A Novel Fluorescence Derivation For Determining The Activity Of Key Enzyme In Apoptosis

Apoptosis is a basis biological phenomena in cells, playing an important role of removing unnecessary or abnormal cells in organisma multicellularis.It acts important roles for evolution of organisms, stability of internal environment and development of multiple systems. Although the detail mechanism of apoptosis is not very clear, caspase(cysteine aspartate-specific protease) was determined to acts indispensable roles in apoptosis. Actually, the process of apoptosis is cascade amplification of caspase irreversibly hydrolyzing substrates.Up to date, at least 14 caspases were found. Homology between caspases molecules is very high, and the structure is similar that all caspases are cysteine family protease.Caspases are key roles in apoptosis which is very important for determining the progress of apoptosis. Recently, researchers of our lab found that peptides are able to make condensation reaction with catechol and sodium metameriodate at pH 7.5,which produces a single fluorescent product with an excitation wavelength of 390nm and an emission wavelength of 490nm.It is more important that fluorescent product is not produced in carbohydrate (such as free amino acid, glucose and galactose), polyamine (such as spermine) and DNA base (such as adenine, guanine, thymine and cytosine) at the same condition.Thus, a novel and selective fluorescence reaction appears to be a promising strategy due to its high selectivity for the detection of active caspases and provides a convenient alternative for many applications.In the section one, conditions for fluorescence derivation of oligopeptide were optimized.On the basis of optimum conditions of fluorescence reaction, the analysis conditions of high performance liquid chromatography were optimized. Optimum fluorescence derivation system consisted of 100μ-L of test solution,100μL of catechol(10 mmol/L),50μL of sodium periodate(12 mmol/L) and 50μL of sodium borate (300 mmol/L,pH 7.0), and the mixture was heated at 120℃for 10 min. Optimum chromatographic conditions was column of CAPCELL PAK C18 MGS5 (5μm,150 mm×4.6 mm I.D.).The mobile phase consisted of a mixture of sodium borate (250 mmol/L,pH7.5,20%) and water (80-0%) and methanol (0-80%) at gradient elution.The elution time was 40 min at a flow rate of 1.0 mL/min at 30℃. A fluorescence detector was set at an excitation wavelength of 390nm and an emission wavelength of 490nm.In the section two, leu-enkephalin was determined by a novel and selective fluorescence reaction and compared to ultraviolet detection and OPA derivation. Leu-enkephalin is a very important endogenous peptide. The sequence of amino acids was Tyr-Gly-Gly-Phe-Leu.In vitro study, the retention time was 25 min.There was good linear relationships (r=0.9999) within the range of 0.5～50.0 mg/L.The lower limit of quantification was 0.25 mg/L.In vivo study, the drug-free serum did not interfere with the determination of leu-enkephalin.There was good linear relationships (r=0.9998) within the range of 0.5～50.0 mg/L. The lower limit of quantification was 0.5 mg/L.There were many endogenous interfering substances in blood plasma. Ultraviolet detection and OPA derivation could not efficiently detect the concentration of leu-enkephalin.Section three is the most important part of our research, in which a novel fluorescence reaction can be used to detect the activated caspase 3 and 8 in Hela cells and A549 cells undergoing apoptosis.Hypothermia-ultraviolet (UV) irradiation, mitomycin C (MMC) and camptothecin were used to induce apoptosis in each cell line, respectively. It was designed to add serin which is hydrophilic amino acid to N-terminal of special recognition sequence, which is a part of enzyme substrate. Serin could raise the water solubility of substrate.The extended sequence could increase the specificity of substrate. Two oligopeptides LG and ALG were added to C-terminal of recognition sequence, which were proved to have high derivation efficiency. In this research, the commonly used kits to detect caspase 3 and 8 activation were used as the control of fluorescence reaction.Our data have demonstrated that the ratio of relative activation is similar between these two methods, which reveals that the novel fluorescence reaction is reliable.Therefore, the higher selectivity of fluorescence reaction indicated by recombinant caspase activation assays makes it a highly reliable method for the detection of active caspases.