@article{oai:ir.soken.ac.jp:00004139,
 author = {桑島, 邦博 and HARUSHIMA, Yoshiaki and KUWAJIMA, Kunihiro and SUGAI, Shintaro},
 issue = {4},
 journal = {Biopolymers, Biopolymers},
 month = {Apr},
 note = {The effect of Ca2+ ion on structural fluctuation of a milk Ca2+-binding protein, α-lactalbumin, under native conditions was investigated by comparing hydrogen-exchange reactions of tryptophan residues in the apo-form without Ca2+ and in the holo-form at 1 mM CaCl2 at pH 7.0 in the presence of 0.1M Na+. The reactions were followed by measuring time-dependent absorption changes at 298–300 nm due to the 2H-1H exchange of the tryptophan imino protons and were found to be biphasic under all the conditions examined. Two of the four tryptophan protons are insensitive to Ca2+ concentration and show a relatively fast exchange rate. The other two protons are much more extensively protected (a protection degree of 103–105) and are markedly affected by the presence of Ca2+. Examinations of the temperature dependence and pH dependence of the individual exchange rates have been utilized for elucidating the exchange mechanism. The fast protons show a low activation energy reaction with so-called EX2 kinetics. The exchange reaction of the slow protons is accompanied by a high activation energy, and the exchange mechanism of the protons depended on the presence or absence of stabilizing Ca2+ ions—the EX1 kinetics for the apo-protein and the EX2 kinetics for the holo-protein at 1 mM Ca2+. The exchange reaction in the thermally unfolded state was also found to be biphasic, but the fast phase, which has an exchange rate in the fully exposed state, becomes predominant with decreasing temperature. By taking this fact and using a structural unfolding model of hydrogen exchange, the present results are fully consistent with thermodynamic parameters of the thermal transition and kinetic parameters of refolding reactions induced by concentration jumps of guanidine hydrochloride obtained in previous studies. It is demonstrated that the reaction of the slow protons in the native state is mediated by a transient global unfolding equivalent to the “thermal” unfolding under a native condition and that switching of the exchange mechanism from the EX1 to EX2 kinetics results from acceleration of the refolding rate with an increase in Ca2+ concentration. The transient global unfolding takes place even under a strongly native condition, e.g., at a temperature 20° below the beginning of the thermal transition.},
 pages = {629--644},
 title = {Hydrogen exchange of the tryptophan residues in bovine α-lactalbumin studied by uv spectroscopy},
 volume = {27},
 year = {1988}
}