Chemical evidence that proteolytic cleavage causes the heterogeneity present in human ceruloplasmin preparations.

Abstract

Nine samples of human ceruloplasmin [iron(II):oxygen oxidoreductase; EC 1.16.3.1] prepared by different procedures were examined for heterogeneity; gel electrophoresis showed that 7 contained a number of components with MW ranging from 20,000-130,000 and 2 contained largely a single component of MW 130,000. Digestion of a single-component preparation with plasmin produced fragments with MW 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 MW, which corresponded to a component present in a multicomponent ceruloplasmin preparation. The 20,000 MW fragment appears to correspond to the so-called .alpha.-subunit of L-chain of human ceroluplasmin. Chemical evidence is provided that ceruloplasmin is a single-chain protein and that the so-called subunits are fragments. The 20,000 MW fragment contains a single cysteine; amino acid sequence studies showed 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 Cu containing proteins.