The efficiency and the performance of size exclusion (SEC) and ion exchange (IEC) chromatography in the separation of a number of metal-binding proteins have been studied.  The proteins were the following: superoxide dismutase (SOD), albumin, carbonic anhydrase, ferritin, transferrin, haemoglobin, myoglobin, conalbumin, aminopeptidase cytochrome C, ceruloplasmin, tyrosinase, dopamine-ß-hydroxylase, plasma amine oxidase, lactoferrin and alkalyne phosphatase. The last six proteins were eliminated after several attempts at analysing them without success. The parameters optimized for the separation of the proteins by SEC were: eluent type, pH of the eluent and concentration of the salts added. SEC was found to be especially suitable for the separation of  ferritin from the other proteins. The two different ion exchange modes: cation exchange and anion exchange, have been investigated. Anion exchange chromatography allowed the separation of cytochrome C, myoglobin, carbonic anhydrase, haemoglobin, superoxide dismutase, transferrin, albumin and aminopeptidase. Cation exchange was useful in the analysis of proteins with a high isoelectric point (pI), such as cytochrome C. Preliminary studies have been carried out on the separation of different glycosilated isoforms of transferrin by anion exchange, leading to promising results. IEC was sucessfully applied to the determination of transferrin in human serum.

 

The orthogonality of SEC and IEC is discussed.

 

Measurements have been made using electrothermal atomic absorption spectrometry (ETAAS) to determine the amount of Fe, Cu and Zn bound to the proteins studied.