title: Heme and pH-dependent stability of an anionic horseradish peroxidase.
abstract: Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation. However ejection of the heme group leads to an irreversible unfolding behavior at pH 4, while at pH 7 and 10 refolding is still observed. This is putatively correlated with the titration state of the heme pocket. Thermal transitions of HRPA1 showed scan rate dependence at the three pH values, showing that the denaturation process was kinetically controlled. The denaturation process was interpreted in terms of the classic scheme, N<-->U-->D and fitted to far-UV CD ellipticity. A good agreement was obtained between the experimental and theoretical T(m) values and percentages of irreversibility. However the equilibrium between N and U is probably more complex than just a two-state process as revealed by the multiple T(m) values.
authors.affiliation: Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av Rovisco Pais, Lisboa 1049-001, Portugal.
authors.forename: Ana Sofia L
authors.initials: AS
authors.lastname: Carvalho
authors.forename: Eduardo Pinho e
authors.initials: EP
authors.lastname: Melo
authors.forename: Bruno Sommer
authors.initials: BS
authors.lastname: Ferreira
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Neves-Petersen
authors.forename: Steffen B
authors.initials: SB
authors.lastname: Petersen
authors.forename: Maria Raquel
authors.initials: MR
authors.lastname: Aires-Barros
mesh_terms: D000838:Anions; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004795:Enzyme Stability; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000838:Anions; D014364:Tryptophan; D006735:Horseradish Peroxidase