RESUMO
The effect of succinimidyl carbonates activated methoxypolyethylene glycol (mPEG-SC) on the catalytic properties and conformation of native trypsin and dynamic high-pressure microfluidisation (DHPM) induced unfolded trypsin was studied. The thermal stability of unfolded trypsin was enhanced more significantly than that of native trypsin between 45 and 70°C. The autolysis analysis indicated that modified unfolded trypsin was markedly more resistant to autolysis compared to modified native trypsin between 40 and 180min. Upon mPEG-SC conjugation, the Km value of the enzyme decreased by about 2-fold, and the catalytic efficiency (Kcat/Km) increased by about 3-4-fold. Moreover, the increased thermal stability of unfolded trypsin might be due to the lower surface hydrophobicity and the higher hydrogen bond formation after mPEG-SC modification, which was reflected in the decrease of UV absorbance, the quenching and blue shift of fluorescence spectra, as well as the increase of ß-sheet content.
Assuntos
Polietilenoglicóis/química , Tripsina/química , Biocatálise , Temperatura Alta , Cinética , Conformação Proteica , Dobramento de Proteína , Estabilidade ProteicaRESUMO
Few reports have focused on the effect of citric acid on thermodynamics and conformation of polyphenoloxidase (PPO). In this study, variations on activity, thermodynamics and conformation of mushroom PPO induced by citric acid (1-60mM) and relationships among these were investigated. It showed that with the increasing concentration of citric acid, the activity of PPO decreased gradually to an inactivity condition; inactivation rate constant (k) of PPO increased and the activation energy (Ea) as well as thermodynamic parameters (ΔG, ΔH, ΔS) decreased, which indicated that the thermosensitivity, stability and number of non-covalent bonds of PPO decreased. The conformation was gradually unfolded, which was reflected in the decrease of α-helix contents, increase of ß-sheet and exposure of aromatic amino acid residuals. Moreover, two linear relationships of relative activities, enthalpies (ΔH) against α-helix contents were obtained. It indicated that changes of activity and thermodynamics might correlate to the unfolding of conformation.
Assuntos
Agaricales/enzimologia , Catecol Oxidase/química , Ácido Cítrico/farmacologia , Proteínas Fúngicas/química , Agaricales/química , Catecol Oxidase/antagonistas & inibidores , Catecol Oxidase/metabolismo , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Cinética , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , TermodinâmicaRESUMO
OBJECTIVE: To investigate the effect of Uncaria rhynchophylla total alkaloids (RTA) pretreatment on the voltage-gated sodium currents of the rat hippocampal neurons after acute hypoxia. METHOD: Primary cultured hippocampal neurons were divided into RTA pre-treated and non-pretreated groups. Patch clamp whole-cell recording was used to compare the voltage-gated sodium current amplitude and threshold with those before hypoxia. RESULT: After acute hypoxia, sodium current amplitude was significantly decreased and its threshold was upside. RTA pretreatment could inhibit the reduction of sodium current amplitude. CONCLUSION: RTA pretreatment alleviates the acute hypoxia-induced change of sodium currents, which may be one of the mechanisms for protective effect of RTA on cells.
