Viktor Boris

Classic fluorometrically-based kinetics assays have proved indispensable in the quest to elucidate the catalytic function of the human histidine nucleotide binding protein (hHINT1). As researchers continue to probe this enzyme’s role in vivo, like its involvement modulating how opioids function in the central nervous system, improving existing in vitro fluorometric assays of hHINT1 has become advantageous. Here we demonstrate the successful application of a high throughout take on a classic hHINT1 competitive inhibition assay to aid in the study of hHINT1’s catalytic function which has traditionally been a slow process. The assay was performed by preparing a 96 well microplate with eight different concentrations of the hHINT1 enzyme substrate tryptamine 5’- adenosine monophosphoramidate. The plate reader was programmed to quickly take a sample which was injected by hand using a micropipette, shake the sample, and record a fluorescent kinetic trace for one minute. The rates of reaction at varying substrate concentrations were then plotted to construct a Michalis-Menten plot. This was repeated with different concentrations of a published competitive inhibitor of hHINT1. This method was able to determine the inhibition constant Ki for the inhibitor which was confirmed to be accurate in comparison with published isothermal titration calorimetry data. This study demonstrates the efficacy of this new high-throughput tool for studying hHint1 enzymology. More generally, this study also demonstrated the power of automation in experimental design for the study of enzymes involved in medically relevant pathologies like hHINT1 and pain.