JOHN H. NORTHROP 489 



pound formed between the enzyme and substrate, since if this were 

 true a greater amount of substrate would be required to saturate a 

 greater amount of enzyme. The experiment, however, is not con- 

 clusive, since if it is assumed, as was done by Michaelis and Menten, 

 that the amount of substrate combined with the enzyme is negligibly 

 small, then the difference in concentration of substrate necessary to 

 saturate different amounts of enzyme would be entirely too small to 

 detect experimentally. As far as the relation between the rate of 

 hydrolysis and the concentration of enzyme or substrate is concerned, 

 therefore, the facts may be accounted for by the assumption of an 

 intermediate compound. 



It has been shown in a preceding paper^ that the inhibiting action 

 of the products of the reaction on the trypsin is in quantitative agree- 

 ment with the assumption that the enzyme and the inhibiting sub- 

 stance combine to form a compound which is inactive and that the 

 rate of hydrolysis is proportional to the concentration of uncombined 

 trypsin. It has also been shown that the same assumption will 

 account quantitatively for the protective action of the inhibiting 

 substances when the spontaneous inactivation of the enzyme is fol- 

 lowed. The fact that the inhibiting substance protects the enzyme 

 from decomposition is strong evidence that the inhibiting substance 

 combines with the enzyme. In the presence of the substrate, however, 

 the enzyme becomes inactivated at the same rate as the "pure" 

 enzyme^ (see Ringer).'' These facts render it unlikely that the en- 

 zyme is combined with the substrate. The present paper contains 

 the results of experiments planned to determine whether or not the 

 action of the enzyme with different concentrations of substrate and 

 of inhibiting substances can be accounted for on the assumption of a 

 compound between the enzyme and substrate. The observed facts 

 cannot be accounted for on the basis of the formation of a compound 

 between enzyme and substrate, if it be assumed that this compound 

 is governed by the law of mass action. 



8 Northrop, J. H.,/. Gen. Physiol., 1921-22, iv, 266. 

 » Ringer, W. E., Z. physiol. Chem., 1921, cvi, 107. 



