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.
