Electron paramagnetic resonance studies of spin-labelled ethidium bromide DNA interactions

Spin-Labelled Ethidium Bromide (SLEB) was prepared in order to study its interactions with natural DNA in the form of fibres . The technique of electron paramagnetic resonance was used in this thesis. Knowledge of the conformational transition pathway of natural DNA for given counterion concentration as a function of relative humidity was utilised in the study of effect DNA confomation on the binding of SLEB. To aid interpretation of the results the relevant background material was reviewed. In order to attempt to extract geometric information on binding computer ERR lineshape simulations were used. To facilitate this a microcomputer spectrometer control system was designed and implemented. This allowed spectra to be acquired in digital form and transfered to the mainframe computer. Two schemes for magnetic field control were investigated, one based on a commercial NMR magnetometer, and a superior pulsed NMR field locking magnetometer developed in this laboratory. In order to obtain lineshapes undistorted by dipolar broadening it is advantageous to use fibres with a high phosphate to drug ratio (P/D), however spectrometer sensitivity becomes a limiting factor. A review of noise in spectrometer systems is included. The use of a microwave low-noise preamplifer to reduce the system noise figure was investigated. An attempt to construct a loop-gap resonator was made and justified theoretically. A 35GHz spectrometer was constructed and a cavity designed and built to allow the humidity to be varied. The system was made compatible with the control system. Spectra recorded and simulated at this frequency should help confirm those obtained at 9GHz. The results obtained from P/D«70 fibres with a 0.5mM NaCl concentration show the SLEB is in a disordered state from 33% to 75% relative humidity. Spectral changes occur in the range 75% to 98% consistant with intercalation. In this humidity range a transition to the B-form is expected

21st Rocky Mountain Conference on Analytical Chemistry

Abstracts and meeting program from the 21st annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Rocky Mountain Section of the Society for Applied Spectroscopy and the Rocky Mountain Chromatography Discussion Group. Held in Denver, Colorado, July 30 - August 1, 1979

COMPUTER APPLICATIONS IN QUANTITATIVE EPR SPECTROSCOPY OF METALLOPROTEINS (FERRITIN, APOFERRITIN, IRON)

This dissertation examines the use of computers in quantitative EPR spectroscopy. The computers used ranged from hand held calculators to large main frame systems. Applications discussed are protein assay calculations, an EPR minicomputer interface and software system and the modification of an existing EPR simulation program to include corrections for strains in the g and A tensors. The modification permits more accurate linewidth simulation for lines with large M(,I) values. The computer interface and software allows for the collection of EPR spectra, which can then be stored, scaled, added, subtracted (for comparison) and double integrated. The program enhances weak signals by signal averaging. Double integration was used to assist in the study of early iron binding in horse spleen apoferritin. Iron(II) was added to apoferritin followed by oxidation by a variety of methods. In all cases an iron(III) EPR signal was observed at g\u27 = 4.3 which was attributed to mononuclear Fe(III) bound to the protein; this signal increased until 0.5 equivalent per subunit of added iron. In another experiment increasing amounts of Tb(III) were added to apoferritin solutions. Subsequent addition of 0.5 equivalent of iron(II) per subunit resulted in an Fe(III) signal that decreased as a function of added Tb(III). It was also found that ultracentrifugation of commercial ferritin yields a light, low iron content, fraction which showed a majority of the iron signal intensity relative to the heavy, iron rich, fraction. These results suggest that iron core starts to form at an initial binding site that lies between two adjacent subunits resulting in a 0.5 equivalent of binding site per subunit and that this site also serves as the nucleus of core formation within the ferritin molecule. As the core grows beyond 0.5 equivalents per subunit more of the mononuclear sites are converted into growing core. At 0.5 equivalents per subunit double integration shows that only 20% of the added iron is EPR active suggesting a majority of the added iron is present as polymeric iron (core) species

Application Of Digital Signal Analysis, Mass Data Acquisition and Processing Techniques, and Automated Experiment Protocols to the Study of Cardiac Cell Membrane Electrophysiology, with Mathematical Modeling

Traditional methods of collecting, analyzing and storing data from cardiac cell membrane electrophysiology experiments have become increasingly cumbersome and unwieldy as experimental protocols have become more sophisticated and complex. A global approach to collecting, analyzing, refining and storing electrophysiologic data, as well as a new approach to mathematical modeling of cell membrane single ion channel kinetics, was developed. This utilizes a comprehensive microcomputer based system of software with specialized analog and digital electronics for data acquisition, analysis and archiving. Unique discrete signal processing techniques for characterizing the electronic recording system, including specialized hardware and software adapted for minimizing distortions in biosignal recordings, are discussed in detail

Spectroscopic Studies of Bacterial Iron-Sulfur Proteins (Electron Paramagnetic Resonance, Magnetic Circular Dichroism).

Iron-sulfur proteins play a vital role in metabolism; mediating such life-sustaining processes as aerobic and anaerobic respiration, nitrogen fixation, and photosynthesis. This work employs low temperature magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), and UV-visible spectroscopy to characterize the iron-sulfur clusters of the following bacterial proteins: Azotobacter vinelandii ferredoxinI, Thermus thermophilus ferredoxin, Escherichia coli nitrate reductase and the rubredoxin and ferredoxin from Clostridium pasteurianum. Novel 3Fe-xS clusters were identified in A. vinelandii FdI, T. thermophilus Fd, ferricyanide treated C. pasteurianum Fd, and E. coli nitrate reductase. The uniformity of the magnetic and electronic properties of these clusters in both the oxidized and the reduced states indicates a common iron-sulfur core structure for this type of cluster. E. coli nitrate reductase is the first example of an active enzyme which contains a 3Fe-xS cluster. This argues against the currently prevailing hypothesis that all such clusters are isolation artifacts. This work has also developed the potential of low temperature MCD for the detection and characterization of iron-sulfur clusters in multicluster enzymes. Studies on the well-characterized Clostridial proteins demonstrated that MCD magnetization curves provide a selective method of obtaining ground state g-values, spin states, and estimations of the polarizations of the electronic transitions for randomly oriented samples. Moreover, zero field splitting parameters were obtained by a detailed study of the temperature dependence of individual MCD transitions. This has led to a more detailed understanding of the complex Kramers\u27 and non-Kramers\u27 ground states exhibited by reduced 3Fe-xS clusters (S = 2) and oxidized and reduced rubredoxin (S = 5/2 and S = 2 respectively)

An in situ study of cytochrome bd: a ubiquinol oxidase of 'Escherichia coli'

An in situ study was conducted of the cytochrome bd ubiquinol:oxygen oxidoreductase (cytochrome b558-b595-d) of Escherichia coli grown anaerobically on glycerol with fumarate as respiratory oxidant. Nitrite reacts with and is reduced by the oxidase, resulting in the formation of NO adducts to haems b595 and d. The kinetics of formation of these species indicate that the affinity of haem d for nitrite is higher than that of haem b595. CO also binds to the oxidase, resulting in the formation of CO adducts to haems d and b595. Binding titrations indicate that the affinity of haem d for CO is higher than that of haem b595. The steady state kinetics of the oxidase reaction in the presence of nitrite or CO are cooperative with respect to oxygen binding, suggesting that both haems d and b595 are involved in the reduction of oxygen. E.p.r. studies of the ferric oxidase indicate the presence of two high spin haem signals, one rhombic and one axial, which are assigned to haems b595 and d, respectively. These signals titrate potentiometrically with midpoint potentials similar to those published on the basis of optically followed titrations for haems b595 and d. The high spin ferric haem spectra are affected by oxygen, CO, cyanide, and pH. A low spin ferric haem signal is observed at g=3.3 and is assigned to haem b558. The sidedness with respect to the cytoplasmic membrane of ligand binding haems of the oxidase was determined by investigating the effect of the exogenous paramagnetic probe DyEDTA on the e.p.r. properties of the ferrous haems d-NO and b595-NO. These haems are located towards the inner aspect of the cytoplasmic membrane at around 8 and 12A° below the surface, respectively. Overall, the data supports a functional model for cytochrome bd with two oxygen binding sites, haems d and b595, forming the binuclear centre of the oxidase reaction. Possible mechanisms of this reaction are discussed

Hampton University/American Society for Engineering Education/NASA Summer Faculty Fellowship Program 1986

Since 1964, the National Aeronautics and Space Administration (NASA) has supported a program of summer faculty fellowships for engineering and science educators. In a series of collaborations between NASA research and development centers and nearby universities, engineering faculty members spend 10 or 11 weeks working with professional peers on research. The Summer Faculty Program Committee of the American Society of Engineering Education supervises the programs. Objectives: (1) To further the professional knowledge of qualified engineering and science faculty members; (2) to stimulate and exchange ideas between participants and NASA; (3) To enrich and refresh the research and teaching activities of participants' institutions; (4) to contribute to the research objectives of the NASA center. Program Description: College or university will be faculty members appointed as Research Fellows to spend 10 weeks in cooperative research and study at the NASA-Langley Research Center. The Fellow will devote approximately 90 percent of the time to a research problem and the remaining time to a study program. The study program will consist of lectures and seminars on topics of general interest or that are directly relevant to the Fellows' research project. The lecturers and seminar leaders will be distinguished scientists and engineers from NASA, education or industry

Aerospace medicine and biology: A continuing bibliography with indexes (supplement 353)

This bibliography lists 238 reports, articles, and other documents introduced into the NASA Scientific and Technical Information System in August 1991. Subject coverage includes: aerospace medicine and psychology, life support systems and controlled environments, safety equipment, exobiology and extraterrestrial life, biotechnology, human factors engineering, and flight crew behavior and performance