Protein chromatography with pyridine- and alkyl-thioether-based agarose adsorbents

In an attempt to identify the part of the ligand of 3-(2-pyridylthiol)-2-hydroxypropylagarose that is responsible for the specific adsorption of immunoglobulins and alpha 2-macroglobulin from serum, nine agarose derivatives were prepared: (I) 3-(N-2-iminopyridyl)-2-hydroxypropyl-, (II) 3-(4-pyridylthio)-2-hydroxypropyl-, (III) 3-(2-pyridylthio-N-oxide)-2-hydroxypropyl-, (IV) 3-(2-pyridylthio)-2-hydroxypropyl-, (V) 3-(ethylthio)-2-hydroxypropyl-, (VI) 3-(n-butylthio)-2-hydroxypropyl-, (VII) 3-(2-aminoethylthio)-2-hydroxypropyl-, (VIII) 3-(2-hydroxyethylthiol)-2-hydroxypropyl- and (IX) 3-(N-2-pyridyl-2-one)-2-hydroxypropylagarose. The selective adsorption of the above serum proteins to these derivatives was analysed by chromatography. The electron distributions were calculated for three of the investigated pyridine derivatives in order to establish whether there is any relationship between the electron distribution in the molecule and the absorption properties of the pyridine derivatives. By optimizing the preparation methods for the different derivatives, the possible side-reactions were minimized and checked. The adsorbed serum proteins were determined by the Ouchterlony technique and electrophoresis. The concentration of human serum albumin in the different fractions was determined by conventional radioimmunological methods. These data make it possible to classify the adsorbents according to their selectivity and hydrophobic thiophilic behaviour.