We investigate the feasibility of using rapid mixing and chemical relaxation by temperature jump to detect intermediates and their reaction kinetics for the telomerase catalyzed reaction on the cell nucleus. We consider only (desoxyribo-) nucleotide additions to the telomerase–telomere complex and the translocation along the extended single-stranded DNA chain. Since telomerase is not sufficiently available to conduct ordinary mixing experiments, we use 2-photon laser absorption microscopy in a volume of less than 1 m3 (nm3-range), observing fluorescence changes due to nucleotide-binding with attached fluorophors. We distinguish between experiments on the surface of a cell nucleus and on the surface of an object glass and use both stopped flow and constant flow. Two models are considered, one incorporating a fast isomerization of the initial telomere–telomerase complex. We simulate the reactions numerically. The differences between stopped flow and constant flow are minimal. Diffusion of reactants restricts the time range available for stopped-flow experiments, so constant-flow experiments are preferred. We show that most reaction steps can be individually investigated by judicious selection of fluorophors bound to one of the three nucleotides. This mode also permits distinction between the two models. Interference by single-molecule kinetics implies that some experiments must be repeated to obtain averaged data.
- CHEMICAL RELAXATION,
- RAPID MIXING,
- TWO-PHOTON ABSORPTION
Available at: http://works.bepress.com/tjalling_ypma/12/