Biophysics Senior Project Abstracts
Synthesis of Rare Earth Crystalline Thermometers for Low Temperature Metalloprotein EPR Measurements
Michael Spilatro, 2007
Electron Paramagnetic Resonance (EPR) spectrometers are used to study the active sites in metalloproteins. Because a temperature gradient exists along the sample, rare earth ethyl sulfate crystals are used as thermometers by providing accurate temperature readings within the sample cavity via EPR measurements. Three methods were researched to develop the most efficient and reproducible procedure. The best candidate was crystals synthesized with the trivalent cerium ion, Ce3+, doped within the diamagnetic host yttrium ethyl sulfate, [Ce: Y(C2H4SO4) * 9H2O] (Ce:YES), from the hydrolysis of diethyl sulfate in the presence of rare earth carbonates. Crystals grown from this method were tested for their expected EPR signals. EPR spectroscopy of these Ce:YES crystals did exhibit the expected angular dependence, although they did not have the excited state signal at temperatures above 2 K. Research is being done on two new candidates, crystals of cerium doped in magnesium double nitrate, [Ce2Mg3(NO3)6 * 24H2O] (CeMN) or cerium doped in lanthanum trifluoromethanesulfonate, [Ce: LA(CF3SO3)3 * 9H2O] (Ce:LaTFMS).
Synthesis of Ce:YES Crystalline Thermometers from Alcohol Solutions to Determine the Temperature of EPR Metalloprotein Samples
Hikmat Daghestani, 2006
Advances in biomechanical and biophysical research have tremendously increased over the years. Such advances include Electron Paramagnetic Resonance (EPR) spectroscopy, which is a valuable tool in studying the structure of the active site of metalloproteins and their biomechanical functions at low temperatures. For advanced studies, the exact temperature of the system must be known. However, Like many other techniques, EPR has its setbacks; the design of the spectrometer introduces a temperature gradient along the sample tube providing inaccurate Temperature of the sample. EPR spectra of cerium doped in yttrium ethylsulfate (Ce:YES) crystals have been demonstrated as an effective method in determining the temperature of a sample when employed in the sample. Due to the inaccessibility of starting materials from previous synthesis methods, studies were conducted in order to synthesize the crystals from rare earth tri-chlorides with sodium ethylsulfate in alcohol solutions. EPR measurements were conducted on these crystals and spectra were compared with expected results. Sharp derivative signals, from the | ± 5/2> sates were observed at g=3.7 at 2 K when the c-axis of the crystal was parallel to the applied magnetic field. These signals significantly decreased in intensity and broadened when the temperature was raised to 20 K. These results have demonstrated that crystals do not have the correct symmetry resulting in a change in the crystal field. The change in crystal field created a strong spin-lattice relaxation in the crystals. Electron transitions in the excited | ± 1/2> states were not observed due to the strong spin-lattice relaxation times. The current synthesis method was deemed ineffective in producing the expected signals; therefore other methods should be studied. A thermometer device from D.E.R. epoxy was also developed in order to incorporate three crystals into a protein sample to accurately determine the temperature of the sample without interfering with the sample or the cavity.
Electron Paramagnetic Resonance of Color Centers in Precious Gems
Adam Duncan, 2006
In collaboration with the Carnegie Museum in Pittsburgh Allegheny College undertook the investigation of the damage done to precious gems by display lighting over long periods of time. The gems studied in the experiment were barite and topaz. Earlier research by Joseph S. Gallagher (Class of 2001) revealed that color center formation could be detected by Electron Paramagnetic Resonance before optical evidence was present. One factor that was not taken into account was determining the principle axes of the gems being investigated. This factor had lead to some ambiguity in the earlier experimental results. This original goal of the research was to study color-center formation further. However, it was soon realized that without proper orientation of the crystals the experimental results could not be interpreted properly. The research was then redirected to focus on deeper investigation of crystal structure of the crystal samples. This will then allow one to look at the EPR spectra from these crystals under various rotations (every 30 degrees) to establish a proper coordinate system for the definition of the principal axes of the crystals. The two crystal samples were given designated axes for reference. The two crystals were then scanned on their a and c axes. The results showed that each crystal displayed axial symmetry. In both cases the designated axes for both crystals match the results from the data. A principle axis for both crystals was determined. From these results one can now study the effects of radiation on precious gems without ambiguity in the results.
Analysis of an Unknown Purple Protein using Electrochemical Electron Paramagnetic Resonance Techniques
Jonathan Schmitt, 2006
Researchers at Structural Proteomics have isolated and purified a protein with a deep purple color, nicknamed "deep purple". EPR and redox titration techiniques were used to study the identity of this unknown purple protein. EPR revealed g-values consistent with a high spin ferric ion with several different ligand symmetries. However, these results were not conclusive and further analysis had to be done. Electrochemical Redox Titration was performed on this protein in order to identify the nature of the high spin ferric ion. After the protein was fully reduced the g-values 6.19,4.8,and 3.79 had been eliminated. However, the g-values 8.25,4.3,2.1, and 2.05 showed little response throughout the reduction process. The reduction process proved that ferric ions were present in the protein. Using the spectra, along with the rhombograms, it was found that the protein had three high-spin ferric ion species at the active site. Each of the three iron ions had varying degrees of axial symmetry.
Synthesis of Rare Earth Crystalline Thermometers for Low Temperature Metalloprotein EPR Measurements
Colby Mangini, 2004
Electron Paramagnetic Resonance (EPR) spectrometers have advanced research of metalloproteins at low temperatures in the fields of Biophysics and Biochemistry. However, such research requires an accurate determination of the temperature of the protein. The designs of today's spectrometers introduce a temperature gradient along the sample preventing this accurate determination. The temperature of single crystals of the trivalent cerium ion, Ce3+, doped with the diamagnetic host yttrium ethyl sulfate, Ce:Y(C2H5SO4)3*9H2O (Ce:YES) can be determined to an exceedingly high degree of accuracy via EPR measurements. When placed in a metalloprotein sample, the Ce:YES crystals can act as thermometers by providing an accurate temperature of the sample. The research being done is to develop and refine a repeatable growing method for the Ce:YES crystals and to test such crystals for their expected EPR signals. To minimize interactions between the crystals and the protein sample, the crystals are coated in a D.E.R. 332 epoxy resin. The coated crystals are then mounted on a 1.0mm diameter aluminum wire that is incorporated into the protein sample. The purpose of the multi-crystal crystalline thermometer is to assure that the temperature extracted from the device is taken throughout the entire sample cavity.
Electron Paramagnetic Resonance Spectroscopy of Heme Peroxidase Proteins and Model Peroxidase Complexes
Derek Segesdy, 2004
Electron Paramagnetic Spectroscopy has been used to study a range of biological and chemical samples containing heme prosthetic groups. Two peroxidases proteins, horseradish peroxidases and ascorbate peroxidases have been studied extensively. The physical characteristics of the heme iron have been determined using EPR. In HRP, a quantum-mixed spin state was identified to be a mixture of high spin and intermediate spin at a ratio of 3 to 1 respectively. Studies of APX protein revealed that both high and low spin iron(III) states in axial symmetry are present. Met-myoglobin samples of the dehydrated and hydrated forms were analyzed giving spectra of low and high spin iron(III) in axial symmetry respectively. One model complex, Mn(acac)3 was characterized. The Mn ion exists in the 2+ state with a nuclear spin of 5/2 giving rise to hyperfine structure. The study of these samples as well as their preparation and operation of the spectrometer have been used as preliminary research for the Biochemistry Junior Seminar, "Biochemical Investigations", at Allegheny College.
Rare Earth Crystalline Thermometer for Low-Temperature Metalloprotein Samples
David Kirkwood, 2003
The single rare earth ethyl sulfate (RES) crystal can be used as a thermometer in metalloprotein samples and low temperature EPR experiments. Single rare earth crystals of cerium yttrium ethyl sulfate (Ce:YES) were synthesized and grown. The two lowest Kramer doublets of the cerium3+ ions in these crystals produce strong EPR resonance signals. The intensity of the EPR signals from the two lowest doublets of the Cerium3+ ion are proportional to the Boltzmann constant and therefore provide information about the temperature of the crystal. By placing these crystals within a metalloprotein sample, the determination of the crystal temperature will in turn allow for accurate measurement of the sample temperature. The crystals were studied with an ESR 900 spectrometer. The angle between the crystal c-axis and the direction of the applied external magnetic field was varied to determine the behavior of resonance signal positions with respect to this variation. With the c-axis parallel to the applied external magnetic field, the temperature was varied from 2.0 K to 40.0 K, to determine the dependence of signal intensity on temperature. The Ce:YES crystals were found to behave similarly to studies completed on structurally similar lanthanum trifluoromethanesulfonate (La:TFMS) crystals. The observed results suggest a number of problems that need to be addressed. The results suggest a deviation from the expected C3h symmetry of the crystal lattice. The deviation suggests the possibility of synthesis and growth problems, a dependence on Ce3+ concentration, or erroneous crystal orientation within the resonance cavity. Anti-symmetric resonance signals and non-derivative signals, suggest the magnetic field was swept to quickly across the resonance condition for the Ce:YES crystals.
Electron Paramagnetic Resonance of Metalloproteins and Model Complexes
Tracy Moore, 2003
No abstract available.
A New Cavity for Q-Band EPR Studies of Metalloproteins
William Gunderson, 2002
A Q-Band EPR Spectrometer, at Carnegie Mellon University was designed to examine metalloproteins. This unique spectrometer allows orientations of the static magnetic field both perpendicular and parallel to the microwave magnetic field. This allows the observation of transitions in both Kramers and non-Kramers ions. An inherent limitation of this spectrometer is the low quality factor, Q, limiting the sensitivity of the instrument. To improve the Q a new cavity has been designed that will only allow orientations of the static field perpendicular to the microwave field. This cavity was constructed and tested. The Q of the new cavity is an improvement upon the original design and will allow us to study metalloproteins with very weak signals. This new cavity will also allow for the introduction of ENDOR capabilities to the spectrometer, which will provide a better understanding of the ligands of active sites of metalloproteins.
Electron Paramagnetic Resonance Spectroscopy of Color Centers
Joseph Lewis, 2002
A previous comprehensive senior study showed that when gem crystals are irradiated by intense visible light over a long period of time, color centers form. The gems of interest were topaz celestite, fluorite, inesite, and barite. As an extension of this senior study, preliminary work was done for an in depth study of the angular dependence of the EPR signal. An EPR goniometer was designed so that angle between the gem's principal axes and the external magnetic field could be altered accurately and externally. The apparatus was designed in a modular format to allow for easy maintenance. Additionally, I used an optical goniometer to find the relative angles between the faces of a topaz crystal. With the angles between faces we can connect the principle axes to the whole crystal. So when the crystal is attached to the EPR goniometer we can look at when the external field is parallel to one of the principal axes. The work done orienting the crystal was also useful in providing a detailed procedure for future reference.
Electron Paramagnetic Resonance of Color Centers in Single Crystals of Precious Gems
Joseph Gallagher, 2001
Allegheny College in collaboration with the Carnegie Mellon Research Institute and Carnegie Museum in Pittsburgh undertook this project in an attempt to investigate a few of the concerns the museum was having concerning its precious gem exhibit. Speculation existed concerning the damage that the intense radiation over prolonged periods of time was having on the precious gems. The gems of interest were topaz, celestite, fluorite, inesite, and barite. This radiation is that which is seen illuminating the gems in the museum's exhibit. Irradiation was simulated by exposing the crystals under a Xe arc lamp for two weeks. The museum's concerns centered primarily around alterations in the color of the gems. This is an important point considering the aesthetic value of precious gems is often a matter of their color. Both unirradiated and irradiated gems were analyzed using an X-band (~9 GHz) EPR spectrometer at the Mellon Institute at Carnegie Mellon University. Samples were run at room temperature, 300 K, and low temperature 2 K - 300 K. The results illustrated the ability of our technique to provide the possible nuclei in the environment of the color center. Furthermore, the fluorite crystal showed that irradiation created color centers. The other three gems did not show ample evidence to indicate that color center formation had occurred following irradiation, thus demonstrating the limitations of our technique which are addressed in the study.