The studies on the mechanism of the enzyme action was mainly based on the assumption of the formation of enzyme-substrate complex and on the mathematical treatment of its kinetics to calculate the reaction constants. On the other hand, the complex could not be demonstrated experimentally except for a few enzymes.
Nakamura has proposed a method, namely the crossing paper electrophoresis, to detect the enzyme-substrate complex and with many enzymes he and his coworkers have succeeded to demonstrate the complexes on the filter paper. In the experiment described here, the enzyme-substrate complex of papain with benzoyl-L-argininamide and casein was demonstrated by this technique.
The complex was formed not only in the optimal pH range (pH 5.0-7.0), but also in the pH (pH 3.0 and 9.0) at which no enzymic activity was detected. Moreover, with inactive papain, as well as mercuripapain and PCMB-inhibited papain, the complex formation could be proved with ease. These findings indicate that pH values, inhibitors and activators influence only the activity state of papain but not the binding with substrate. They stand against the theory, that the active SH-group of papain should play a definite role to bind the substrate covalently.
It was concluded, therefore, that there should be two separate centers on the papain molecule: the one is the binding site for substrate, and the other, active site of the enzyme.

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