Quantification of β-CD Modification on Rluc

The amount of β-CD modified on a Rluc was determined by a sulfuric acid-phenol assay with slight modification [18 (link)]. The detailed results can be found from the supporting documents. In a typical test, a 30 μL of ice-colded glucose standard samples or β-CD-Rluc solution was mixed with 100 μL of concentrated sulfuric acid in microplate wells. 20 μL of aqueous 5% phenol solution was then added into the well. The plate was floated uncovered on near boiling (>90 °C) water bath for 5 min for color development, followed by cooling on ice for another 5 min. A microplate reader (Tecan M200 multimode microplate reader) was used to collect the absorbance of each well at 490 nm. The concentration of standard solution was plotted with the absorbance of each solution. The molar concentration of β-CD was thus calculated. The number of β-CD per protein was obtained by dividing the molar concentration of β-CD over the molar concentration of protein Rluc.