Characterisation of cortisol-bovine serum albumin conjugates by chromatofocusing

Abstract

Cortisol-bovine serum albumin conjugates were prepared at steroid: protein molar ratios in the range 2–24, by coupling cortisol-3-(O-carboxymethyl)oxime to the protein, using the N-hydroxysuccinimide method. These conjugates were characterised by size-exclusion chromatography on Sephacryl S-200 Superfine and chromatofocusing on PBE-94. All conjugates contained a variable fraction of polymers that became more pronounced at low steroid: protein molar ratios. Chromatofocusing separations showed the presence of an envelope of peaks, for each conjugate, that were eluted at various pH values in the range 4.35–3.60. No direct relationship was observed between the pH of elution and the degree of substitution of the protein with cortisol molecules, so that chromatofocusing appears to be inadequate for the efficient separation of conjugates with a defined degree of substitution with steroid molecules.