A General Fluorescence Turn‐On Sulfotransferase Assay through the Detection of Free Phosphate Ions by Using A Calcein/Ce³⁺ System

Abstract

Biomolecule sulfation catalyzed by sulfotransferases (STases) plays a profound role in numerous biological processes. However, the exact biofunctions of the sulfation modifications still remain largely unknown partially because STases are difficult to assay. Most of the existing STase assays are targeted to discriminate the property changes of the sulfated substrate against the un‐sulfated ones, which typically suffer from several drawbacks due to the labile sulfated products, inhibitory effect of the 3'‐phosphoadenosine‐5'‐phosphate (PAP) by‐product as well as the lack of generality. To address such issues, herein, we have developed a versatile and general fluorescence turn‐on strategy for assaying STase activities. Unlike traditional STase assays, this work is targeted to measuring the phosphate ion (Pi) released from the enzymatic degradation of PAP by‐product during the sulfation reaction, which can lead to fluorescence enhancement of a calcein/Ce3+ system. Since PAP is the common by‐product of all kinds of STase reactions, this assay is universally applicable to the varying kinds of STases. In this regard, the intrinsic instability of the sulfated substrates will no longer influence the assay accuracy. Furthermore, the inhibitory effect of PAP on the STase reaction, which is common in traditional STase assays, are effectively removed in this study, thereby allowing more accurate determination of the enzyme activity.