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Hydrogen exchange of the tryptophan residues in bovine α‐lactalbumin studied by uv spectroscopy
Author(s) -
Harushima Yoshiaki,
Kuwajima Kunihiro,
Sugai Shintaro
Publication year - 1988
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.360270407
Subject(s) - chemistry , tryptophan , kinetics , activation energy , lactalbumin , ion exchange , crystallography , ion , absorption (acoustics) , inorganic chemistry , amino acid , chromatography , organic chemistry , biochemistry , physics , quantum mechanics , acoustics
The effect of Ca 2+ ion on structural fluctuation of a milk Ca 2+ ‐binding protein, α‐lactalbumin, under native conditions was investigated by comparing hydrogen‐exchange reactions of tryptophan residues in the apo‐form without Ca 2+ and in the holo‐form at 1 m M CaCl 2 at pH 7.0 in the presence of 0.1 M Na + . The reactions were followed by measuring time‐dependent absorption changes at 298–300 nm due to the 2 H‐ 1 H 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 Ca 2+ concentration and show a relatively fast exchange rate. The other two protons are much more extensively protected (a protection degree of 10 3 –10 5 ) and are markedly affected by the presence of Ca 2+ . 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 EX 2 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 Ca 2+ ions—the EX 1 kinetics for the apo‐protein and the EX 2 kinetics for the holo‐protein at 1 m M Ca 2+ . 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 EX 1 to EX 2 kinetics results from acceleration of the refolding rate with an increase in Ca 2+ 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.