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. 1980 Oct;32(1):561–575. doi: 10.1016/S0006-3495(80)84990-3

Hydrogen isotope exchange kinetics of single protons in bovine pancreatic trypsin inhibitor.

C K Woodward, B D Hilton
PMCID: PMC1327353  PMID: 7248461

Abstract

The exchange kinetics of the slowest exchanging BPTI beta-sheet protons are complex compared to model peptides; the activation energy, E alpha, and the pH dependence are temperature dependent. We have measured the exchange kinetics in the range pH 1--11, 33--71 degrees C, particularly the temperature dependence. The data are fit to a model in which exchange of each proton is determined by two discrete dynamical processes, one with E alpha approximately 65 kcal/mol and less than first order dependence on catalyst ion, and one with E alpha 20--30 kcal/mol and approaching first order in catalyst ion. The low activation energy process is the mechanism of interest in the native conformation of globular proteins and involves low energy, small amplitude fluctuations; the high activation energy process involves major unfolding. The model is simple, has a precedent in the hydrogen exchange literature, and explains quantitatively the complex feature of the exchange kinetics of single protons in BPTI, including the following. For the slowest exchanging protons, in the range 36 degrees--68 degrees C, E alpha is approximately 65 kcal/mol at pH approximately 4, 20--30 kcal/mol at pH greater than 10, and rises to approximately 65 kcal/mol with increasing temperature at pH 6--10; the Arrhenius plots converge around 70 degrees C; the pH of minimum rate, pHmin, is greater than 1 pH unit higher at 68 degrees C than for model compounds; and at high pH, the pH-rate profiles shift to steeper slope; the exchange rates around pHmin are correlated to the thermal unfolding temperature in BPTI derivatives (Wagner and Wüthrich, 1979, J. Mol. Biol. 130:31). For the more rapidly exchanging protons in BPTI the model accounts for the observation of normal pHmin and E alpha of 20--30 kcal/mol at all pH's. The important results of our analysis are (a) rates for exchange from the folded state of proteins are not correlated to thermal lability, as proposed by Wuthrich et al. (1979, J. Mol. Biol. 134:75); (b) the unfolding rate for the BPTI cooperative thermal transition is equal to the observed exchange rates of the slowest exchanging protons between pH 8.4--9.6, 51 degrees C; (c) the rates for exchange of single protons from folded BPTI are consistent with our previous hydrogen-tritium exchange results and with a penetration model of the dynamic processes limiting hydrogen exchange.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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