Amyotrophic lateral sclerosis (ALS), or more commonly known as Lou Gehrig’s disease, is a neurodegenerative disease that usually causes eventual respiratory failure due to motor neuron death. Approximately 90% of all ALS cases are classified as sporadic ALS, meaning that there is no known cause. The remaining 10% of cases are classified as familial ALS, and of these, 2% have been linked to the gene for SOD1. This gene encodes the copper-zinc superoxide dismutase protein, an antioxidant enzyme which catalyzes the reduction of the superoxide radical (O2–) to hydrogen peroxide, which can then be further broken down into water and diatomic oxygen via hydrogen peroxidases. In vivo, metals bound to the wild-type protein are the catalyst for the reduction of superoxide, but certain mutants exhibit different metal-binding properties. In our experiments, we explored these properties through a variety of applications. I produced the S134N mutant in E. coli expression bacteria, in addition to the previously generated variants, and then purified the proteins using hydrophobic interaction and ion exchange chromatography. Metals were removed from SOD1 via the standard EDTA dialysis method. Metal content was assayed using an inductively coupled plasma-optical emission spectrometer (ICP-OES). Apoproteins were selectively titrated with metals to form the Cu2Co2SOD derivatives, with metal-binding monitored by UV-Visible spectrophotometry. Results from these experiments have furthered the body of evidence suggesting that proper metallation is possible under the correct experimental conditions. Furthermore, these studies contradict previously obtained results about metal-binding, possibly suggesting an issue of a thermodynamic versus kinetic product. Future experiments will seek to elucidate this, and provide answers to these questions.
Sean Lehman, ’10 Sioux Rapids, IA
Majors: Biochemistry and Molecular Biology, Integrated Psychosocial Theory
Sponsor: Cynthia Strong