Increasing the binding strength of proteins to PEI coated supports by immobilizing at high ionic strength

The immobilization of proteins was performed by ionic adsorption on agarose supports coated with polyethyleneimine of high molecular size (PEI) (600 kDa). The immobilization processes were carried out at different ionic strengths (5, 50, 100, 300, 500 and 700 mM NaCl). At NaCl concentrations lower than 100 mM, the immobilization proceeded very rapidly in all cases, while in some cases at concentrations over this value; the adsorption rate was significantly decreased. In all cases, the activity of the enzymes remained over 90% during the immobilization process. Thus, considering the immobilization rate, it seems to be better to use low ionic strength. However, desorption of proteins that had been adsorbed at low ionic strength started when using only moderately high ionic strength. When the enzymes were adsorbed at higher NaCl concentrations the strength of the adsorption was in all cases much higher than when it was performed under the usual low ionic strength. This may be explained assuming that at high ionic strength, proteins can only become immobilized on areas of the protein which are able to simultaneously yield a very intense multipoint adsorption, even permitting the penetration in the polymeric bed structure, while using low ionic strength any area of the protein with negative charges may be involved in the adsorption.