Background
Enzyme assays have widespread applications in drug discovery from plants to natural products. The appropriate use of blanks in enzyme assays is important for assay baseline-correction, and the correction of false signals associated with background matrix interferences. However, the blank-correction procedures reported in published literature are highly inconsistent. We investigated the influence of using different types of blanks on the final calculated activity/inhibition
Conclusion
The choice of blanks and blank-correction methods contribute to the variability of assay results and the likelihood of underestimating the enzyme inhibitory potential of a test sample. This highlights the importance of standardising the use of blanks and the reporting of blank-correction procedures in published studies in order to ensure the accuracy and reproducibility of results, and avoid overlooked opportunities in drug discovery research due to inadvertent underestimation of enzyme inhibitory potential of test samples resulting from unsuitable blank-correction. Based on our assessments, we recommend method six [RD - (Su + SaB)] as a suitable method for blank-correction of raw data in enzyme assays.
Results
In the three assays analysed here, using only a buffer blank underestimated the enzyme inhibitory potential of the test sample. In the absorbance-based α-glucosidase assay, enzyme inhibition was underestimated when a sample blank was omitted for the coloured plant extracts. Similarly, in the fluorescence-based α-amylase and lipase assays, enzyme inhibition was underestimated when a substrate blank was omitted. For all three assays, method six [Raw Data - (Substrate + Sample Blank)] enabled the correction of interferences due to the buffer, sample, and substrate without double-blanking, and eliminated the need to add substrate to each sample blank.