Comparative evaluation of DNS, PAHBAH, and BCA colourimetric assays for quantifying reducing sugars

Colourimetric assays for quantifying reducing sugars are widely used, including for assessing glycoside hydrolase activities. Consequently, there is a need for reliable high-throughput methodologies. Several protocols have been described in literature, with the most commonly used being the dinitrosalicylic acid (DNS), bicinchoninic acid (BCA) and p-hydroxybenzoic acid hydrazide (PAHBAH) assays. This study presents a comparative evaluation of these assays. Our results show that both the DNS and PAHBAH assays overestimate reducing sugar concentrations mainly due to saccharide degradation under alkaline conditions (pH ≥ 12.9) and high temperatures (100 °C) during the assays. To evaluate the impact of this overestimation, we experimentally measured glycoside hydrolase activity. All three assays overestimated reducing sugar concentrations compared to a reference method, though to varying extents. The BCA assay exhibited the least overestimation, likely due to minimal saccharide degradation under its milder alkaline conditions. It was therefore the most reliable method among those tested.