A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50 °C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 50 °C, and those of lysozyme, RNase A and β-lactoglobulin at pH 8 and 40 °C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.
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