Study on the affinity characteristics of proteins on the immobilized metal affinity chromatography column
Using frontal chromatography to determine the affinity binding constant (KPML) and the number of affinity binding sites (ΛPML) 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 KPML(Lys) > KPML(Cyt-C) > KPML(RNase) > KPML(BSA), and the values of KPML on the IDA-Cu(II)-silica column were larger than those on the IDS-Cu(II)-silica column, while the ΛPML values on the IDA-Cu(II)-silica column were smaller than those on the IDS-Cu(II)-silica column. The results of the KPML 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 ΛPML, the parameter KPML 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.