Rapid one-step purification of native dimeric ALS-associated human Cu/Zn superoxide dismutase from transgenic rat tissues.
Mutated Cu/Zn superoxide dismutase (SOD1) was the first proven cause of amyotrophic lateral sclerosis (ALS) and was the basis for the first animal model. Many approaches, including transgenic and knock-out animals, cell models, and in vitro studies using recombinant hSOD1 mutants and wild-type, have been employed in an attempt to elucidate the gained toxic function. However, a thorough characterization of the properties of hSOD1 mutants produced in vivo has yet to be carried out, primarily due to the lack of a procedure capable of purifying the enzyme from relevant tissues in a manner that avoids potential artifacts. Here we report a new, one-step purification procedure using a semi-preparative polymeric reversed-phase HPLC system, which yields greater than 99% pure enzyme from the spinal cord, and >95% pure from brain, heart, and kidney. This novel approach for purifying 'in vivo expressed' native dimeric SOD1 will facilitate the determination of the true 'as isolated' properties of the enzyme that is responsible for disease, devoid of any expression system, or harsh purification, artifacts. An important new finding related to the specific activity of human SOD1 (normalized to copper content) is also discussed.
Bhogaraju
Venkata K
VK
Department of Pharmacology and Toxicology and the J. Thomas May Center for ALS Research and Translational Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
Levi
Mark S
MS
Reed
Ronald L
RL
Crow
John P
JP
D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003300:Copper / Q000032:analysis; D004195:Disease Models, Animal; D004563:Electrochemistry / Q000379:methods; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000235:genetics; D051381:Rats; D013482:Superoxide Dismutase / Q000032:analysis* / Q000235:genetics
D003300:Copper; C109415:SOD1 G93A protein; D013482:Superoxide Dismutase