RESUMEN
One of the important physicochemical features of the proteins specifically multi-subunit types is their stability at high temperatures. The kinetics of the dissociation and denaturation of proteins possessing at least two subunits has certain challenges because the overall mechanism of dissociation can include hidden reversible and/or irreversible steps (conformational lock). There are numerous proteins related to diseases which are in equilibrium with oligomer forms. This equilibrium plays an important role in holding the activity levels of these proteins in vitro and in vivo. The stability of proteins is an essential milestone to study conformational changes of the proteins in the living cell. This concept is of great importance when proteins are used in biomedicine fields. The quaternary structure of multi-subunit proteins includes properties that affect function and kinetics of denaturation. The kinetics of denaturation may include some hidden stages of conformational transitions in the protein and steps of reversible dissociation of the oligomer. The transforming factors affect different areas especially active centers in the clefts of each subunit. The distinctive agent among all, is the temperature which sequentially destructs independent inter-subunit contact sites. Recent experimental data regarding dissociative mechanism for irreversible thermal denaturation of multi-subunit proteins have been summarized in the present paper.
Asunto(s)
Enzimas/química , Proteínas/química , Calor , Cinética , Modelos Moleculares , Transición de Fase , Conformación Proteica , Desnaturalización Proteica , Estabilidad Proteica , TermodinámicaRESUMEN
The kinetics of thermal inactivation of bovine carbonic anhydrase (BCA) was studied in a 50 mM Tris-HCl buffer, pH 7.8 using p-nitrophenyl acetate as substrate in absorbance of 400 nm by UV-VIS spectrophotometry. The number of conformational locks and inter-subunit amino acid residues of BCA were obtained by thermal inactivation analysis. The cleavage bonds between dimers of BCA during thermal dissociation and type of interactions between specific amino acid residues were also detected. The thermal inactivation curves were plotted in temperatures ranging between 40-70°C. It was shown several phases for inactivation of BCA at 65°C. Analyses of the curves were done by the conformational lock theory. The subunits are dissociated and several intermediates appear during inactivation through increasing the temperature in comparison with native state. Dynamic light scattering measurements was done to study the changes in hydrodynamic radius during thermal inactivation. Three distinct zones were shown in DLS data. Biochemical computation using ligplot is performed to find the inter-subunit amino acid residues for BCA.