While spacer arms have been shown to play an important role in affinity chromatography, no systematic investigations of spacer arms in the purification of transcortin have been reported. Among the five cortisol-agaroses, cortisol-21-succinyl-1,6-hexanediamidoagarose achieved the highest extraction efficiency of transcortin from plasma. The optimal length of the spacer arm for extraction is ca. 12-13 Å. Cortisol-succinyl-agaroses having hydrophobic spacer arms extract transcortin better then those having hydrophilic arms of approximately equal length. Affinity supports are usually synthesized sequentially; cortisol-agaroses thus prepared were found to complicate the purification of transcortin. The problems of nonspecificity and instability associated with these agaroses were eliminated by using reverse addition. A complete ligand-spacer arm, synthesized in a single step by displacing the tosyl group from cortisol-21-tosylate with 1,6-hexanediamine, was coupled with cyanogen bromide-activated agarose. Although the 21-deoxy-21-(ω-amidohexyl) aminocortisol-agarose ranked second in extraction efficiency, its superior stability and low nonspecific adsorption of other proteins make it the prime choice for affinity chromatography of transcortin.
ASJC Scopus subject areas
- Molecular Biology