Spectrophotometric Kinetic Studies and Substrate Inhibition of Hog Kidney Histaminase Activity by an Improved 2,4-Dinitrophenylhydrazine Method

Abstract

We improved the experimental procedure for the measurement of hog kidney histaminase activity using histamine as a substrate on the basis of a spectrophotometric estimation of the 2,4-dinitrophenylhydrazone of imidazole acetaldehyde and studied the steady-state kinetics to obtain the basic data for further investigations of the oxidative deamination of histamine. The initial and mean velocities of the enzymatic reaction were calculated and plotted against the amount of enzyme. It was found that the initial velocity increased linearly. The time tα necessary to reach the extent of reaction α was calculated and plotted against the reciprocal of the enzyme concentration e0. It was found that tα was linearly proportional to 1/e0. From Lineweaver-Burk plots, inhibition by high concentration of substrate was evident, and the v-pS curve was bell-shaped, with a pS maximum at 3.2. Km and V were obtained: Km = 7.7×10-5 M. V = 0.0026 μmol/min (0.00075 μmol/min/mg protein). It was concluded that our DNP method was useful for the measurement of hog kidney histaminase activity using histamine as a substrate, basic steady-state kinetic studies and further investigations of substrate inhibition and inhibitory effect.