Rapid one-step purification of native dimeric ALS-associated human Cu/Zn superoxide dismutase from transgenic rat tissues

Abstract

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.

Key words::

• SOD1
• HPLC purification
• specific activity
• copper
• transgenic animals
• hSOD1-G93A
• hSOD-WT
• rhSOD1-G93A

Acknowledgements

This work was supported by a grant (5R01N S040819)from the National Institutes of Health and research gifts from The Paul Dunn ALS Foundation (Little Rock, Arkansas, USA) and The J. Thomas May Center for ALS Research and Translational Medicine (Little Rock, Akansas, USA).

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.