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Purification and characterization of a small copper carrier found in mammalian blood plasma and in the urine of mice and dogs with copper overload

We recently discovered a small copper carrier (SCC), which in most mammals is mainly bound to larger proteins and can be released by EDTA. It normally accounts for at least 10% of total plasma copper, and more than half the total in Atp7b−/− mice and some Labrador retrievers with copper overload, where it circulates in the “free” form and appears in the urine. This suggests SCC is an alternative means of excreting copper when biliary excretion is inhibited. To begin to isolate and characterize SCC, blood plasma of human volunteers (permitted by the university IRB), pigs, cows, sheep and Labrador retriever dogs were analyzed for their quantities or free and protein‐bound SCC, using 10 and 3kDa ultrafiltration and size exclusion chromatography (SEC) with Superdex 200 and peptide FPLC. By MALDI‐TOF, the main component identified as potentially SCC consistently had a mass of 1329.5 Da (not including Cu), identical to that reported for urinary SCC from Atp7b−/− mice. It eluted between vitamin B12 and NADH in small pore SEC and not with Cu‐EDTA, which aggregated. Two amino acids were identified; a third was blocked by sulfate or phosphate. However, solution NMR indicated the samples were not pure. Preliminary data suggested phenyl HIC and anion exchange might be useful purification steps. Plasma from the pig had the highest levels of total SCC and more than 10% in the “free form”. So it was chosen to identify the best steps for large scale SCC purification.