Fluorescence Analysis Was Used To Detect The Activity Of Phospholipase C And Dnase I

Various types of hydrolases are of vital importance for medical diagnosis,drug research,and industrial production.Through the analysis of the activities and inhibitors of these hydrolase,we can get the biological information of them,so that it can play better in various fields.Several assays have been employed for the determination of such hydrolases activity,and each has its advantages and disadvantage.Fluorescence analysis is a simple,rapid and sensitive detection method.In this paper,the activities of phospholipase C and deoxyribonuclease I were studied.Phospholipases C(PLCs)are phosphodiesterases that hydrolyse the phosphodiester bond between glycerol and phosphate in glycerophospholipids,yielding diacylglycerol(DAG)and a phosphoryl-base,e.g.phosphorylcholine,or phosphorylinositol.Phospholipase C has been widely used in food processing,oil refining,cosmetics and pharmaceutical production.The ability to quantitatively monitor PLC catalytic activity is important.Egg-yolk agar plate medium,p-nitrophenylphosphorylcholine,Inorganic Phosphate Assay,thin layer chromatography,radiometric,titration,turbidimetric or fluorescence detection have been employed for the determination of PLC activity.Deoxyribonuclease I(DNase I)is an important enzyme that cleaves both double-stranded and single-stranded DNA at their phosphate backbone.DNase I is a useful biomarker.Previous studies have shown that patients with prostate cancer and systemic lupus erythematosus exhibit reduced DNase I activity,and patients with myocardial infarction exhibit increased DNase I activity.A number of traditional methods have been used to measure DNase I activity,such as gel-based electrophoresis,ELISA,colorimetric method and single radial enzyme diffusion assay etc.However,these methods have some drawbacks,such as ELISA requires multiple washing steps,or on a single radial enzyme diffusion assay,which requires a long digestion time and an expensive fluorescence detection system.Therefore,it is essential to establish a simple,rapid and convenient method for detecting DNase I activity.In this paper,fluorescent labeled liposomes were used as probes to detect the activity of PLC.The activity of DNase I was detected by fluorescently labeled double-stranded DNA as probe.Respectively design a simple,rapid,sensitive and specific method to monitor the activity and inhibition of PLC and DNase I.The paper mainly consists of the following three chapters:Chapter 1 consists of three parts.The first part is an overview of phospholipase C.This part introduced phospholipase and its substrate,phospholipase C and its detection method.The second part is an overview of the Dnase I,which mainly introduce the nuclease,Dnase I and its detection method.The third part mainly expounds the background,research purpose and research content of this thesis.Chapter 2,a method was established to detect the activity of PLC,which was based on the self-assembly monolayer fluorescent liposomes as probes.The probe was a monolayer fluorescent labeled liposome that were formed from the self-assembly of dipalmitoylphosphatidylcholine(DPPC)and fluorescently labeled phospholipid(Liss Rhod PE).In this probe,the fluorescence quenching of Rhodamine B occured,and the fluorescence intensity decreased.The addition of PLC led to the degradation of liposomes and the release of Rhodamine B,which reduced the degree of self quenching and produced strong fluorescence signals.The activity of PLC could be detected by the change of fluorescence intensity.The fluorescent intensity is proportional to the concentration of PLC in the range of 5 to 300 U/L with a low detection limit of 2 U/L,which indicates the probe provide simple and high sensitivity for phospholipase C detection.In addition to determining the activity of phospholipase C,the probe can be used to study PLC inhibitors.Chapter 3,a fluorescence polarization method using fluorescence marked double stranded DNA as probe for PLC detection was developed.The probe was a FAM labeled double stranded DNA containing 30bp bases.For the probe,the fluorescence polarization value of FAM-dsDNA is relatively large,which is due to the large molecular weight.The addition of DNase I led to the hydrolysis of double stranded DNA,which reduced the molecular weight of probe and produced the change in fluorescence polarization value.The fluorescence polarization responses based on FAM-dsDNA could be used for the detection of DNase I.The fluorescence polarization value was proportional to the concentration of DNase I in the range of 10 to 500 U/L with a low detection limit of 6 U/L.This work demonstrated that the developed method can not only provide promising platform for monitoring DNase I,but also be used to study DNase I inhibitors.