An optimized method for estimating glutaminase activity in biological samples

Spectrophotometric methodologies have been used to assess glutaminase activity, for which coloured complexes have been developed that measure spectrophotometry across the visible spectrum using different reagents. The present paper describes a precise, simple and reliable procedure for quantifying glutaminase activity, which is a key enzyme in glutamine hydrolysis and also involved in glutamine metabolism regulation. The procedure presented here measures glutaminase activity by incubating glutaminase enzyme at 37 °C for 20 min with a glutamine substrate dissolved in a buffer (pH 8.6). The enzymatic reaction contains suitable activity of glutamate oxidase, which acts to convert glutamate to hydrogen peroxide and 2-oxoglutarate. To terminate the enzymatic activity, a working solution containing pyridine-2,6-dicarboxylic (PDA) acid and ammonium vanadate (AV) was added following incubation. Oxo-peroxo-pyridine-2,6-dicarboxylato-vanadate (OPDV), a stable orange-coloured chelate complex measuring 435 nm spectrophotometrically, was produced by the interaction between the generated hydrogen peroxide and the supplied reagent. Using the response surface methodology (RSM) as an indicator of the assay's accuracy, we employed the Box-Behnken design (BBD) to improve the method's design (the OPDV-Glutaminase assay). Improvement factors were the volume of working reagent solution (PDA/AV), volume of glutamate oxidase solution (GO), and incubation time. In matched samples, this novel method was verified against a Bland-Altman plot assessment of glutaminase activity using the indophenol methodology. A correlation value of 0.99 between the two methods' comparisons showed that the novel protocol was equally applicable to the reference method.