The overall goal of our research is to understand the chemistry of transition metals in biological systems. Toward this end, we employ a variety of chemical and biochemical methods and develop instrumentation and approaches to data analysis as outlined below.
 

Spectroscopic Techniques:

Electron Paramagnetic Resonance (EPR) Spectroscopy

Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

Electron-Nuclear Double Resonance (ENDOR) Spectroscpy

Circular Dichroism (CD)

Fourier Transform Infrared (FT-IR) Spectroscopy

Fluorescence Spectroscopy

NMR Spectroscopy

UV-Visible Spectroscopy

UV Difference Spectroscopy

Mössbauer Spectroscopy

Mass Spectrometry

Atomic Absorption Spectroscopy
 
 

EPR spectra of the vanadyl ferritins

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Biochemical Techniques

Gel Permeation Chromatography

Ion Exchange Chromatography

HPLC

Salt Precipitation

Flow and Equilibrium Dialysis

Ultrafiltration

SDS and Native Gel Electrophoresis

Work with Radio Isotope Labeled Compounds

Protein Digestion

Protein Sequencing by Mass Spectrometry

Carbohydrate Analysis

Culturing and Growing Bacteria for Production of Recombinant Proteins.

Biorad Protein Assays

Enzyme Assays

Analytical Ultracentrifugation
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Figure: Chromatogram of plasma showing vanadium-48 binding to the transferrins.

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Kinetic Studies

Iron oxidation and hydrolysis kinetics using electrode oximetry and pH stat.

Stopped-flow Kinetics

Rapid-freeze Quench EPR Kinetics

Enzyme Kinetics

Kinetics of Iron Uptake and Removal from Transferrin and Ferritin
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Figure:  Lineweaver-Burk plot of Zn(II) inhibition of  Fe(II) oxidation in ferritin.

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Thermochemistry

Differential Scanning Calorimetry (DSC) of Protein Stability.

Isothermal Titration Calorimetry (ITC) of Metal Binding to Proteins.
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Figure: Isothermal titration calorimetry of Tb(III) binding to apoferritin.

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Instrumentation Development

Construction and modification of X-band and Q-band ENDOR spectrometers.

Construction and modification of microwave bridges for EPR spectroscopy.

Construction of special microwave resonance cavities for EPR/ENDOR.

Development of kinetic apparatus for simultaneously measuring dioxygen and proton consumption, or production, in chemical reactions.

Assembly of L-band and X-band EPR Spectrometers
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Figure: Schematic of ENDOR Spectrometer.

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Data Analysis

Development of equations for analysis of binding and kinetic data.

Fitting of binding or kinetic data using multivariable least-squares techniques and the software program Origin.

EPR and ESEEM spectral simulations using the Spin Hamiltonian formalism.