Thermodynamic characterization of the palm tree Roystonea regia peroxidase stability

Abstract

The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization.

Peroxidase from soybean meal: obtention, purification and application in reduction of deoxynivalenol levels

“…In comparative terms, the PO from SBM demonstrates greater thermal stability than lignin peroxidase and manganese peroxidase from Bjerkandera adusta, by Ertan et al, 31 with values of 36 and 14 kJ mol -1 for ∆H°, and 77 and 70 kJ mol -1 for ∆G°, respectively. According Zamorano et al, 32 a ∆G° of 100.32 kJ mol -1 for the purification of PO from real palm leaves (Roystonea regia). These values are lower than those in literature, demonstrating low sensitivity of the PO from SBM to thermal alterations, even when considering a variable rate of temperature.…”

Steady-state kinetics of <i>Roystonea regia</i> palm tree peroxidase

“…The reactions, initiated by the addition of RPTP, were performed at 25˚C in 20.0 -30.0 mM universal buffer, containing variable concentrations of the reducing substrate at fixed H 2 O 2 concentration and viceversa. The reactions were carried out at the optimal pH for each substrate, 6.9 for guaiacol, 3.5 for catechol, 4.0 for ferulic acid, 3.0 for ABTS and 6.0 for o-dianisidine and o-phenylendiamine [43].…”

“…The concentration of peroxidase was measured spectrophotometrically at 403 nm, using the experimentally determined extinction coefficient value of 60.8 ± 2.3 mM -1 ·cm -1 for the protein monomer [43]. To determine the microscopic rate constants and other kinetic parameters for the oxidation of the substrates by RPTP in the presence of H 2 O 2 , the mathematical treatment of Morales and Ros-Barceló [44] was applied.…”

“…peroxidases from other plants, such as peanut [10], barley [11], tea [12], Arabidopsis thaliana [13], and palm trees [14][15][16][17][18].…”

“…Despite representing different structural groups, kinetic models for the hydrogen peroxide-mediated inactivation of horseradish peroxidase (HRP) [33], ascorbate peroxidase (APX) [38], peroxidase from the Royal Palm Tree (RPTP) [16], microperoxidase-11 [39] and Chamaerops excelsa peroxidase (CEP) [40] are similar in the sense that they are time-dependent and exhibit saturation kinetics. From the inactivation stoichiometry, it has been concluded that for APX only 2.5 molecules of hydrogen peroxide are required per active site for the inactivation form to be generated [38], in contrast to the 265 molecules required for HRP [33].…”