Some studies of the chromatographic properties of gels ('artificial antibodies/receptors') for selective adsorption of proteins

Although conventional imprinting involving the use of charged functional mo nomers has been used by many authors for selective adsorption of small mole cules, it has not been very successful with high-molecular-weight substance s, for example proteins (I macromolecules, such as proteins are difficult t o apply as templates.,."; H. S. Andersson, Doctoral Thesis, Tryck i Hogskol an Kalmar, 1999, p. 8.). Four years ago we therefore introduced an alternat ive method based on the polymerization of non-charged monomers (acrylamide and NN'-methylenebisacrylamide) in the presence of the protein of interest. The selectivity of the gels for proteins was high (for instance, myoglobin from horse was adsorbed by a column designed to be selective for this prot ein, whereas, myoglobin from whale was not adsorbed) and they can therefore be regarded as 'artificial antibodies(or 'artificial receptors'). This paper deals with improvements of the chromatographic properties of the se gels. For example, by modifying the polymerization conditions the protei n (hemoglobin) capacity, as well as the flow rate were increased fourfold. This was achieved by entrapment of the selective soft polyacrylamide gel in the pores of a rigid inert gel by letting the monomers and the protein dif fuse into the pores of agarose beads (Sepharose(TM)) before starting the po lymerization. The gel formed was cut into pieces. The agarose beads were fr eed from the surrounding polyacrylamide gel by stirring. This technique is universal and is recommended also for molecular imprinting studies of small molecules. Another universal method has been introduced for rapid screening of potenti al monomers and gels for the preparation of selective adsorbents. This very simple method is based on the assumption that the absorption maximum of a protein changes when the protein interacts with the free monomers or the ad sorbent synthesized from the monomers (and does not change when the protein does not interact). Preliminary docking experiments indicate that selectiv e adsorption of the protein by the polyacrylamide matrix is based primarily on hydrogen-bonding and dipole-dipole interactions. The strengths of these interactions can be varied by choosing different gel matrices (for removal of a given protein the interactions should be very strong, whereas they sh ould be weaker for chromatographic analysis).