Studies On The Dissociation Of Porcine Hemoglobin Molecules Induced By Urea And Guanidine Hydrochloride

The dissociation of porcine hemoglobin molecules induced by urea and guanidine hydrochloride was studied through using intrinsic fluorescence emission spectrum, native-PAGE and high-performance size-exclusion chromatography. Through the analysis of the thermodynamic equilibrium between denaturant molecules and porcine hemoglobin molecules, based on the association-dissociation equilibrium, a theoretical model was built to quantitatively describe the dissociation of denaturant-induced porcine hemoglobin molecules. Through the theoretical model, two characteristic parameters k and m which were used to describe the mutual transition between different types of subunits could be obtained. The characteristic parameter k indicates the thermodynamic equilibrium constant of the dissociation of porcine hemoglobin molecules from one type of subunits to another; m indicates the average number of denaturant molecules associated with a porcine hemoglobin subunit during the dissociation process.In the dissociation of urea-induced porcine hemoglobin molecules, when the denaturant concentration was equal to or more than 5.0 mol/L, part of them started to dissociate from original tetrameric molecules to dimeric molecules, and with the increasing of the urea concentration in denaturation solution, the dimeric molecules continuously increased and in this process, k and m were 4.18×10-9 L·mol-1 and 1.69, respectively; in the dissociation of guanidine hydrochloride-induced porcine hemoglobin molecules, when the guanidine hydrochloride concentration was equal to or more than 3.5 mol/L, part of them started to dissociate from their tetrameric molecules into their dimeric and further into monomeric molecules, and with the increasing of the guanidine hydrochloride concentration in denaturation solution, the monomeric molecules continuously increased, dimeric molecules continuously decreased and tetrameric molecules had not changed much, and in this process, k1=7.82×10-7 L·mol-1, k2=3.56×10-9 L·mol"1, m1=1.17 and m2=3.06; by comparing the dissociation induced by urea and guanidine hydrochloride and the characteristic parameters k and m, we found that the dissociated capability of guanidine hydrochloride was stronger than urea. At the same time, by comparing the structural changes and the biological activity changes of the porcine hemoglobin molecules under different denaturant concentrations in denaturation solutions, we found that the biological activity changes of the porcine hemoglobin molecules were faster than their structural changes during their dissociation processes.Two characteristic dissociation parameters k and m we proposed in our research not only can quantitatively describe the distribution and mutual transformation of the subunits in the dissociation of porcine hemoglobin molecules induced by denaturant molecules in different concentrations, but also can describe the degree of the dissociation, providing a more simple and exact theoretical model to describe the dissociation of proteins.