# Impacts of Tween-20, Glycerol, and Trehalose on Hyaluronidase Activity: Insights from Microscale Thermophoresis and Capillary Electrophoresis

## TL;DR

This study examines how common additives like Tween-20, glycerol, and trehalose affect hyaluronidase activity and enzyme behavior using advanced analytical techniques.

## Contribution

The study introduces a novel labeling method for hyaluronidase compatible with MST and systematically evaluates the impact of three additives on enzyme activity.

## Key findings

- Tween-20 had no effect on hyaluronidase activity or MST signals at concentrations between 0.005 and 0.05%.
- Glycerol reduced hyaluronidase activity at concentrations above 2%.
- Trehalose enhanced catalytic activity even at low concentrations and affected enzyme fluorescence.

## Abstract

Additives such as surfactants (Tween-20) and cryoprotectants (glycerol and trehalose) are often used in enzymatic assays to improve the quality and long-term stabilization of proteins. However, these additives can affect the enzymatic activity and the enzyme’s affinity for active compounds, such as inhibitors, and must be considered during assay design since a slight shift in enzyme behavior may compromise the reliability of the results. In this study, the effects of Tween-20, glycerol, and trehalose on hyaluronidase (Hyal) were systematically evaluated by assessing their influence both directly—through microscale thermophoresis (MST) signals of the labeled enzyme (Hyal*)—and indirectly, by monitoring the formation of the final product of the degradation of hyaluronic acid, tetrasaccharide (Tet), using capillary electrophoresis (CE/UV). Hyal was labeled for the first time with ATTO-647 NHS ester, a commercial dye compatible with MST. Efficient labeling was achieved in a phosphate-based buffer without loss of catalytic activity. Tween-20 showed no impact on MST signals nor on enzymatic performance when used between 0.005 and 0.05% (v/v). Glycerol also did not interfere with MST measurements; however, it significantly reduced catalytic activity at concentrations above 2% (v/v). Trehalose affected Hyal* fluorescence in a concentration-dependent manner and enhanced catalytic activity even at 0.02% (v/v).