Enzyme Microb. Technol., 40, 696-703 (2007)
Carvalho, A. S. L., Ferreira, B. S., Neves-Petersen, M. T., Petersen, S. B., Aires-Barros, M. R., Melo, E. P.
Horseradish peroxidase A2 is the most abundant anionic peroxidase in horseradish roots. Steady-state fluorescence, Circular Dichroism and Differential Scanning Calorimetry were used to study the thermal stability of HRPA2 at pH values of 4, 7 and 10. Changes in the intrinsic protein probes follow the increasing order: tryptophan fluorescence, secondary structure, and heme group environment at pH 4 and 7 showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. At pH 10, the heme environment is the first structural region to suffer conformational alterations concurrently with local tryptophan environmental changes. The denaturation process was analyzed by the classic scheme, NnUgD used to fit thermal denaturation, followed by far-UV CD ellipticity. This model described well the experimental Tm values, their dependence on the scan rate and the percentages of irreversibility after thermal denaturation. Thermal denaturation is partially reversible at pH 10 and 7 while a 100% irreversible process was observed at pH 4. Despite the accurate fit of data by this pathway intermediate states must populate the equilibrium between N and U as changes in the intrinsic protein probes were not coincident.
Keywords: horseradish peroxidase A2; thermal denaturation; pH-dependent stability; isoenzymes
|Back to: Publications||BioEngineering Research Group|