Study On Unfolding And Subsequent Refolding Behaviors Of Saurida Myosin Subjected To PH-shifting

pH-shifting, a novel surimi production method, can improve protein yield and functionalproperties of muscle proteins to a certain extent. However, the relationship between thefunctional properties of muscle proteins and their structures was not well elucidated. In thiswork, saurida myosin was selected as a model system. First of all, the unfolding behaviorof saurida myosin was investigated over a wide range of pH values (2.0-12.0), as well asfurther analysis of the effect of different types of acids and alkalies under extreme pHconditions on myosin unfolding behavior. Then, the folding behavior of acid-andbase-induced myosin at various salt concentrations and conformational changes of myosinunfolding by two types of acids and alkalies and subsequent refolding through twopathways were also studied. This study may provide a theoretical reference for the utilityof pH-shifting. Meanwhile, an understanding of how myosin unfold at low or high pH andrefold on subsequent pH readjustment on the molecular level may provide us keyinformation, which enables us to produce different protein structures with differentfunctional properties. The main results are as follows:1. One major myosin heavy chain band at200kDa, followed by3light chains at26,22,18kDa were observed on SDS-PAGE gel. Protein purity was97%, meeting therequirements of follow-up study. Solubility of myosin was greater than any other pHs atextreme pHs. Hydrophobicity reached its minimal at isoelectric point (pI). Total sulfhydrylcontents reached maximum values (24.9mol/105g and11.3mol/105g) under near-neutralconditions and disulfide bond formation of myosin was promoted under extreme pHconditions. At600mmol/L KCl, the isoelectric point of saurida myosin was shifted to theacidic direction (5.0â†'4.0). Total sulfhydryl contents were less under alkaline pHs than thatat acdic pHs. At extremes acidic and alkaline pHs, the trend of α-helicity appeared to besimilar to the solubility of myosin, while hydrophobicity reversely correlated with. At lowpHs, surface hydrophobicity (ANS-S0) values at60mmol/L KCl were higher than those at600mmol/L KCl. Furthermore, myosin regained helixed at high KCl concentration.2. ANS-S0values of strong acid treated myosin increased in the following order:hydrochloride acid (HA)~sulfuric acid (SA)> phosphoric acid (PA); For organic acids,the order was: citric acid (CA)> lactic acid (LA)> acetic acid (AA). Tryptophanfluorescence of acid-treated myosin was not significantly affected by different acids. Theα-helix content of myosin treated by LA was significiently different from other acids, but no significant differences among other acids treatment. Total sulfhydryl contents ofacid-induced myosin decreased with decreasing pHs. At pH=2.0, total sulfhydryl content ofthree kinds of organic (AA, LA and citric CA) induced myosin was less than other that ofthree strong acid treatment. Under alkaline conditions, with an increase in pH value,ANS-S0values and tryptophan fluorescence of myosin decreased. Myosin treated withammonia water showed the minimum ANS-S0value and tryptophan fluorescence intensity.Myosin treated by ammonia water gave lower ANS-S0value, but with more α-helicalcontent and fluorescence intensity than did the other two alkali treatment. There was nosignificant difference between conformational changes of myosin subjected to KOH andNaOH treatment. Ammonia water treatment resulted in minimum myosin denaturation.3. In the range of0.4-0.6mol/L KCl, solubility of myosin under three pH conditionswere more than91.6%. When KCl concentrations>0.6mol/L, the solubility of myosindecreased under three pH conditions, and reduced slowly at neutral and alkaline pH, whileappeared insoluble at acidic pH. When KCl concentrations was at0-0.04mol/L, ANS-S0atextreme acid and alkaline pH was greater than the neutral value (p<0.05). Saltconcentration>0.6mol/L, under both the alkaline and neutral conditions, the ANS-S0values of myosin increased with increasing KCl concentration, and basic conditions gaverise to more ANS-S0values. At pH2.0, with increasing salt concentration, the totalsulfhydryl decreased slowly. With KCl concentration>0.6mol/L, the total sulfhydrylcontent at pH7.0decreased, while under alkaline conditions it appeared to increase slowly.Electrophoresis analysis showed increasing salt concentrations (0-0.6mol/L) under basiccondition promoted the formation of macromolecular myosin polymers. Both pH and saltconcentration had an interactive effect on α-helix content of myosin. Under neutralconditions, the α-helical content was low at0-0.2mol/L KCl; while under neutral andalkaline conditions, when KCl concentration was greater than0.6mol/L, the α-helicalcontent increased with increasing salt concentration. Micro DSC analysis showed twosharp endothermic peaks between30-60oC and salt increased myosin thermostability. Saltresulted in less myosin unfolding at acidic pH and more unfolding at both neutral andalkaline pH.4. The hydrophobicity increased on refolding of myosin, while tryptophan fluorescenceintensity, total sulfhydryl content and α-helicity were found to decrease compared to thecontrol (p<0.05); Refolding myosin from alkaline condition led to higher ANS-S0valueand α-helical content than from acid condition. There was no significant differencebetween total sulfhydryl of myosin unfolded by two strong alkalics after refolding. TheANS-S0values and α-helix content of CA-treated myosin were lower than HA-treated group after myosin refolding. Total sulfhydryl content of myosin between HA and CA wasnot significantly different; ANS-S0and α-helical content of IP refolding group myosinwere higher than those of NP refolding treatment, but no significant difference intryptophan fluorescence intensity. Base type, pH and refolding pathway, each was closelycorrelated with alkali-induced unfolding and susequent refolding conformation of myosin.Tryptophan fluorescence intensity of myosin treated by NaOH was higher than that ofKOH treated group, but with lower α-helix content. IP refolding groups showed greaterANS-S0and less α-helical content than the NP refolding groups with no significantdifference in tryptophan fluorescence intensity and total SH content. Refolding led to lowertransition temperature and no significant difference was observed in the first transitiontemperature between acid and base treatment, but not the same as the second transitiontemperature. Under the same pH conditions, IP refolding treatment gave rise to higherthermal transition temperature than that of NP refolding treatment.