Monitoring Of Proteinase Activation In Cell Apoptosis By Capillary Electrophoresis

Capillary electrophoresis (CE) is a micro separation technique for analysis, with many advantages of high sensitivity, high resolution, high speed, and minute sample cost. It has been applied in many fields, such as biology, medicine, pharmaceutics, environment and food, especially in the field of life sciences. Protein science is now one of focuses of life sciences, in which much attention has been paid to the activity of enzyme. Many important diseases are involved with abnormal activity of the enzymes.Caspase is a family of death-specific proteinase, which functions in two major signaling pathways, surface death receptors mediated and mitochondria initiated pathways. No matter which pathway the apoptosis is initiated through, Caspase-3 should be activated as a symbol at the protein level. But the activation mechanism of Caspase-2 is still unclear. In this thesis, CE was demonstrated as a novel means to monitor the activations of Caspase-3 and Caspase-2 in HeLa cell apoptosis. Compared with the reaction rate of Caspase-3, the reaction rate of Caspase-2 had a distinct jump. CE had highly potential applicability for the screen and assessment of anti-cancer drug.To monitor the activation of Caspase-3 during cell apoptosis, a unique fluorescent probe, ECFP-DEVD-DsRed, was engineered in HeLa cells as a substrate of proteinase Caspase-3. Molecular imaging with in vivo fluorescence resonance energy transfer was conducted to evaluate the stability and reliability of this fusion protein probe expressed in HeLa cells. With treatment of a certain dose of cisplation of HeLa cells, apoptosis was initiated, and then Caspase-3 was activated that could specifically recognize the DEVD site and would subsequently cleave the constructed fluorescent probe into two pieces of protein fragments. Analyzing the cell lysates with CE in vitro at a series of time points quantitatively gave a clear description of activation process of Caspase-3 in cell apoptosis. Several parameters that influenced CE separation quality were optimized, such as buffer type, pH value, concentration and applied voltage. The employment of two color fluorescent proteins significantly simplified the separation and identification of residues of the cleavage reaction.To characterize dynamics of Caspase-2 activation in cell apoptosis, a fusion protein, ECFP-VDVAD-DsRed, was specially designed and expressed in HeLa cells as the substrate of proteinase Caspase-2. In this substrate, the sequence VDVAD could be specifically recognized and cleaved by Caspase-2 as soon as its activation was initiated with treatment of a certain dose of cisplatin to HeLa cells, which led to a break of the substrate into two fragments. Analyses of the cell lysates using CE in a time course of the apoptosis illustrated the dynamics of Caspase-2 activation. It showed that the employment of fusion fluorescent protein greatly facilitated both CE separation and identification of the analytes. This result from cell colony by CE was compared with that from single cell achieved by optical imaging.