title: Discrimination between the four tryptophan residues of MM-creatine kinase on the basis of the effect of N-bromosuccinimide on activity and spectral properties.
abstract: Rabbit muscle cytosolic creatine kinase (MM-CK) has been treated with N-bromosuccinimide, a reagent known to oxidize selectively the indole moiety of tryptophan residues of proteins in acidic conditions. Inactivation of the enzyme is achieved by modification of one residue per monomer. NBS treatment decreases the ultraviolet absorbance at 280 nm and the intrinsic fluorescence of the protein. From these data it can be deduced that the quantum yields of the four tryptophan residues of each monomer are different due to the more or less hydrophobic environment of each of them and that at least two of them are sufficiently close to Cys 282 to allow fluorescence energy transfer to an extrinsic fluorophore bound to this residue. The accessibility to iodide of the tryptophans has been evaluated during guanidinium chloride denaturation. These data allowed us to acquire a new insight into the environment, the contribution to intrinsic fluorescence and the role in enzymatic activity and fluorescence resonance energy transfer of the tryptophan residues of CK and to tentatively assign a position in the sequence to each of them.
authors.affiliation: Biomembranes et Enzymes Associés, URA 1535 Centre National de la Recherche Scientifique-Université Lyon I, Villeurbanne, France.
authors.forename: E
authors.initials: E
authors.lastname: Clottes
authors.forename: C
authors.initials: C
authors.lastname: Vial
mesh_terms: D000818:Animals; D001974:Bromosuccinimide / Q000494:pharmacology*; D003402:Creatine Kinase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007527:Isoenzymes; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D014364:Tryptophan; D003402:Creatine Kinase; D001974:Bromosuccinimide