Thermal and Conformational Stability of Seed Coat Soybean Peroxidase

Abstract

Soybean peroxidase (SBP) obtained from the soybean seed coats belongs to class III of the plant peroxidase superfamily. Detailed circular dichroism and steady state fluorescence studies have been carried out to monitor thermal as well as denaturant-induced unfolding of SBP and apo-SBP. Melting of secondary and tertiary structures of SBP occurs with characteristic transition midpoints, T(m), of 86 and 83.5 degrees C, respectively, at neutral pH. Removal of heme resulted in greatly decreased thermal stability of the protein (T(m) = 38 degrees C). The deltaG degrees (H2O) determined from guanidine hydrochloride-induced denaturation at 25 degrees C and at neutral pH is 43.3 kJ mol(-1) for SBP and 9.0 kJ mol(-1) for apo-SBP. Comparison with the reported unfolding data of the homologous enzyme, horseradish peroxidase (HRP-C), showed that SBP exhibits significantly high thermal and conformational stability. We show that this enhanced structural stability of SBP relative to HRP-C arises due to the unique nature of their heme binding. A stronger heme-apoprotein affinity probably due to the interaction between Met37 and the C8 heme vinyl substituent contributes to the unusually high structural stability of SBP.

Thermal Unfolding of Soybean Peroxidase

“…apoSBP was prepared by the acidbutanone procedure (11). Concentrations of the enzyme samples were determined spectrophotometrically using ⑀ 403 ϭ 94.6 mM Ϫ1 cm Ϫ1 for SBP and ⑀ 280 ϭ 24 mM Ϫ1 cm Ϫ1 for apoSBP (6). The GdnHCl concentration in the stock solution was determined using refractometer according to the reported method (2).…”

“…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 the inability to estimate their fractional contribution to the observed spectroscopic property and thus their concentrations. If, however the number, accumulation, and stability interval of the intermediates are too small, making their presence insignificant, the analysis with the two-state model may provide thermodynamic parameters that can be very close to their true values (6,7).…”

“…It contains 326 amino acids with a mean molecular mass of ϳ37 kDa (8,9) and belongs to the class III of the plant peroxidase superfamily. The structure is stabilized by a heme prosthetic group, four disulfide bonds, and two calcium ions apart from the stabilization by interresidue interactions (6,10). It is highly thermally stable, showing a characteristic transition midpoint, T m ϭ 86°C, for the unfolding of backbone at neutral pH (6).…”

“…2 The Gibbs' free energy of unfolding ⌬G o (H 2 O,25°C) determined from the guanidine hydrochloride denaturation at neutral pH is 43.3 Ϯ 2.4 kJ mol Ϫ1 for SBP (6). We observed in our previous study that although the unfolding of SBP induced by guanidine hydrochloride (GdnHCl) occurs via a simple twostate pathway, that induced by heat proceeds through partly folded intermediates whose accumulation is too small to form separate transitions (6). In other words, thermal unfolding of SBP is a single step transition that involves multiple intermediates.…”

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

“…The T m of RmAFP1 at pH 7.4 was determined by CD spectroscopy to be 28.5 C. This method is widely used for this purpose, 20,21,[25][26][27][28] and has the advantage over DSC that it circumvents the question of heating rate. With DSC the T mApp was calculated to be 45.1 C at 1 C/min.…”