Peptide sequences obtained from hen ovotransferrin are compared with the complete amino acid sequence of the protein deduced from a cDNA sequence (Jeltsch and Chambon, preceding paper). Of the 705 positions of the whole protein 605 can be matched by the peptide sequences. Some possible discrepancies between the two methods are pointed out. The two halves of the chain show marked similarities in their sequences with 37 identical residues. The positions of the 15 disulphide bridges are shown; there are 6 homologous bridges in each half of the molecule and 3 extra bridges which occur only in the C-terminal half. The terminal residues of the halfmolecule fragments obtained by limited proteolysis are identified. The two domains are joined by a 9-residue connecting peptide. Sequence variability has been found at 9 positions. The sequence of hen ovotransferrin IS compared with the partial sequence available for human transferrin. From this some tentative conclusions about the identities of the metal-binding residues and about the evolution of transferrin are reached.The iron-binding protein transferrin is present in all vertebrate species. The closely related protein lactoferrin (or lactotransferrin) appears to be restricted to mammals. The amino acid sequences of these proteins have been studied for many years in order to elucidate their structure, evolution and ironbinding properties. Brew and his colleagues [1,2] have determined about 80 % of the sequence of human serum transferrin and have discovered that the N-terminal and C-terminal halves of the chain have similar sequences, with about 40 identical residues. X-ray crystallography [3] and partial proteolysis experiments [4,5] show that each half of the chain folds up into a largely independent, compact domain which carries a specific metal-binding site. Since the domains can be isolated as single-chain fragments after partial proteolysis it can be inferred that the disulphide bridges are located within the domains, none passing between them. These data suggest that the present-day bilobar protein has evolved by gene duplication and subsequent fusion from a smaller ancestral protein, which apparently no longer exists. Partial sequence data on human lactoferrin show that this protein is homologous to transferrin and that it too possesses internal duplication of structure [6,7]. Lactoferrin may, thus, have arisen by a further duplication of the transferrin gene in the ancestors of the mammals.In birds expression of the transferrin gene in the oviduct, which is under steroid control, leads to the synthesis of ovotransferrin (conalbumin), which differs from serum transferrin only in the nature of the attached glycan [8,9]. The sequences of cysteic-acid-containing peptides from digests of performicacid-oxidized ovotransferrin have been determined [lo] ; 34 unique cysteic acid residues were identified and some of the disulphide bridges were characterized after pepsin digestion of the unoxidized protein. A 102-residue sequence, which includes the point of attachment of ...

Nine samples of human ceruloplasmin [iron (II).oxygen oxidoreductase; EC 1.16.3.1] prepared by different procedures have been examined for heterogeneity; gel electrophoresis showed that seven contained a number of components with molecular weights ranging from 20,000 to 130,000, and two contained largely a single component of molecular weight 130,000. Digestion of a single-component preparation with plasmin produced fragments with molecular weights similar to those found in the multicomponent preparations. Amino-terminal analysis, peptide mapping, and amino acid analysis showed that plasmin digestion generated a fragment of 20,000 molecular weight, which corresponded to a component present in a multicomponent ceruloplasmin preparation. The 20,000 molecular weight fragment appears to correspond to the so-called a-subunit or L-chain of human ceruloplasmin. Chemical evidence is thus provided that ceruloplasmin is a single-chain protein and that the so-called subunits are fragments.The 20,000 molecular weight fragment contains a single cysteine; amino acid sequence studies have shown that the sequence in the vicinity of this residue is similar to that around the single cysteine residue in plant plastocyanins and bacterial azurins, which are small, blue, copper-containing proteins. Despite Ryd6n's evidence (1, 2) for a single-chain structure for human ceruloplasmin (ferroxidase) [iron(II):oxygen oxidoreductase; EC 1.16.3.1], several authors continue to propose subunit structures (3, 4). The dilemma is posed by the following quotation from a recent review on ceruloplasmin (5); on the question of subunits the authors conclude "one, two, four, eight-only the future will tell." Furthermore, although many spectroscopic and magnetic investigations have been made of ceruloplasmin copper, little is known about its copper-binding sites or their possible structural similarity to sites in other copper-containing oxidases or electron transport proteins. In this paper we give chemical evidence for a single-chain structure for ceruloplasmin and report an amino acid sequence that may be involved in copper binding.We have found that ceruloplasmin prepared by different procedures contains varying amounts of discrete subunit-like fragments. Limited plasmin digestion of essentially undegraded ceruloplasmin has shown that cleavage by this enzyme, or by one with a similar specificity, is responsible for the variety of molecular forms reported for ceruloplasmin. We have also shown that plasmin produces a fragment of 20,000 molecular weight (Mr) that appears to correspond to the so-called a-chain (4) or L-chain (3). We have almost completely determined the amino acid sequence of the corresponding 20,000 Mr fragmentThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5377 isolated from partially degraded ceruloplasmin. A single cysteine residue is located in the COO...

The (6) and probably corresponds to the a subunit proposed by Simons and Bearn (4) and the light chain of Freeman and Daniel (5).The relation of Cp F5 to the structure of the intact ceruloplasmin chain has to be deduced from indirect observations because of the small amount of single-chain ceruloplasmin available to us and the strong interaction of the fragments. From the kinetics of the proteolytic cleavage of intact ceruloplasmin and the chemical properties of the fragments, we have suggested that Cp F5 is from the COOH terminus of the single chain (8). We have obtained a unique amino acid sequence and conclude that Cp F5 is probably an intact domain; it appears to be attached to the COOH-terminal end of the ceruloplasmin chain by a labile interdomain peptide bond. Prediction of the secondary structure by the empirical method of Chou and Fasman (9, 10) leads to a model in which approximately 30% of the residues occur in f3 sheets, 25% in a helices, and 45% in j turns and other structure.Because the ceruloplasmin preparation studied is derived from a pool of plasma from more than 10,000 donors from the ethnically mixed American population, the finding of a unique amino acid sequence indicates that the frequency of genetic polymorphism is too low to interfere with sequence determination of this fragment.In a previous report (8)