Fluorescence Analysis Of Protein Kinase And Phosphatase Activity Based On Metal Organic Framework

Protein is the executant of life activities.Enzyme proteins with catalytic function are closely related to the growth and development of human cells,metabolism,and other life activities.Reversible phosphorylation regulated by protein kinase and protein phosphatase plays an important role in signal transduction.The abnormal activity of two enzymes will lead to a variety of diseases in the human body.The analysis and detection of protein kinase and protein phosphatase will greatly promote the related basic research and drug research targeting the corresponding enzymes.Therefore,the development of detection methods with simple operation,low cost and high sensitivity has important research significance and broad application prospects.We studied a fluorescence method for activity analysis and inhibitor screening of protein kinase A?PKA?based on titanium-based metal organic framework?MOF,Ti-MIL125-NH₂?.This method took advantage of the highly specific recognition of phosphate groups by Ti-MIL125-NH₂.In the presence of PKA,the fluorophore-labeled substrate was phosphorylated,and then the generated phosphopeptide could specifically bind to the titanium sites of Ti-MIL125-NH₂.This resulted in fluorescence enrichment,which could be efficiently detected by spectrofluorophotometer.Under optimal conditions,including acetonitrile proportion,ATP concentration,elution condition,and co-incubation time of material with phosphorylation system,the method presented a linear relationship in the experimental range of 0.00005-0.01 U?L^-1?R²=0.992 6?,and the limit of detection was 0.00003 U?L^-1?3?,n=11?.Furthermore,protein kinase Akt1 was tested to verify the universality of this method.Correspondingly,the linear range was 0.0005-0.05?g mL^-1?R²=0.989 7?.The method was also successfully used in MCF-7 cell lysates for kinase activity analysis.These results show that the proposed high sensitivity fluorescence method based on MOF is a relatively comprehensive tool for detecting PKA activity and has potential application value in kinase-related basic research and kinase targeting drug research.We also studied a fluorescence method for activity analysis and inhibitor screening of protein tyrosine phosphatase 1B?PTP1B?based on titanium-based metal organic framework?MOF,Ti-MIL125-NH₂?.This method was based on the specific recognition of phosphate groups by Ti-MIL125-NH₂.In the absence of PTP1B or adding PTP1B inhibitor,the phosphorylated substrate in experimental system would be fully captured by the titanium site of Ti-MIL125-NH₂,and the final fluorescence intensity was the highest.When PTP1B was added,the phosphate group of the substrate would be specifically cut off by PTP1B,and the final fluorescence intensity would gradually decrease with the increase of PTP1B concentration.After experimental optimization,including reducing non-specific adsorption,L-aspartic acid concentration,phosphorylated substrate concentration,Ti-MIL125-NH₂ concentration,and elution condition,the method had a linear relationship in the range of 0.00006-0.002?g mL^-1?R²=0.993 7?,and the limit of detection was 0.00003?g mL^-1?3?,n=11?.At the same time,the inhibitor screening experiment also achieved satisfactory results.In addition,fetal bovine serum was used to simulate the complex environment of biological system.The recovery rate of sample was in the range of 91.9-103.7%,and the corresponding RSD was in the range of0.72-1.21%.These results show that the proposed method is an effective tool for the study of PTP1B,which shows great potential in the basic research of PTP1B and related drug research.