Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Steady state fluorescence and fluorescence anisotropy studies of ligand binding and kinetics
Abstract
A thermostated fluorescence micro-stopped flow apparatus was constructed for measuring changes in total fluorescence and fluorescence anisotropy in a reaction volume of 0.04 ml. Precise measurements of fluorescence anisotropy were obtained using laser excitation modulated by a variable retardance photoelastic modulator. The temperature was regulated by a Peltier effect heater-cooler at 0.01$\sp\circ$C over 30 minutes. Using a gold mirror as a reference, the intrinsic precision of the anisotropy measurements was found to be 6.5 $\times$ 10$\sp{-5}$ based on 10 measurements. Data were acquired and stored both in a general purpose interface for the IBM PC-XT computer and in a Nicolet digital oscilloscope interfaced to the computer. The computer programs for data acquisition and control of the anisotropy apparatus, the SLM lifetime apparatus, and the Peltier device are given in the dissertation, together with all electronic modules developed in the laboratory. It was discovered that Guanine Nucleotide Exchange Factor (GEF) contained bound NADPH that appeared to function as an allosteric regulator in the initiation of protein synthesis. Fluorescent ribonucleotides were synthesized with DANSYL bound to the 2$\sp\prime(3\sp\prime$) site on the ribose. These compounds were used to study binding to protein initiation factor eIF-2 and to Histone H1. Fluorescent probes based on fluorescein were also synthesized. Their utility depended on discovering an effective means for preventing photobleaching. It was discovered that ascorbate was an effective agent for preventing photobleaching, but in higher concentrations, both static and dynamic quenching of fluorescence occurred. A sensitive assay was developed for the protein Avidin based on a fluorescein labeled biocytin. This allowed 10 nM avidin to be detected. The kinetics of the binding of labeled biocytin to avidin were also determined. The association rate constant was 5.5 $\times$ 10$\sp{+6}$ M$\sp{-1}$s$\sp{-1}$ at 20$\sp\circ$C, with an activation energy of 11.6 kcal/mole, clearly not characteristic of diffusion limited association.
Subject Area
Biochemistry|Biophysics
Recommended Citation
Mueser, Timothy C, "Steady state fluorescence and fluorescence anisotropy studies of ligand binding and kinetics" (1989). ETD collection for University of Nebraska-Lincoln. AAI8925251.
https://digitalcommons.unl.edu/dissertations/AAI8925251