Heme and pH-dependent stability of an anionic horseradish peroxidase.
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.
Carvalho
Ana Sofia L
AS
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av Rovisco Pais, Lisboa 1049-001, Portugal.
Melo
Eduardo Pinho e
EP
Ferreira
Bruno Sommer
BS
Neves-Petersen
Maria Teresa
MT
Petersen
Steffen B
SB
Aires-Barros
Maria Raquel
MR
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
D000838:Anions; D014364:Tryptophan; D006735:Horseradish Peroxidase