Jaroslava Turková

Effect of concentration of immobilized inhibitor in the biospecific chromatography of pepsins

Abstract A specific sorbent for porcine pepsin containing 0.85 μmol of e-aminocaproyl- l -Phe- d -OCH3 per gram of dry carrier (hydroxyalkyl methacrylate copolymer) sorbed 29.4 mg of pepsin gram of dry sorbent, which means that 99% of immobilized inhibitor molecules participated in the specific complex formation with the isolated enzyme. With increasing amount of bound inhibitor this fraction decreased sharply (only 26% for 4.5 μmol). A specific sorbent with a content of 155 μmol/g appeared to be unsuitble for the affinity chromatography of pepsin (possibility of formation of multiple non-specific bonds between isolated enzyme and specific sorbent). The sorption of chicken and human pepsin was found to be lower thant that of porcine pepsin. The cause is seen in differences between the equilibrium constants of the individual enzyme—immobilized inhibitor complexes. The amount of sorbed chickchen pepsin increased after reaction with o-nitrobenzenesulphenyl chloride. Using experimentally determined curves representing the dependence of the amount of sorbed enzyme on the content of immobilized inhibitor, it is possible to estimate the order of magnitude of the equilibrium constant of the respective specific complex.