Papain-Collodion Membranes. I. Preparation and Properties

A number of papain-collodion membranes were prepared by adsorbing papain on collodion membranes and cross-linking with bisdiazobenzidine- 2,2′-disulfonic acid. The thickness of the enzyme layers could be controlled by adjusting the amount of papain in the adsorption solution. The swollen collodion membranes had an adsorption capacity for papain of 67.5 mg/cm³. The adsorbed papain was found to form a monomolecular layer on the pores of the collodion matrix. A quantitative analysis of the adsorption process has been carried out. Three- layer papain membranes (300-500 μ thick) consisting of two enzyme layers (70 μ thick) separated by a collodion layer, two-layer papain membranes consisting of an enzyme layer (~120 μ) and a collodion layer (200 to 300 μ), and a one-layer papain membrane (70-120 μ) were made by the adsorption techniques developed. The permeability and filtration coefficients of the papain-collodion membranes were determined and compared with the corresponding coefficients of collodion membranes devoid of papain. Pore radii of 280 Å were found for the one-layer papain membranes prepared; pore radii of 300 Å were found for the collodion membranes used as matrix. The pH- activity profiles for a three-layer papain membrane using five different substrates (benzoyl-L-arginine ethyl ester, benzoylglycine ethyl ester, benzoyl-DL- arginine p-nitroanilide, benzoyl-L-arginine amide, and acetyl-L-glutamic acid diamide) differed from one another and from the normal bell-shaped curves obtained for native papain. The shape of the pH- activity curves for the papain membrane was found to depend on the nature of the products liberated (including H⁺ and NH₄⁺) and on the kinetic parameters determining the rate of hydrolysis. When benzoyl-L- arginine ethyl ester and benzoylglycine ethyl ester were used as substrates the pH established locally in the papain membrane as a result of enzymic activity was found to be more acid by 2-4 units than the pH of the external solution. A theoretical approach based on the rapid establishment of a steady state in the membrane is suggested to explain the various pH- activity profiles observed.