On monolayers of proteolytic enzymes and proteins

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Abstract
When solutions of pancreatin were introduced under monolayers of proteins such as egg-albumin or caseinogen, proteolytic hydrolysis of the latter proceeded. The reaction could be followed qualitatively visually and quantitatively by observing the concomitant changes in phase boundary potentials. The reaction kinetics of the proteolytic digestion of protein monolayers followed closely those previously observed for the bulk phase reaction both in respect to dependence on the pH of the solution and the order of the reaction. The proteinase fraction of pancreatin was responsible for the removal of some 10% of the protein in the case of egg-albumin and of 15% in the case of caseinogen; the carboxypoly-peptidase fraction removed a further 25 and 16% respectively. The proteinase and carboxypolypeptidase fractions of pancreatin when purified by the precipitation or by the absorption process formed protein-like films, which were themselves capable of being hydrolyzed by pancreatin without loss of surface activity. The characteristics of films of trypsin were found to be indistinguishable from those of albumin over the pH region in which they exhibited no tryptic activity; over the active region they formed solutions of low surface phase boundary potentials. When pancreatin solutions were digested by pepsin at pH 2 and the solution restored to pH 8, while no tryptic activity was observed for bulk phase reaction, monolayers of egg-albumin and caseinogen were readily digested. Solutions of trypsin when boiled at pH 8 likewise exhibited a marked surface proteolytic activity. The properties of the surfaces of solutions containing both enzyme and protein were ex-amined during the course of the reaction. It is concluded that in the tryptic enzymes the "free enzymes" can be separated from their protein-like carriers but when free can only react with proteins when presented to them in a suitable form such as a monolayer at an air-water interface. To render the "free enzyme" active for protein solutions it must be adsorbed on a protein in a particular state or configuration. Experimental evidence is given that the disappearance of a protein film on an enzyme containing substrate is due to digestion only and not due to the adsorption of some constituent of the enzyme of lower surface tension.

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