Study on the affinity characteristics of proteins on the immobilized metal affinity chromatography column

Abstract

Using frontal chromatography to determine the affinity binding constant (K_PML) and the number of affinity binding sites (Λ_PML) was demonstrated by the accuracy and precision affinity experiments of proteins (BSA, RNase, Cyt-C and Lys) on the iminodisuccinic acid metal chelate copper (IDS-Cu(II)-silica) column. The results displayed that R² > 0.98 and RSD < 2% for the two binding parameters. In order to further prove the universality of frontal chromatography, the affinity characteristics of the four proteins on the iminodiacetic acid metal chelate copper (IDA-Cu(II)-silica) column were investigated in turn, and the results of the affinity parameters were compared with those of the IDS-Cu(II)-silica column. The results showed that the binding constants of the four standard proteins on both metal chelate copper columns followed K_PML(Lys) > K_PML(Cyt-C) > K_PML(RNase) > K_PML(BSA), and the values of K_PML on the IDA-Cu(II)-silica column were larger than those on the IDS-Cu(II)-silica column, while the Λ_PML values on the IDA-Cu(II)-silica column were smaller than those on the IDS-Cu(II)-silica column. The results of the K_PML value were consistent with the affinity chromatographic behaviors of the proteins on the two columns, which proved the feasibility of the FC method again. Compared with Λ_PML, the parameter K_PML could better reflect the affinity strength of the proteins on the immobilized metal affinity chromatography (IMAC) column. The present work provided a fast and effective method for determining the binding parameters between proteins and IMAC columns, and also provided a theoretical basis for the separation and purification of proteins using the IMAC system.