(416c) Affinity Enhanced Gel Permeation Chromatography of Proteins upon Modification with Multi-Ligand Affinity Carriers

A multi-ligand affinity carrier based on a polyethylene glycol derivative modified with iminodiacetic acid (IDA) was prepared as an affinity macroligand. PEG-(IDA Cu (II))64, a 700,000 molecular weight multi-armed chelators was used as an affinity macroligand to investigate its interaction with proteins and its behavior in gel permeation chromatography. The multi-arm PEG chelator loaded with metal ions (e.g., Cu) is effective in interacting in batch mode with protein mixtures consisting of histidine containing proteins such as bovine hemoglobin, horse heart cytochrome C and ovalbumin. Due to the larger number of histidine residues on the hemoglobin surface the metal affinity binding of the multiligand was more selective towards this biomolecule. After binding, the radius of giration of the molecule-complex is apparently increased due to the large molecular size of the multi-PEG molecule. Next, the mixture of proteins along with the complex hemoglobin-multiPEG ligand was introduced into a gel permeation chromatography column. Due to the ?enhanced? molecular size of the bound hemoglobin its retention time in the column was effectively reduced leaving the column considerably faster than the rest of the unbound proteins, allowing with this an efficient protein fractionation. The preparation of such multiligand chelating-derivatives as well as binding, separation and elution studies will be presented.