Construction of a Turn Off-On-Off Fluorescent System Based on Competitive Coordination of Cu2+ between 6,7-Dihydroxycoumarin and Pyrophosphate Ion for Sensitive Assay of Pyrophosphatase Activity.

Lingzhi Zhao,Chunye Liu,Chenxiao Zhang,Liu Zhao,Yanqing Miao

doi:10.1155/2016/4306838

Abstract

The detection of pyrophosphatase (PPase) activity is of great significance in diagnosing diseases and understanding the function of PPase-related biological events. This study constructed a turn off-on-off fluorescent system for PPase activity assay based on PPase-regulated competitive coordination of Cu2+ between a water-soluble fluorescent probe 6,7-dihydroxycoumarin (DHC) and pyrophosphate (PPi). The probe DHC can coordinate with Cu2+ and consequently display on-off type fluorescence response. Furthermore, the in situ formed nonfluorescent Cu2+-DHC complex can act as an effective off-on type fluorescent probe for sensing PPi due to the higher coordination reactivity between Cu2+ and PPi than that between Cu2+ and DHC. The subsequent addition of PPase to the mixture containing Cu2+, DHC, and PPi leads to the fluorescence requenching of the system again (an off state) because PPase catalyzes the hydrolysis of PPi into orthophosphate in the reaction system. Under the optimum conditions, the decrease of the fluorescence intensity of DHC-Cu2+-PPi system was linear with the increase of the PPase activity in the range from 0.1 to 0.3 U. The detection limit was down to 0.028 U PPase (S/N = 3). Moreover, the as-established system was also applied to evaluate PPase inhibitor. This study offers a simple yet effective method for the detection of PPase activity.

Highlights

As one kind of the most important biological anions, pyrophosphate (P2O74−, PPi) is one of the by-products of adenosine triphosphate (ATP) hydrolysis and can be used as a potential biomarker for the clinic diagnosis and therapy of familial chondrocalcinosis or calcium pyrophosphate crystal deposition disease [1,2,3,4]
The addition of PPase to the Cu2+-PPi complex containing DHC obviously leads to the fluorescence quenching again (Figure 1(b), green curve) because Pi, which is produced from PPasecatalyzed hydrolysis of PPi, cannot chelate Cu2+ to form the complex
The addition of PPase to the Cu2+-PPi complex containing DHC obviously leads to the fluorescence quenching again because Pi, which is produced from PPase-catalyzed hydrolysis of PPi, cannot chelate Cu2+

Summary

Introduction

As one kind of the most important biological anions, pyrophosphate (P2O74−, PPi) is one of the by-products of adenosine triphosphate (ATP) hydrolysis and can be used as a potential biomarker for the clinic diagnosis and therapy of familial chondrocalcinosis or calcium pyrophosphate crystal deposition disease [1,2,3,4]. Various analytical methods and techniques have been developed for PPase activity assay, such as radioactive labelbased chromatography, colorimetry assay, and fluorescence methods a [10,11,12,13,14,15,16] Most of these methods are based on monitoring the change in the amount of the PPi substrate or Pi product. An effective fluorescence method for highly sensitive assay of PPase activity has been developed in the physiological buffer, which utilizes a simple competitive coordination reactivity of Cu2+ between DHC and PPi. initially, the strong fluorescence emission peak of DHC was observed at 475 nm. To the best of our knowledge, the as-designed turn off-on-off fluorescence sensing system is convenient without complicated protocols and exhibits excellent PPi sensing selectivity over other anions and structural analogues and excellent performances for sensitive assay of PPase activity in the physiological buffer compared to that of the existing methods, which paves a fluorescence route to the fast and simple clinic detection of PPi, the hydrolytic activity of PPases, and screening of PPase inhibitors

Experimental

Results and Discussion

Conclusion