Affiliation:
1. State Key Laboratory of Elemento-organic Chemistry Tianjin Key Laboratory of Biosensing and Molecular Recognition College of Chemistry Nankai University Tianjin 300071 China
2. Department of Chemical and Biological Physics Weizmann Institute of Science Rehovot 76100 Israel
Abstract
AbstractNitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the solo thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long‐time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron‐electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu.
Funder
National Natural Science Foundation of China
Subject
General Chemistry,Catalysis,Organic Chemistry
Cited by
1 articles.
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