Sorption and elution conditions

In the binding of the complementary sites of the affinity ligand and of the isolated substances, ionic bonds or hydrogen bonds, hydrophobic interactions and Van der Waals-London forces may participate to various extents. Therefore, the optimum conditions for sorption and desorption will vary in particular instances. In general, the starting conditions for sorption should be selected so as to cause maximum sorption of the substance to be isolated. The choice of the starting buffer is completely dependent on the optimum conditions of a specific complex formation 

The practical utilization of pH during the sorption of neutral proteinase from Bacillus subtilis on Sepharose with attached glycyl-D-phenylalanine (through a spacer 23 atoms long) is shown in Fig. 4.7.5. Walsh et al. [62] demonstrated that between pH 5 and 6.5 neutral proteinase was effectively adsorbed and thus separated both from subtilisin and other proteins present in the culture filtrate. At higher pH values, an effective separation of neutral proteinase from subtilisin does not take place. 

Effect of ionic strength of buffer on j8-galactosidase binding on to a column of agarose substituted with -aminophenyl-j8-D-thiogalactopyranoside(bed dimension, 114x 17 mm flow-rate, 2.0 ml-min -cm ). 

Ionic strength Protein bound (mg) Activity bound (units) Specific activity (units/ntg) 

Neutral proteinase is set free from the complex with the immobilized affinant by increasing the pH to values at which the binding of the substrate is already weak, but at which the enzyme is still not denatured. The optimum pH for the elution of neutral proteinase depends not only on its affinity towards the affinant and on the pH dependence of this affinity, but also on the concentration of the immobilized inhibitor. Neutral proteinase is a metalloenzyme and is therefore inhibited by 

---