Folding
and aggregation of proteins profoundly influence their functions.
We have investigated the effects of thermal history, concentration
and pH on the denaturation and refolding of lysozyme by using ultrasensitive
differential scanning calorimetry (US-DSC) and sedimentation velocity
(SV) via analytical ultracentrifugation (AUC). The former is sensitive
to small energy change whereas the latter can differentiate the oligomers
such as dimer and trimer from individual protein molecules. Our studies
reveal that the degree of denaturation irreversibility increases as
heating times increases. The denaturation temperature (<i>T</i><sub>d</sub>) and enthalpy change (Δ<i>H</i>) are
influenced by heating rate since the denaturation is not in equilibrium
during the heating. We can obtain <i>T</i><sub>d</sub> and
Δ<i>H</i> in equilibrium by extrapolation of heating
rate to zero. In a dilute solution, no aggregation but unfolding happens
in the denaturation. However, when the concentration is above a critical
value (∼15.0 mg/mL), lysozyme molecules readily form trimers
or other oligomers. Lysozyme molecules unfold into stretched chains
at pH > 6.0, which would further forms large aggregates. The formation
of aggregates makes the refolding of lysozyme impossible
