Premium
An antibody‐based affinity chromatography tool to assess Cu, Zn superoxide dismutase (SOD) G93A structural complexity in vivo
Author(s) -
Palacios Florencia,
Cota Germán,
Horjales Sofía,
Lima Analía,
Battistoni Julio,
SoteloSilveira José,
Marín Mónica
Publication year - 2010
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200900106
Subject(s) - superoxide dismutase , in vivo , chemistry , affinity chromatography , antibody , biochemistry , enzyme , biology , immunology , microbiology and biotechnology
‘Conformational diseases’ are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1 G93A mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue‐specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody‐based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSOD G93A rats provided evidence about heterodimers rSOD–hSOD G93A formation in both extracts. Moreover, when characterized by 2‐DE and MALDI‐TOF/TOF MS, the extracted hSOD G93A showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post‐translational modifications of hSOD1 produced in various tissues.