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J. Biol. Chem., Vol. 277, Issue 43, 40717-40721, October 25, 2002

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Thermal Unfolding of Soybean Peroxidase
APPROPRIATE HIGH DENATURANT CONCENTRATIONS INDUCE COOPERATIVITY ALLOWING THE CORRECT MEASUREMENT OF THERMODYNAMIC PARAMETERS^*

Amisha Kamal JanardhananPillai Kizhakkedathu, Mahammed Nazeerunnisa, and Digambar V. Behere

From the Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India

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 two-state 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.

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