by atomic absorption measurements or EPR spectroscopy. No such difference between apoprotein and the boiled native enzyme was observed using the superoxide dismutase assay. 

In blood, copper is found in both red cells and plasma. Copper of the red cells is tightly bound to a colorless protein (mol wt approximately 33,000) containing 2 atoms of copper per molecule (erythrocu-prein). Erythrocuprein has an isoelectric point of 5.3 and presents no oxidative properties. Its function in the physiology of the cell is not clear, although there is a rapid exchange between the red cell copper and the plasma copper [47]. 

The average concentration of copper in blood is 1.10 mg/1 in men and 1.23 mg/I in women. More than 90% of copper in the body is found in the blood plasma. The major copper-binding substance in blood plasma is monomeric glycometaUoprotein ceruloplasmin from a group of a2-globulins (132 kDa), which is composed of 1046 amino acids and contains about 7-8% of sugar components. Ceruloplasmin has a blue colour and in the normal state one molecule of ceruloplasmin contains six atoms of copper (at full saturation additional binding sites may be occupied by an additional two copper atoms). Blood plasma contains about 300 mg/1 of ceruloplasmin. In erythrocytes, copper occurs in another protein called erythrocuprein (31 kDa) and in the enzyme superoxide dismutase. 

An essential trace element for which there is a daily requirement of 2.5 mg. A normal adult contains about 100 mg. In plasma, most copper is carried by a specific copper transport protein, caeruloplasmin. Copper is also found in certain copper storage proteins erythrocuprein (in erythrocytes), cerebrocuprein (in the brain) and hepatocuprein (in the liver). 

Copper is present in a number of mammalian proteins and the characteristics of these have been summarized by Scheinberg and Sternlieb [3]. Amongst the copper proteins identified in man, but of unknown function, are cerebro-cuprein I, a protein extracted from normal human brain by Porter and Ainsworth [4], liver copper-protein [5], and erythrocuprein [6]. Tyrosinase, a protein of 0.25% copper content is to be found wherever melanin is present in the body, it catalyses the oxidation of tyrosine to dopa and accelerates the conversion of dopa to dopa quinone, the initicJ stages in the conversion of tyrosine to melanin. Lack of this enzyme is not, however, associated with deficient production of pressor amines, another pathway being present in the adreneil gletnd for the hydroxylation of tyrosine [7]. 

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