Microscale thermophoresis for studying protein-small molecule affinity: Application to hyaluronidase

Abstract

Studying the binding affinity between large biomolecules (i.e. enzyme) and small molecules (molecular weight less than 1000 Da) is an extremely challenging task when using conventional techniques because of the difficulty associated with the detection of the minuscule variations caused by the binding. Microscale thermophoresis (MST) is a sensitive novel emerging biophysical tool allowing a rapid and accurate determination of the dissociation constant (Kd) of enzyme - small molecule system. For the first time, a systematic MST study was carried out to evaluate Kd between hyaluronidase (Hyal), a model enzyme, and two small flavonoid compounds known as potent Hyal's inhibitors, epigallocatechin gallate (EGCG) and apigenin-7-glucoside. In the optimized conditions, hyaluronidase fluorescent labelling (Hyal*) was achieved by direct labelling with RED-NHS NT647 dye using a phosphate ammonia buffer (20 mM NaH2PO4 + 50 mM NH3) + 77 mM NaCl at pH 6.6. This latter ensured a high labelling efficiency while preventing Hyal* precipitation (pI of hyaluronidase is equal to 8.6). Pre-capillary CE/UV enzymatic assays of Hyal* demonstrated the preservation of its enzymatic activity even though the tested concentration was very low (50-fold times lower than control enzymatic assay). Fluorescence signal intensity and shape as well as MST traces have shown that phosphate ammonia buffer required the addition of surfactant, Tween-20 at 0.05%, to efficiently improve the protein solubility and limited aggregate formation using standard uncoated capillaries. MST results revealed the absence of affinity between Hyal* and the tested flavonoids regardless of the incubation time and the tested inhibitory concentration range unless the hyaluronidase substrate, hyaluronic acid (HA) is added to the preparation. Kd values were estimated to be 163 µM for Hyal*/HA/EGCG and 157 µM for Hyal*/HA/apigenin-7-glucoside. The repeatability of the assays was excellent as confirmed by the very low standard deviation on Kd values.