Parallel Adsorption of Crystalline Pepsin and Peptic Activity Upon Casein and Ovalbumin. ∗

Abstract

Waldschmidt-Leitz and Kofrányi 1 found that by repeatedly shaking a solution of crystalline pepsin with small quantities of suspension of cantaloupe seed globulin they could adsorb the peptic activity while the crystalline pepsin-protein was left in solution. They accordingly claim that Northrop's 2 crystalline pepsin is simply a case of an enzyme adsorbed upon a protein. Their evidence for the non-adsorption of the pepsin-protein was obtained by determining the weight of dry matter left after centrifuging off the suspended material and by blank determinations made with cantaloupe globulin alone.

It is unfortunate that these authors did not employ a specific test for the protein of crystalline pepsin, for their centrifuged solutions may have contained not only crystalline pepsin, but also cantaloupe globulin in quantity not indicated by blank determinations, as well as digestion products. By the employment of a test which is specific for crystalline pepsin-protein, provided other proteins are not present in the solution, I have been able to demonstrate that when the peptic activity of a solution of crystalline pepsin is completely adsorbed upon casein or coagulated ovalbumin the pepsin-protein is also completely adsorbed. Furthermore, I have found that the rates of adsorption of peptic activity and of pepsin-protein are the same. These findings conclusively disprove the work of Waldschmidt-Leitz and Kofrányi unless one is willing to make the highly improbable assumption that crystallized cantaloupe seed globulin possesses some peculiar action upon pepsin which is not exerted by casein or by denatured ovalbumin.

Casein and coagulated ovalbumin were chosen as adsorbents because of their insolubility at pH 4.8. Edestin could not be used since it was too soluble below pH 5 and it was not desirable to subject pepsin to solutions less acid than this. Coagulated edestin formed large lumps which could not be pipetted in suspension. The casein was prepared by washing 10 gm. of Merck's casein repeatedly with dilute acetate buffer of pH 4.8. The casein was then mixed with 90 cc. of water and 10 cc. of 0.5 M acetate buffer of pH 4.8. The ovalbumin was prepared by heating 1000 mg. of recrystallized, dialyzed ovalbumin in 90 cc. of water for 15 minutes, cooling and adding 10 cc. of acetate buffer. Both proteins were suspended by shaking just before pipetting. The casein was lumpy and difficult to pipette.