A New Method of Determination of Hydroxamic Acid by Its Urease Inhibition and Application to Biochemical Studies

Abstract

Based on the characteristic of hydroxamic acid that it is a potent and specific inhibitor of urease activity, investigations were performed for the establishment of an enzymatic method for the determination of hydroxamic acid and its application to the assays of some enzyme activities. Crude urease prepared from jack beans was preincubated with the sample solution containing 0.5-10 nmol of hydroxamic acid under the standard conditions and the residual urease activity was measured colorimetrically. A linear correlation was observed between logarithmic concentrations of hydroxamic acid and the inhibition percentage ranging from 20 to 80%. According to the standard relation obtained, hydroxamic acid was determined or detected with higher sensitivity in the following examples: 1) distribution of nicotinohydroxamic acid in the tissues of rats after oral administration, 2) stability of caprylohydroxamic acid in animal food to which the compound had been added as a growth-promoting agent, 3) qualitative detection of hydroxamic acid on a paper chromatogram, 4) applications of this method to the assays of the formation of acetyl CoA by the pyruvic oxidase system or of acetyl phosphate by the acetokinase system. This new method had advantages in easiness and sensitivity over the general method hitherto used, which is based on the ferric-hydroxamic acid complex formation in an acidic solution. However, the power and the rate of inhibition were dependent upon the acyl residue of hydroxamic acid and therefore authentic hydroxamic acid was necessary for the determination.