Abstract
For most multidomain proteins the thermal unfolding transitions are accompanied by an irreversible step, often related to aggregation at elevated temperatures. As a consequence the analysis of thermostabilities in terms of equilibrium thermodynamics is not applicable, at least not if the irreversible process is fast with respect the structural unfolding transition. In a comparative study we investigated aggregation effects and unfolding kinetics for five homologous alpha-amylases, all from mesophilic sources but with rather different thermostabilities. The results indicate that for all enzymes the irreversible process is fast and the precedent unfolding transition is the rate-limiting step. In this case the kinetic barrier toward unfolding, as measured by unfolding rates as function of temperature, is the key feature in thermostability. The investigated enzymes exhibit activation energies (E(a)) between 208 and 364 kJmol(-1) and pronounced differences in the corresponding unfolding rates. The most thermostable alpha-amylase from Bacillus licheniformis (apparent transition temperature, T(1/2) approximately 100 degrees C) shows an unfolding rate which is four orders of magnitude smaller as compared with the alpha-amylase from pig pancreas (T(1/2) approximately 65 degrees C). Even with respect to two other alpha-amylases from Bacillus species (T(1/2) approximately 86 degrees C) the difference in unfolding rates is still two orders of magnitude.
Highlights
Thermal stability of proteins includes thermodynamic as well as kinetic stability
The impact of aggregation and heating rates on the apparent transition temperatures has been described for several cases in previous studies (6 – 8, 23)
It is quite often stated that heating rates of 1 °C per minute are reasonable to measure the unfolding transition of mesophilic proteins, it was demonstrated in various studies that equilibrium was not reached
Summary
Enzymes—␣-Amylase from Bacillus licheniformis (BLA, purchased from Sigma), from Bacillus amyloliquefaciens (BAA, from Fluka), from Bacillus subtilis (BSUA, from Fluka), and from Aspergillus oryzae (TAKA, from Sigma) was obtained as lyophilized powder. A further ␣-amylase from pig pancreas (PPA, from Roche Applied Science) was obtained in ammonium sulfate solution. Powders were dissolved in buffer (for details see below), and all enzymes were purified by the use of a desalting column (Econo-Pac 10 DG, Bio-Rad). In the case of CD-spectroscopy respective buffers were used, but with 10 mM Mops. Excitation waveing; ⌬S, entropy change; ⌬G#N, free energy difference between ground state and transition state; Ea, activation energy; r, heating rate ku, unfolding rate constant; kf, (re-)folding rate constant; ki, rate constant for the irreversible process; t1⁄2, half-life time; fN, fraction of folded protein; Mops, 3-(N-morpholino)propanesulfonic acid
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