Purification and structural stability of white Spanish broom (Cytisus multiflorus) peroxidase.

New plant peroxidase has been isolated to homogeneity from the white Spanish broom Cytisus multiflorus. The enzyme purification steps included homogenization, NH(4)SO(4) precipitation, extraction of broom colored compounds and consecutive chromatography on Phenyl-Sepharose, HiTrap™ SP HP and Superdex-75 and 200. The novel peroxidase was characterized as having a molecular weight of 50 ± 3 kDa. Steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the structural stability of C. multiflorus peroxidase (CMP) at pH 7.0. Thus changes in far-UV CD corresponded to changes in the overall secondary structure of enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. It is shown that the process of CMP denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N ⟶ kD, where k is a first-order kinetic constant that changes with temperature following the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Thermal stability of matrix protein from Newcastle disease virus.

The thermal stability of the matrix protein (M protein) of Newcastle disease virus (NDV) has been investigated using high-sensitivity differential scanning calorimetry (DSC) at pH 7.4. The thermal folding/unfolding of M protein at this pH value is a reversible process involving a highly cooperative transition between folded and unfolded monomers with a transition temperature (Tm) of 63 °C, an unfolding enthalpy, ΔH(Tm), of 340 kcal mol(-1), and the difference in heat capacity between the native and denatured states of the protein, ΔCp, of 5.1 kcal K(-1) mol(-1). The heat capacity of the native state of the protein is in good agreement with the values calculated using a structure-based parameterization, whereas the calculated values for the hypothetical fully-unfolded state of the protein is higher than those determined experimentally. This difference between the heat capacity of denatured M protein and the heat capacity expected for an unstructured polypeptide of the same sequence, together with the data derived from the heat-induced changes in the steady-state fluorescence of the protein, indicates that the polypeptide chain maintains a significant amount of residual structure after thermal denaturation.

Purification, crystallization and preliminary crystallographic analysis of peroxidase from the palm tree Chamaerops excelsa.

Plant peroxidases are presently used extensively in a wide range of biotechnological applications owing to their high environmental and thermal stability. As part of efforts towards the discovery of appealing new biotechnological enzymes, the peroxidase from leaves of the palm tree Chamaerops excelsa (CEP) was extracted, purified and crystallized in its native form. An X-ray diffraction data set was collected at a synchrotron source and data analysis showed that the CEP crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 70.2, b = 100.7, c = 132.3 Å.

Suicide inactivation of peroxidase from Chamaerops excelsa palm tree leaves.

The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H(2)O(2) required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r=4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase.

Zinc-, cobalt- and iron-chelated forms of adenylate kinase from the Gram-negative bacterium Desulfovibrio gigas.

Adenylate kinase (AK) from the sulphate-reducing bacterium Desulfovibrio gigas (AK) has been characterized earlier as a Co(2+)/Zn(2+)-containing enzyme, which is an unusual characteristic for adenylate kinases from Gram-negative bacteria, in which these enzymes are normally devoid of metal ions. AK was overexpressed in E. coli and homogeneous Co(2+)-, Zn(2+)- and Fe(2+)-forms of the enzyme were obtained under in vivo conditions. Their structural stability and spectroscopic and kinetic properties were compared. The thermal denaturation of Co(2+)- and Zn(2+)-forms of AK was studied as a cooperative two-state process, sufficiently reversible at pH 10, which can be correctly interpreted in terms of a simple two-state thermodynamic model. In contrast, the thermally induced denaturation of Fe(2+)-AK is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Practically identical contents of secondary-structure elements were found for all the metal-chelated-forms of AK upon analysis of circular dichroism data, while their tertiary structures were significantly different. The peculiar tertiary structure of Fe(2+)-AK, in contrast to Co(2+)- and Zn(2+)-AK, and the consequent changes in the physico-chemical and enzymatic properties of the enzyme are discussed.

Thermal stability of peroxidase from Chamaerops excelsa palm tree at pH 3.

The structural stability of a peroxidase, a dimeric protein from palm tree Chamaerops excelsa leaves (CEP), has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism and steady-state tryptophan fluorescence at pH 3. The thermally induced denaturation of CEP at this pH value 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, leading to the conclusion that in solution CEP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of CEP 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.

Effect of acetonitrile on Cynara cardunculus L. cardosin A stability.

The kinetics of the structural changes affecting cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculus L., in the presence of a mixture of acetonitrile (AN) in water (W) was studied. Incubation of cardosin A with 10% (v/v) AN resulted in a gradual increase in protein helicity, accompanied by changes in the tertiary structure, seen by changes in the intrinsic fluorescence of tryptophan. Differential scanning calorimetry (DSC) revealed that the temperature of denaturation of cardosin A decreased upon the addition of AN. With longer incubation times, the small chain of cardosin A denatured completely, consequent exposure of the single tryptophan residue accounting well for the observed spectral shift intrinsic fluorescence of the protein. Enzymatic activity assays demonstrated that the kinetically determined unfolding of the small chain of cardosin A does not result in loss of the activity of this enzyme.

Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas.

A novel adenylate kinase (AK) has recently been purified from Desulfovibrio gigas and characterized as a Co(2+)/Zn(2+)-containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these enzymes are normally devoid of metals. Here, we studied the conformational stability of holo- and apo-AK as a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy. The thermal unfolding of AK is a cooperative two-state process, and is sufficiently reversible in the 9-11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic model. The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the highly sensitive but integral DSC-method.

Thermal stability of peroxidase from the african oil palm tree Elaeis guineensis.

The thermal stability of peroxidase from leaves of the African oil palm tree Elaeis guineensis (AOPTP) at pH 3.0 was studied by differential scanning calorimetry (DSC), intrinsic fluorescence, CD and enzymatic assays. The spectral parameters as monitored by ellipticity changes in the far-UV CD spectrum of the enzyme as well as the increase in tryptophan intensity emission upon heating, together with changes in enzymatic activity with temperature were seen to be good complements to the highly sensitive but integral method of DSC. The data obtained in this investigation show that thermal denaturation of palm peroxidase is an irreversible process, under kinetic control, that can be satisfactorily described by the two-state kinetic scheme, N -->(k) D, 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. On the basis of this model, the parameters of the Arrhenius equation were calculated.