Investigating the Cu Chemistry of the Superoxide Dismutase IV Mutant Gautam Mankaney
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Investigating the Cu Chemistry of the Superoxide Dismutase IV Mutant Gautam Mankaney Mentor: Dr. Joseph Beckman Oregon State University Howard Hughes Medical Internship Amyotrophic Lateral Sclerosis • Neurodegenerative Disease • Affects 30,000 Americans What is Superoxide Dismutase? •Enzyme •Scavenges superoxide •Homodimer •2 Cu Atoms 2 Zn Atoms The Mechanism H 2O 2 SOD AND ALS •105 Missense Mutations • Two Theories 1. Aggregation 2. Aberrant Oxidative Chemistry The Aberrant Oxidative Chemistry Theory •SOD losses its affinity for the Zn atom Zn Loss of Zn O2- SCAVENGING ABILITY LEVELS OF SUPEROXIDE, O2REVERSE REACTION FORMING O2- Nitration of tyrosine leads to motor neuron degeneration Aberrant Cu Theory and Recent Findings • Jiou Wang, John Hopkins University* • Keith Nylin, Oregon State University *Jiou Wang, et al. Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature . 2003 THE GOALS OF MY PROJECT 1.Will the Quad Mutant bind Cu? 2.If it does bind Cu, is the enzyme active as an SOD? Expressing and Purifying the QUAD-Mutant Transformation Plasmid encoding for human Quad/C111S SOD Select Colony to Induce Production of Protein Escherichia coli Obtain the Protein Purification SDS • DNase • Precipitate other proteins • Dialyze to remove salts Crude Cell Extract SOD Fast Performance Liquid Chromatography (Anion Exchange Column) SDS Different Fractions High Salts Wt Standard TESTING FOR SOD ACTIVITY Two Ways to test for Activity: -show IV mutant can scavenge for O2-show IV mutant can produce O2- IF QUAD HAS ACTIVITY, THEN IT MUST HAVE A Cu ATOM Cu •Source of O2- SOD ACTIVITY ASSAY* SOD competes with another compound •Native gel – non-denaturing gel • Riboflavin, Nitroblue tetrazolium, EDTA riboflavin Formazan *Ching-Nen Chen, et al. Assay of superoxide dismutase activity by combining electrophoresis and densitometry. 2003 THERE IS SOD ACTIVITY Cu Activity β-Me CuSO4 pH: 9.5 8.0 β-Me 7.0 5.0 9.5 Activity IV 8.0 7.0 5.0 Zn(-) Standard COMPETITION ACTIVITY ASSAY* SOD Xanthine + Xanthine Oxidase Cytochrome C O2 O2- Cytochrome C (reduced) -Cytochrome C reduced absorbs at 550nm -Rate at which peak forms is measured (Abs/time) *JM McCord and I. Fridovich. CuZn and Mn Superoxide Dismutase Assay. 1988 Competition Assay Positive for SOD Activity Cytochrome C SOD Reduction % Inhibition Amount of IV SOD added (ng) Rate of Cytochrome C Reduction (Abs/sec) % Percent Inhibition 0 µg 0.0234 9 ng 0.0210 10.3% 18 ng 0.0189 19.2% 27 ng 0.0165 29.5% 45 ng 0.0116 50.4% ------ Review of Results QUAD mutant is active as an SOD TWO ACTIVITY ASSAYS Cu atom Future Research Activity assay measuring the production of superoxide Measure Cu concentration and the QUAD mutants affinity to bind Cu Investigate the Triple mutant Many Thanks Thank you: Dr. Joseph Beckman Keith Nylin Beckman Lab Dr. Kevin Ahern Dr. Indira Rajagopal Howard Hughes Medical Institute PAR Assay* Nitrilotriacetic acid – chelates Zn GuanidineHCl Absorbance – 500nm PAR Zn Cu PAR NTA PAR Cu Zn Cu PAR Measure concentrations of Zn and Cu 4-pyridylazoresorcinol: -Affinity for Zn & Cu -Abs @ 500nm TIME Zn Zn Cu PAR Cu EDTA PAR PAR PAR ASSAY INDICATES NO METALS IN SOD SAMPLE STANDARD CURVES SOD METAL CONTENT Zn = .218% Or ≈ 2 Zn atoms / 1000 SOD molecules Cu = .437 Or ≈4 Cu atoms / 1000 SOD molecules The Aberrant Oxidative Chemistry Theory Ran in acetonitrile Impurity There should be a peak at 15699 mass units [Cu,Zn(-) Quad] Apo-Quad Mutant
