A Method for the Determination of Enterokinase.

Abstract

Any method of assay for enterokinase is indirect, and consists in a determination of the amount of trypsin which the enterokinase can produce from a given amount of trypsinogen. The amount of trypsin is determined by means of its digestive action on some suitable protein substrate. The methods used by Waldsehmidt-Leitz, 1 and Linderstrom-Lang and Steenberg 2 consist in a determination of that amount of kinase which will just give maximal activity to a given quantity of trypsinogen or only one-half of the maximal activity in a given period of time, usually 30 minutes.

Such methods of assay involve all of the variables and possible errors of a trypsin assay plus a great number involved with the activation process. A brief study has shown that many gross errors are easily incurred during activation. Activation was found to occur in the presence of the alkaline protein substrate so that any method of partial activation before addition of the protein substrate is complicated by the additional amount of activation which occurs subsequently. The amount of kinase which will impart maximal activity to a given quantity of trypsinogen is not a clean-cut quantity. As one adds increasing amounts of enterokinase to a constant quantity of trypsinogen a maximum value is almost never reached. It is reached very gradually so that a small error at any point in the determination would be magnified in the final estimation.

In assaying enzymes it is customary to have present an excess of substrate. Assuming the action of enterokinase to be that of an enzyme and since activation occurs during digestion and at a slower rate than at the neutral pH usually used for activation, it seemed logical to attempt to assay by using an excess of trypsinogen with no activation with the added enterokinase, other than that occurring simultaneously with the digestion.