title: Kinetic study on myoglobin refolding monitored by five optical probe stopped-flow methods.
abstract: The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped-flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of > 1 x 10(2) s-1, (4.5-9.3) s-1, and (2-5) x 10(-3) s-1. In the fastest phase, a substantial amount of secondary structure (approximately 40%) is formed within the dead time of the CD stopped-flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a "heme pocket precursor" recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand-exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group.
authors.affiliation: Laboratory of Biodynamics, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Kanagawa, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Chiba
authors.forename: A
authors.initials: A
authors.lastname: Ikai
authors.forename: Y
authors.initials: Y
authors.lastname: Kawamura-Konishi
authors.forename: H
authors.initials: H
authors.lastname: Kihara
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006736:Horses; D007700:Kinetics; D008786:Metmyoglobin / Q000031:analogs & derivatives / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014508:Urea
chemical_list: D009211:Myoglobin; C007177:cyanometmyoglobin; D008786:Metmyoglobin; D014508:Urea