Informace o publikaci
Caspase 3 chemiluminescence activity determination in apoptotic cells and design of caspase-3 sensor
| Autoři | |
|---|---|
| Rok publikování | 2012 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development of tumor or autoimmune disorders. In some instances knowledges of this pathway can be used for inhibit or prevent cancer development and other diseases. One of the earliest and most consistent observed features of apoptosis is the induction of a series of cytosolic proteases - caspases. Active caspases cleave numerous intracellular proteins and contribute to apoptotic cell death. Caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp on their substrates and hydrolyze peptide bonds after aspartic acid residues. It is known from literature that in one apoptotic cell about 1.6x10(-19) mol of caspase can be activated. Various techniques for the determination of caspase 3 in free cells or tissue samples are commercially available. Caspase 3 activity is usually assayed by a chemiluminescence reaction. The best commercial methods reach the limit o detection higher than 1 pg of caspase 3. We have developed a special device for the determination of caspases activity in apoptotic cells. The activity of caspase 3 is determined by the system based on Luciferin/Luciferase chemiluminescence (CL) reaction. The luciferin modified with tetrapeptide sequence (DEVD) specific to the recognition for caspase 3 is cleaved to form free luciferin, which immediately reacts with luciferase to produce light. Laboratory built luminometer is based on Sens-Tech P25 USB photomultiplier tube (PMT) working at digital photon counting mode. The sample is placed in a highly polished cell made of stainless steel. The sensitivity of the device proved to be by three orders of magnitude better than the commercially available technologies. The objective of this paper is the design of a fluorescent sensor based on Forster resonance energy transfer (FRET) for the determination of caspase 3 in cell nucleus or cytoplasm as a result of the apoptotic process. We designed two fluorescent caspase 3 sensors. |