Amyotrophic lateral sclerosis (ALS), 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. We produced the human wild-type, A4V and G93A mutants in the bacterium E. coli, and then purified the proteins using phenyl-sepharose hydrophobic interaction column chromatography and DEAE Sephadex ion exchange chromatography. Metals were removed from SOD1 via IDA-Sepharose ion exchange column chromatography, as well as by the standard EDTA dialysis method. The protein demetallated by the EDTA dialysis method was later remetallated, and a spectroscopic analysis performed. In vitro mutagenesis was performed to create the D101N and L38V mutants of SOD1. The L38V mutant was then sequenced to confirm that the correct amino acid change took place.
Sean Lehman, ’10 Nevada, IA
Majors: Biochemistry and Molecular Biology, Integrated Psychosocial Theory
Emmanuel Koli, ’08 Accra, Ghana
Major: Biochemistry and Molecular Biology
Sponsor: Cynthia Strong