The kinetics of the thermal deactivation of transglutaminase from guinea‐pig liver

Abstract

By incubating native (N) transglutaminase from guinea-pig liver at various temperatures and assaying it at 2572, two steps in the irreversible deactivation process to the denatured form (D) have been found. The fitting of the data to the equations of two possible models (the two-steps model and the two-isoenzymes model) is only compatible with the first one (N ---* X + D). It is shown that the structure of the active intermediate, X, depends on the deactivation temperature and on the thermal history of the enzyme. This may mean that transglutaminase exists in a large number of microstates. Surprisingly, the activation energy of deactivation is lower than that of activity (36.6 & 3.4 against 47.2 2.2 kJ . mol-I). By deactivating transglutaminase at a constant temperature (55°C) and assaying it at variable temperatures, the activation energy of the intermediate, (X55), has been determined to be 40.2 _+ 5 kJ . mol-l, of the same order of magnitude as the native form.Among several agents assayed, only Ca2+ had a positive effect on the thermal stability of this enzyme. At 40°C, transglutaminase was quite stable in the presence of Ca2+ (in its absence, the half-life was 65 min) and at 45 "C, its thermostability had been considerably increased, the half-life being raised from 47 min to 275 min.Transglutaminase is a Ca2 +-dependent enzyme responsible for post-translational modifications of proteins. It allows the cross-linking of proteins like fibrin, collagen and uteroglobulin [l -51. It is a tool for in vitro modifications ofproteins and especially for making gels [6 -81. Although the kinetics of the reversible thermal deactivation of proteins (and especially enzymes) has been extensively studied [9], the kinetics of the irreversible process has been the object of only a few papers [lo -121. To increase our knowledge in this basic area, we have carried out such a study on the transglutaminase from guinea-pig liver. This basic study may also be of some interest on potential uses of this enzyme. Knowledge of the kinetics of its irreversible deactivation may be essential for the development of rational approaches to enzyme stabilization, e. g. enhancement of enzyme thermostability by such agents as CaZ + . Therefore, the aim of this paper was to study the effect of temperature on the activity and on the stability of transglutaminase from guinea-pig liver and the role of Ca2+ in the enhancement of its thermostability. MATERIALS AND METHODS Purification of ' transglutaminaseEnzyme was extracted and purified from guinea-pig liver according to the method of Brookhart et al. [13], slightlyCorrespondence to J

Transglutaminase cross-linking to enhance elastic properties of soy protein hydrogels with intercalated montmorillonite nanoclay

“…The temperature corresponding to the onset of G 0 and G 00 decrease (53°C) coincided with the ca. 55°C deactivation temperature of mTGase (Nury et al, 1989). The decrease of G 0 and G 00 at 53-80°C may have resulted from the reorganization of enzymatically cross-linked structures.…”

Calcium Ion-Dependent Increase in Thermostability of Dextran Glucosidase fromStreptococcus mutans

“…In the absence and the presence of CaCl 2 , the denaturation kinetics of SmDG were not described by a single exponential decay, but by the two-step irreversible denaturation model, which was used to describe the deactivation of B. licheniformis -amylase 24) and several other enzymes. 25,26) The model postulates that a fully active native enzyme (N) is transformed into a completely inactive form (D) via an active intermediate (X) through two irreversible steps. The deactivation intermediate of SmDG in the absence of any additional salt retained 39% of the activity of intact SmDG.…”

“…24) This model has been used to describe the thermal denaturation of Bacillus licheniformis -amylase, 24) and has been employed for other enzymes. 25,26) We applied the two-step irreversible denaturation model to evaluate the thermostability of SmDG and to investigate the effect of calcium ions on thermostability. The thermal denaturation was described well by the model, and the presence of Ca 2þ enhanced thermostability.…”

Effects of the regulatory ligands calcium and GTP on the thermal stability of tissue transglutaminase

“…To understand the relevance of degradative effects in the biology of transglutaminase, we decided to study the effects of regulatory ligands on the in vitro thermal stability of tissue transglutaminase, complementing previous investigations on the sensitivity to proteinases (Casadio et al 1999) and to chemical denaturants (Cervellati et al 2009). The issue of thermal stability of tissue transglutaminase has been marginally dealt with previously, mostly in relation to exposition to shifts in pH (Lichti et al 1985;Nury et al 1989;Nury and Meunier 1990;Bergamini et al 1999) but not in relation to effects of physiologically relevant ligands. We now discuss the new results we have obtained in relation to transglutaminase stability and unfolding by combined approaches aimed to correlate inactivation and structural perturbations of the protein.…”