Thermally induced conformational changes in horseradish peroxidase

Abstract

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic schemewhere 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.Keywords: horeseradish peroxidase; differential scanning calorimetry; intrinsic fluorescence; circular dichroism; irreversible denaturation.Horseradish peroxidase (HRP) belongs to the superfamily of the heme-containing plant peroxidases (EC 1.11.1.7), which has been divided into three classes [1], supported in the first instance by comparison of amino-acid sequence data and confirmed by more recent data on crystal structures [2]. Plant peroxidases, including HRP, comprise class III of the superfamily. Although the function of peroxidases is often seen primarily in terms of the conversion of H 2 O 2 to H 2 O, this should not be allowed to mask their wider participation in other reactions, many of which are biologically significant. Despite the enormous interest in peroxidases owing to their broad practical applications in biotechnology, the data concerning their structural stability are sparse. Although several publications have addressed the thermal stability of peroxidases [3±8], to date the mechanism of the process of thermal denaturation remains unclear. It is known that the biological functions of proteins depend on the correct folding of their native structure and that loss of this folded structure leads to an unfolded, inactive state. Consequently, the study of protein stability is important both from the academic and applied points of view.Factors affecting conformational stability have been studied most intensively in proteins under reversible conditions [9±15]. Nevertheless, it is well known that for different reasons many proteins cannot refold in vitro after denaturation such as proteolytic digestion [16], aggregation, loss of prosthetic group, the cis/trans izomerization of certain proline residues [17,18] or chemical modifications [19]. Generally, the thermal denaturat...

Microneedles with Intrinsic Immunoadjuvant Properties: Microfabrication, Protein Stability, and Modulated Release

“…7). Thermal inactivation of HRP can be conveniently monitored using a chromogenic substrate system providing a reliable correlate with protein stability, as defined by thermally induced conformation changes (24,25). In such system we also have not observed any aggregation in HRP solutions both in the presence and absence of polymer (size-exclusion HPLC, data not shown), which is in accord with earlier findings (26).…”

Use of fluorescence to monitor the incorporation of horseradish peroxidase into a sol–gel derived medium

“…It is reassuring to know that the encapsulated HRP displays catalytic activity as the matrix forming reaction could damage the enzyme, in terms of structure or removal of the heme group. The latter has been shown to occur in acidic conditions (below pH 3) [24]. The form of the curves reflects the fact that as the matrix shrinks the pore interconnections narrow and diffusion of the reactants becomes harder, hence the longer time taken to achieve maximum activity.…”

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

“…Enzyme Activity and Biochemical Characterization HRP concentrations were determined by spectrophotometer considering an extinction coefficient of 102 mM −1 cm −1 at 403 nm [23][24][25][26]. Horseradish peroxidase activity was measured spectrophotometrically at room temperature using phenol/4-aminoantipyrine and H 2 O 2 as substrates.…”