Unfolding Of Bovineinsulin Induced By Guanidine Hydrochloride And Urea

The unfolding of bovine insulin molecular induced by urea and guanidine hydrochloride were investigated with the combined methods of fluorescence spectra, phase diagram, ANS-binding assay, fluorescent quenching and circular dichroism spectra,then the existence and character of partially-folded intermediates were identified and described via the analysis of conformational and secondary structure changes of insulin in various concentrations of denaturants. Based on the mentioned results and the measurement of residual bioactivity ratio in denaturants solution, relative theoretical model was created to quantitatively present the transition and distribution of steady conformational states with the characteristic parameters m and k. Finally, thermal stability of different conformational states were detected with differential scanning calorimetry method and thermo dynamical parameters (entropy and enthalpy) were calculated by plotting of Van t-Hoff curves based on the molecular retention on RPLC. The corresponding results will provide beneficial information for the research of folding of insulin molecular.Fluorescence spectra measurement showed that fluorescence intensity was gradually increasing with the change of denaturant concentration in the unfolding of insulin molecular. The phase diagram and ANS-binding assay indicated that there were two steady intermediates in the guanidine hydrochloride induced unfolding of insulin molecular at denaturant concentration being about1.5mol·L-1and6.0mol·L-1respectively, which follows "four-states model" meanwhile the second intermediate I2exposed the hydrophobic region surface to the solution which could interact with hydrophobic reagent8-anilino-l-naphthalene sulfonic acid (ANS). While in the urea induced unfolding pathway, a partially-unfolded intermediates presented at urea concentration being about2.5mol·L-1, which also existed some ANS accessible hydrophobic region. Circular dichroism spectra have suggested that in the guanidine hydrochloride induced unfolding of insulin, the first intermediate have similar secondary structure ratio to native state while the second intermediate lost most ordered secondary structure and became more flexible. In the unfolding of insulin induced by urea, the intermediate lost about50%secondary structure comparing with native state.Based on the theoretical model of conformational state transition and distribution, it is concluded that in the Gdmcl-induced unfolding pathway, in order to transit from one state to another, the number of denaturant moleculars an insulin molecular interact with were m1=1.01、m2=5.16、 m3=0.63respectively and thermodynamic equilibrium constants in each process were k,=2.97×10-1, k2=3.90×10-4, k3=1.47×10-1.While in the urea induced unfolding process, the corresponding parameters were m1=2.18, m2=5.16and k1=2.7×10-2, k2=1.01×10-6.The parameter m and k were indicators of the stability of all conformational states. Via k and m the distribution of all states in a series concentration of denaturants were well described.Differential scanning calorimetry measurement have demonstrated that the denaturation temperature of insulin conformational states (N,I1,I2, U) in the guanidine hydrochloride induced unfolding pathway were89.1,91.9,72.1,68.5℃, which indicated that N and I1were much stable than I2and U. The enthalpy of each state interacting with C18were-1.794,-2.070,-2.128,-2.095kJ·moL-1,△H0<0,showed that the insulin unfolding was a heat release process and I2released more energy. AS0*>0demonstrated that the insulin molecular gradually changed from an ordered state to disordered ones. In the guanidine hydrochloride induced unfolding of insulin,AG0<0, a spontaneous process was suggested to the insulin unfolding. While in urea induced unfolding process of insulin, the denaturation temperature of native, intermediate, unfolded state were89.1,60.2、37.5℃,the insulin structure turned from a relatively compact state to be a loose state and the molecular thermal stability was decreasing. Furthermore, thermodynamics characteristic parameters:△Ho<0,△S0*>0,△G0<0suggested that the unfolding pathway was a spontaneous process that insulin molecular unfolded from ordered to disordered structure.