CERULOPLASMIN, HUMAN

Definition

A copper-containing alpha globulin in blood plasma that contains type 1, type 2, and type 3 copper cen- ters in which the type 2 and type 3 centers are near each other, constituting a trinuclear copper cluster. It may play a part in red blood cell production and oxygen reduction.

Pharmaceutical Applications

Typically, 50% of the daily copper intake is absorbed in theGI tract and transported to the liver from where it is transported to the peripheral tissue bound to ceruloplasmin, acopper-binding glycoprotein. A smaller amount of copper is also bound to albumin. Excess copper is mainly excreted in bile into the gut and then the faeces.
Ceruloplasmin (ferroxidase I) and ferroxidase II, two copper-based enzymes, can oxidise ferrous iron to ferric iron. Ferric iron can then be transported with the help of transferrin,for example, to form red blood cells. Furthermore, a variety of copper-dependent enzymes, such as cytochrome c and superoxide dismutase, work as antioxidants and are involved in the reduction of reactive oxygen species (ROS).
Cancer progression has been linked to increased ceruloplasmin and copper levels in a variety of tissues.Copper deficiency has been considered as an anticancer strategy, but several clinical studies have not been encouraging.

Biochem/physiol Actions

Ceruloplasmin (Cp), the principal carrier of copper in plasma, is thought to be involved in oxidative modification of lipoproteins and participate in the acute phase reaction to stress. Cp is an effective antioxidant for a variety of radicals and has a potent peroxidase activity to decompose hydrogen peroxide in the presence of reduced glutathione. It inhibits the peroxidase activity of myeloperoxidase in a concentration-dependent manner and shows selective binding to myeloperoxidase in vitro. This suggests that ceruloplasmin may participate in the clearance and inactivation of myeloperoxidase in vivo. At physiological (micromolar) concentrations it was found to inhibit relaxation of rabbit aorta induced by endothelium-dependent agonists like acetylcholine or ADP. However, it was found ineffective toward vasodilation due to direct stimulation of smooth muscle cells by nitroglycerin. Cp plays an essential role in iron homeostasis, regulating the efflux of iron from macrophages and the oxidation of Fe2+ to Fe3+. Cp deficiency leads to neurodegenerative disorders, possibly by starving neurons of iron.

Purification Methods

This blue protein is the principal Cu transporter (up to 90% of circulating Cu) and is purified by precipitation with polyethylene glycol 4000, batchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin is thus purified 1640-fold and is homogeneous on anionic polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, isoelectric focusing and low-speed equilibrium centrifugation. It has max at 280, 260nm (A 1cm 14.9, 0.68). [Oestnuizen Anal Biochem 146 1 1985, Cohn et al. J Am Chem Soc 68 459 1946.] Chemokines. These are small proteins formed from longer precursors and are chemo-attractants for lymphocytes and lymphoid organs. They are characterised by having cysteine groups in specific relative positions. The two largest families are the  and  families that have four cysteine residues arranged (C-X-C) and (C-C) respectively. The mature chemokines have ~70 amino acids with internal cys S-S bonds and attract myeloid type cells in vitro. The -family (Lymphotactin) has only two cys residues. The -family (Neurotactin, Fractalkine) has the C-C-X-X-X-C sequence (ca 387 amino acids), binds to membrane and promotes adhesion of lymphocytes. The soluble domain of human Fractalkine ‘chemo-attracts’ monocytes and T cells. Several chemokines are available commercially (some prepared by recombinant DNA techniques), including 6Ckine/exodus/SLC which belongs to the -family with 6 cysteines (110 amino acids, mature protein), as the name implies (C-C-C-C-X.....X-C-C) and homes lymphocytes to secondary lymphoid organs with lymphocyte adhesion antitumor properties. Other chemokines available are C10 (CC) and Biotaxin. Several chemokine receptors and antibodies are available commercially and can generally be used without further purification. [Murphy “Molecular biology of lymphocyte chemo-attractant receptors” in Ann Rev Immunol 12 593 1994.] Chirazymes. These are commercially available enzymes, e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or trans-esterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available from Roche Molecular Biochemicals and are used without further purification.