A New Ammonia Determination Assay Based on the Linear Activation of Horseradish Peroxidase for the Determination of Protein-Glutamine Glutaminase Activities

Abstract

A new horseradish peroxidase (HRP)-based assay for the determination of aqueous ammonia was developed. This new assay utilizes the concentration-dependent, linearly increasing activation effect of aqueous ammonia on the enzyme HRP at alkaline pH values. The special feature of this assay is that the analyte, ammonia, is not directly involved in the reaction catalyzed by the enzyme HRP. Instead, the concentration of the analyte is determined by measuring the increase of the activity of the HRP in the absence and presence of the sample. Accordingly, all relevant assay parameters and concentrations of the components were first carefully evaluated to enable the determination of low aqueous ammonia concentrations while simultaneously achieving a broad linear range. The optimized conditions in the newly developed method for the determination of ammonia were pH 10.0, 4.8 nkat mL⁻¹ HRP, 2.3 mmol L⁻¹ o-dianisidine, and 400 µM hydrogen peroxide. The limit of detection and the limit of quantification of the new method were 0.24 mmol L⁻¹ and 0.36 mmol L⁻¹, respectively. One possible application for the newly developed ammonia determination assay was the determination of protein-glutamine glutaminase (PG) activities. This assay was employed as a two-step assay, starting with the PG reaction conducted at the optimal pH value for the PG used. After this step, the pH was increased to 10, and the ammonia released was measured in a second reaction. The results obtained with this method showed less than 10% variation compared to two established methods.