To clarify the functional role of amino acid residues in copper binding and in the maintenance of ceruloplasmin catalyt,ic activity, the protein was chemically modified by iodination.It is shown that addition of 20 mol iodine per 1 rnol ceruloplasmin mainly produces iodination of tyrosine residues, while the protein modification by higher iodine concentrations (60 rnol and more per 1 mol protein) leads also to iodination of histidine. Iodination of ceruloplasmin is followed by loss of copper and the oxidase activity. There is a correlation between copper loss and ceruloplasmin catalytic activity and iodinated histidine content a t high concentrations of iodine in the medium. It appears that tyrosine residues are involved in maintaining the active conformational state of ceruloplasmin, while histidine residues directly bind copper in its catalytic center. Peptide maps for 131I-labeled ceruloplasmin show that most tyrosine peptides of the protein iodinated a t small iodine concentrations are localized in the region of acid and alkaline peptides. Histidine peptides, whose iodination in accompanied by decrease in the oxidase activity and copper content, are mainly localized in the neutral peptide region. One of the neutral peptides having a well-defined Pauly reaction was isolated in a pure form. N-and C-terminal amino acids of the peptide and its amino acid composition were determined.Ceruloplasmin is a metalloglycoprotein of blood plasma, whose oxidase and ferroxidase activities are determined by the unique arrangement of eight copper atoms in the protein molecule [l,2]. I n spite of the recent progress in the study of ceruloplasmin macromolecular structure [3,4], the functional groups of this protein directly involved in metal binding, as well as groups maintaining the molecular activity, are not well understood. Lack of data concerning the active center hampers further research into the biochemical mechanisms of human copper metabolism involving this protein in the normal and, more especially, in Wilson's disease, a severe genetic disturbance of ceruloplasmin and copper metabolism Using the results of acid-alkaline titration of native and apo-ceruloplasmin (copper-free), it was earlier suggested that tyrosine, lysine and histidine residues might participate in copper binding 181. There is also some evidence indicating a decreased oxidase activity of ceruloplasmin in photooxidation, which points to the role of histidine in copper binding in the protein 19, lo].The purpose of this work was to clarify the functional role of amino acid residues in copper binding and in the maintenance of human ceruloplasmin catalytic activity by using the iodination technique.Unusual Abbreviation. CM-ceruloplasmin, reduced carboxymethylated ceruloplasmin.Enzyme. Trypsin (EC 3.4.4.4). Definition. A,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 530 nm, when measured in a 1-cm pathlength cell. 15-71. MATERIALS AND METHODS Preparation of CeruloplasminPreparations of original ceruloplasm...
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