Use of Added Labeled Substrate Conversion Ratio to Measure Endogenous Substrate Concentration and Enzyme Activity 1

A method for measuring the concentration of an enzyme's substrate was developed from the Michaelis-Menten hypothesis, and its validity demonstrated with the hydrolysis of p-nitrophenylacetate by a-chymotrypsin.

To arrive at a simple method for measuring angiotensin in the blood, we have made use of the competition of endogenous angiotensin and added ¹⁴C labeled angiotensin for plasma angiotensinase (1). Because plasma angiotensinase was not isolated we first tested the validity of the method with a model system. The method requires that both substrate and enzyme concentrations be rate-limiting and the enzymatic reaction be free from significant product inhibition. Under these conditions, an increase in the amount of substrate will result in a decrease in the conversion ratio (the amount of product formed per unit of substrate). If a known amount of exogenous substrate (S∗), identical to endogenous substrate except that it is radioactively labeled in a moiety which gives a labeled product, is added to the otherwise unchanged reaction mixture, then the ratio of radioactive product (P∗) to radioactive substrate is the same as the conversion ratio for all substrate. From this conversion ratio, the amount of (labeled) substrate added, and from the reaction rate measured in the presence of excess substrate, one can calculate the amount of unlabeled (endogenous) substrate present, by the following equation: 2

P _max can be measured by running the reaction with a large excess of labeled substrate, and measuring the labeled product. In addition, both K_m and P _max can be determined by measuring P when the reaction is run in absence of endogenous substrate at different labeled substrate concentrations, using the method of Lineweaver and Burk.

P _max gives a measure of enzyme concentration, and X (P∗/S∗) gives the amount of unlabeled substrate converted to product by the enzyme during the test reaction.