Identification and determination of copper-and zinc-protein complexes in blood plasma after chromatographic separation on DEAE-Sepharose CL-6B

Abstract

Plasma proteins were separated on columns of DEAE-Sepharose CL-6B, using a buffer of constant ionic strength. The identities of proteins in individual fractions were determined by radial immunodiffusion, UV absorbance and the use of purified protein ‘markers.’ Copper and zinc concentrations were determined by atomic-absorption spectroscopy. Mean recoveries of copper and zinc in fractionated plasma were 100.7 ± 4.5 and 100.3 ± 3.8%, respectively. The coefficients of variation of replicate separations of the same sample were 1.3% for caeruloplasmin-copper, 2.7% for albumin-zinc and 6.0% for α₂-macroglobulin-zinc. Zinc was bound mainly to albumin (80–90%) and α₂-macroglobulin (10–20%); the remainder (<3%) was associated with the retinol-binding protein complex and with a low relative molecular mass fraction. No evidence could be found for zinc binding to α₂-HS-glycoprotein in uncontaminated plasma. Copper was associated mainly with caeruloplasmin (85–95%) and albumin (5–15%). A third copper peak, (<3%) showing properties similar to a copper-thionein like protein, was detected in some samples. The albumin-copper complex eluted later than the albumin-zinc complex, suggesting that these two metals bind to albumin by different mechanisms.