Study On Unfolding And Refolding Behaviors Of Tilapia Myosin Subjected To PH-shifting Method

The change of pH is an unavoidable part during the food processing,and one of the fundamental to produce food protein isolates.pH have a big influence on the properties such as solubility,gelation,emulsification of fish peotein,and myosin is responsibility for the functional properties of fish proteins.Changes in the functional properties of proteins are fundamentally caused by the change of protein structure,so it's necessary to fully understang the relationship between myosin structure and pH's change.In this study myosin was extracted from Tilapia using ammonium sulfate precipitation method?purity>92%?,and solubility,surface hydrophobicity,total and active sulfhydryl content,intrinsic tryptophan fluorescence spectra,circular dichroism and m-DSC was determined,changes of conformation of tilapia myosin during pHs-induced unfolding and refolding in three salt concentration?0.02 mol/L,0.15 mol/L,0.6mol/L KCl?was studied.On this basis,artificial chaperone system composed SDS and ?-CD was applied to the refolding of myosin to invesigate the effect and mechanism of artificial chaperone-assiatance refolding of myosin.In order to achieve the puepose of promote the valued-added utlization of low-cost aquatic protein resources and development of new protein resources,and thus result in expanding the application of ythe aquatic protein.The main results are as follows:1 There was an increased surface hydrophobicity,weaker tryptophan fluorescence intensity,decreased total SH content and ?-helix content when the pH of the myosin envoronment deviates from 7.0 at 2.0-12.0,and under extreme pH condition the surface hydrophobicity get maximum and lowest tryptophan fluorescence intensity,which indicated that myosin unfolding to a certain degree,and there was a positive correlation between the degree of unfolding and extent of pH deviates from 7.0.The decreased of surface hydrophobicity when pH change from 3.0 to 2.0 or 11.0 to 12.0 may esplained by proteins would undergo unfolding at high or low pH and a further increase or decrease in pH could make protein refold into molten globular conformation.The decrease of the total SH content and SDS-PAGE further indicated that the formation of polymer?>200 KDa?in myosin solution treated with pHs was the result of the formation of disulfide bonds between exposed-SH of the MHC.Micro DSC analysis showed,for the pH 7.0 samples in0.6 mmol/L KCl,3 typical endothermic transition peaks were observed at 38.4,45.4,47.9;And for the other unfolded myosin,thermal denaturation temperature T_m1 and T_m2)changed slightly,but the corresponding enthalpy incerased,most T_m3 and corresponding enthalpy were lost,indicating chemical denaturation of myosin occurred before thermal treatment.2 In comparison,the surface hydrophobicity and solubility of myosin in pH 7.0increase with the increase of salt concentration,suggesting that the salt ions can enhance the interaction between myosin and water by a salting-in effect.The result of solubility and SDS-PSGE showed that the isoelectric point of myosin in 0.02,0.15,and 0.6 mmol/L KCl occurs near 5.5,5.0,and 4.5,respectively,salt ions has the ability to shift the isoelectric point of myosin towards the acidic side.Compare the unfolding behaviorof myosin induced by pH in three ionic strength?0.02,0.15,0.6 mmol/L KCl?,he surface hydrophobicity decrease and tryptophan fluorescence intensity increase with the increase of salt concentration,which indicated that salt ion has the ability to protect myosin from denaturation.3 Unfolding of myosin induced by different acid?HCl,H₂SO₄,H₃PO₄ and C₆H₈O₇?were studied at extreme acid?pH 2.0,3.0?,surface hydrophobicity and reactive SH content of myosin increased by different acid treatments,tryptophan fluorescence intensity and?-helix content decreased,suggesting acid-induced unfolding of myosin.And at pH 2.0,myosin has a greater degree of unfolding than that at pH 3.0.In comparison,the C₆H₈O₇-induced unfolding degree was the highest at pH 2.0 and the ?-helix content of myosin decreased from 41.7% to 20.5%.However,at pH 3.0,the H₂SO₄-induced unfolding was the highest,and resulted in the significantly decline of solubility.The denaturation of myosin during HCl-induced unfolding is the minimum,and no obvious change of ?-helix content was obtained,indicating that myosin molecules was stable and retaining a “molten globule” state.4 In high ionic strength solutions,unfolded samples were readjustment to pH 7.0,resulted in the decrease of surface hydrophobicity and increase of tryptophan fluorescence intensity,but when compare to the control the refolded myosin especially samples refolded from extreme pH still get higher surface hydrophobicity,lower tryptophan fluorescence intensity and ?-helix content.Indicating that the tertiary structure of the refolded myosin could not return to the native state.The total sulphydryl content of the refolded myosin decreased and the reactive sulphydryl content increased compare to the control,suggesting that exposed sulphydryl groups on refolding were partial recovery.With respect to unfolding of myosin in 0.6 mmol/L KCl,isoelectric point adjustment processing before unfolding make decrease of surface hydrophobicity and Active/Total SH,weakertryptophan fluorescence intensity and higher surface hydrophobicity,which result in a greater denaturation and poor stability.5 While,in physiological and low ionic strength solutions,surface hydrophobicity,tryptophan fluorescence intensity and sulphydryl groups content of refolded myosin were lower than those of control.The ?-helix content of myosin refolding at different condition changes significantly,the main trend as follows: at neutral pH,the ?-helix content was highest and no changes,but at isoelectric point or extreme pH,the ?-helix content was very small.6 Artificial chaperone-assisted refolding myosin were studied,the result shows that the concentration of SDS and ?-CD at extreme acid pH?2.0,3.0?were 4.0 mmol/L and8mmol/L,for extreme alkaline pH?11.0,12.0?,it is 2.0mmol/L and 4mmol/L respectively.Under 0.6 M KCL,the surface hydrophobicity of myosin refolding with artificial chaperone was 4500,whereas without ACA the ANS-S0 was 7000;The result of tryptophan fluorescence intensity and CD of myosin refolding with/without artificial chaperone indicate that artificial chaperone have a good effect on refolding processing in both low and high ion strength,however artificial chaperone promated the denaturation of myosin during refolding in physiological ionic strength.The result also showed that artificial chaperone have a good effect of refolding when myosin unfolding at alkaline.