We have earlier reported that both guanidine hydrochloride (GdnHCl)-induced and heat-induced unfolding of seed coat soybean peroxidase (SBP), monitored by far UV CD, show single step transition. However, although GdnHCl-induced unfolding follows a two-state pathway, the heat-induced denaturation proceeds through intermediates as indicated by the very low cooperativity of transition. In the former case, analysis of the data based on the two-state model gives true thermodynamic parameters, whereas underestimated values are obtained in the latter case. Available complex equations also cannot be applied for the analysis of the thermal unfolding of SBP due to the absence of separate transitions for the intermediates. In the present study, we report a method to obtain true thermodynamic parameters from thermal transition curves of SBP using the two-state model. When SBP is subjected to thermal unfolding at high GdnHCl concentrations (5.8 -6.9 M), cooperative behavior is observed, which allowed the analysis by the twostate model to determine their thermodynamic parameters. We then obtained the thermodynamic parameters in the absence of GdnHCl by extrapolating the graph of linear dependence of ⌬H m on T m to the T m corresponding to 0 M GdnHCl. Another key point for checking the validity of our method was the fact that the unfolded state of SBP generated by either heat or GdnHCl is the same by which we could cross-check our results with that obtained from GdnHCl unfolding. Having obtained the true thermodynamic parameters, we report a detailed thermodynamic study of SBP. Further we address the effect of heme in the thermal unfolding mechanism of SBP.Protein folding/unfolding is one of the most intensely studied aspects of modern biophysics and biochemistry. The denaturant-induced unfolding transitions are measured either spectroscopically or calorimetrically. Far UV CD conforms to the secondary structure of a protein and therefore is used as a tool to monitor protein folding/unfolding. The most common denaturing agents used for the unfolding of proteins are heat, chemical denaturants such as urea and guanidine hydrochloride, and extremes of pH. The simplicity of unfolding transition and its reversibility allow a detailed description of its thermodynamics (1). The two-state model (1-3), which assumes that only the native (N) and unfolded (U) states are present at equilibrium (N ª U), seems to be adequate to describe the unfolding curves to determine the thermodynamic parameters accompanying the unfolding of a number of proteins (4, 5). However, in most cases in which the transition is multistep (stable intermediate states), a complete analysis of the system is possible by utilizing more complex equations (2, 3). If the two-state model is still used in such cases, underestimated values for the thermodynamic parameters are obtained (2, 3). In certain other cases, although the unfolding curve shows a single step, the transition involves intermediates (2, 6, 7), which makes the analysis difficult, almost impossible, due to t...

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