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Coenzyme binding of a folding intermediate of aspartate aminotransferase detected by HPLC fluorescence measurements
Author(s) -
Herold Marzell,
Leistler Bernd
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)81042-k
Subject(s) - chemistry , cofactor , fluorescence , monomer , pyridoxal , folding (dsp implementation) , dissociation (chemistry) , equilibrium unfolding , protein folding , pyridoxal phosphate , enzyme , stereochemistry , crystallography , biochemistry , organic chemistry , physics , quantum mechanics , electrical engineering , engineering , polymer
Equilibrium dissociation and unfolding of dimeric aspartate aminotransferase from Escherichia coli proceeds via two compact monomeric intermediates which have similar hydrodynamic volumes but different fluorescence properties. We probed binding of the coenzyme pyridoxal 5′‐phosphate to these intermediates by coupling fluorescence detection to size‐exclusion HPLC. This procedure gave additionally an internal conformational probe of the unfolding transitions of the enzyme. It was shown that the first intermediate, M, is able to bind the coenzyme, whereas the second intermediate, M, is not. It is likely that M is the correctly folded monomer of the protein.