FAILURE OF SEPHAROSE-PEPSIN AS AN IMMOBILIZED MILK CLOTTING ENZYME

Abstract

Porcine pepsinogen A was attached to CNBr-activated Sepharose 4B and self activated to form Sepharose-pepsin A. Immobilized pepsin is active in hydrolyzing hemoglobin at acidic pH but has very low milk clotting activity. The low milk clotting activity of freshly prepared Sepharose-pepsin can be accounted for by leaching of small amounts of free pepsin during contact of the complex with milk substrate. The failure of bound pepsin to catalyze milk clotting appears to be due to part of the micellar casein in milk substrate being inaccessible to the immobilized rennet and may also be influenced by differences in pH optima with casein substrate for pepsin and Sepharose-pepsin. Porcine pepsin A, which is electrophoretically pure, and casein substrate exhibit a distinct bimodal optimum reaction pH at 2 and 6. The reaction at pH6 is not evident when the products are assayed by A280nm because the specific products formed at this pH exhibit low A280nm. The finding that a pure isoenzyme of pepsin can exhibit a bimodal pH profile for hydrolysis has probably been overlooked by other investigators because of the frequent assay by A280nm of products.