Mechanism Of Transglutaminase-catalyzed Cross-linking Of Oxidatively Stressed Myofibrillar Protein

Microbial transglutaminase （MTG） has been applied cmmerically to improve the texturalproperties of meat products for its catalysis of protein cross-linking through acyl transferreaction between glutamine and lysine residues. However, the quality of meat productsprocessed with MTG is often found variable. Based on the structural changes of MFP induedby oxidation may alter the content of avaliable MTG substrates （e.g. protein unfoldinginduced by oxidation can cause the exposure of more glutamine and lysine residues）, ahopothesis that radical stress affects the cross-linking catalysis of MTG was tested. Theultimate goal of the study is to produce scientific evidence that supports the understanding ofthe relationship between protein structural modification and MTG-mediated cross-linking foroptimized quality of processed meats.Myofibrillar protein （MFP） and myosin were oxidatively stressed in a hydroxylradical-generating system （FeCl3/ascorbic acid/H₂O₂, pH6.25） with different H₂O₂concentrations and then subjected to MTG （E:S=1:20）. With this general oxidizing system,four individual experiments were performed to investigate:（1） effects of MFP oxidation onthe cross-linking degree of MTG catalysis;（2） effects of MFP oxidation on secondarystructure and cross-linking location （myosin subfragments as S1, S2, rod, LMM, HMM） inMFP after MTG catalysis;（3） effects of myosin oxidation on isopeptide bond （-CONH-）formation and the specific mass spectrometry-verified cross-linking sites after MTG catalysis;（4） effects of MFP oxidation on MTG cross-linking degree and functionality of MFP undercommon factors influencing the manufacture of meat products （different salt concentrationsand temperatures）.The relationship between structure changes of oxidized MFP and MTG cross-linkingdegree at high salt concentration （0.6mol/L NaCl） was studied. The results showed thatprotein unfolding, polymerization and the modification of amino acid residues coexistedduring oxidation. The degree of MTG cross-linking was dependent on dominant factors. Fornonoxidized MFP, the cross-linking degree was11.6%. For mildly oxidized MFP （1mmol/LH₂O₂）, protein unfolding was the main facor, which promoted MTG cross-linking reactionbecause of the exposure of more available glutamine and lysine substrates.The cross-linkingdegree of mildly oxidized MFP was improved to27.6%. For deeply oxidized MFP （5and10mmol/L H₂O₂）, polymerization and the modification of amino acid residues dominated, whichblocked MTG cross-linking reaction because of reburying or modifying of many availableglutamine and lysine residues. The cross-linking degree of deeply oxidized MFP was as lowas9.6%.The effect of protein oxidation at high salt concentration （0.6mol/L NaCl） on thesecondary structure of MFP when treated with MTG was determined by CD and FTIR. Theuncoiling of MFP occurred when subjected to either oxidation or MTG cross-linking. Thechange of ɑ-helix induced by MTG was related to oxidation degree: mild oxidation （0.1–1mmol/L H₂O₂）>nonoxidation>advanced oxidation （5–20mmol/L H₂O₂）. The influence ofprotein oxidation on the cross-linking location of MTG was studied using chymotrypsinthrough digesting myosin into different fragments （S1/Rod and HMM/LMM）. The results indicated that the main cross-linking location of nonoxidized myosin was S1. For unfoldedmyosin caused by mild oxitaion （0.1–1mmol/L H₂O₂）, every fragment was the target of MTG,especially S1. For polymerized myosin caused by excessive oxidation （5–20mmol/L H₂O₂）,S2played an important role in MTG cross-linking.Myosin was purified to study the cross-linking degree, cross-linking location andcross-linking sites of differently oxidized myosin and to compare with those of MFP. Theε（γ-G）L result showed that the cross-linking degree of mildly oxidized myosin （1mmol/LH₂O₂） was87.6%, which was significantly higher （P<0.05） than nonoxidized （64.7%） andexcessively oxidized myosin （200mmol/L H₂O₂,33.8%）. The trend of cross-linking degreewas similar with that of MFP. The cross-linking location of oxidized myosin was also similarwith that of MFP: for nonoxidized myosin, the main cross-linking position was S1; for mildlyoxidized myosin, both Rod and S1were easily oxidized, in which S1was the main one; thecross-linking location of excessively oxidized myosin can not be obtained since it can not bedigested strictly into specific fragments （S1/Rod or HMM/LMM）. Unlike excessivelyoxidized myosin, an aggregate which can not be digested into S1and Rod was founed innonoxidized and mildly oxidized myosin. Two possible cross-linked peptides were founed inthis aggregate by MALDI-TOF/TOF MS: IQ（IEFKK）AQAR was formed throughcross-linking between the790glutamine of peptide789–794（IQAQAR） and the803lysine ofpeptide800–804（IEFKK） in myosin head; SNNFQK（LQLK）PR was formed throughcross-linking between the565lysine of peptide560–567（SNNFQKPR） in myosin head andthe1872glutamine of peptide1872–1875（LQLK） in myosin tail.MFP was oxidized at three salt concentrations （0.15,0.45and0.6mol/L NaCl） andsubsequently cross-linked at three temperatures （4,15and30°C）, and then the effect ofprotein oxidation on the MTG cross-linking and gelation was studied. At all saltconcentrations and temperatures, the cross-linking degree of almost all oxidized MFP washigher than the nonoxidized, and increased with elevated salt concentration and temperature.For example, the cross-linking degree of2h-oxidized sample at0.6mol/L NaCl was46.8%when treated with MTG at15°C, higher than the31.6%of nonoxidized sample. It was alsohigher than the27.2%of the corresponding sample at0.15mol/L NaCl and15°C as well asthe18.2%of the corresponding sample at0.6mol/L NaCl and4°C. The effect of proteinoxidation on the gelation of MFP when treated with MTG was determined by gel strength anddynamic rheology. Gels formed by oxidized MFP at0.6mol/L NaCl （92.6%–120.3%） werenotably stronger （P<0.05） than that of nonoxidized protein gels （79.0%） when treated withMTG at4°C. However, excessive cross-linking treatment （e.g. improving temperature） washarmful to the network structure of gels. The improvement of gel strength caused by MTGcross-linking at4and15°C in MFP oxidized at0.15M NaCl was lower than the nonoxidized.The final storage modulus change of oxidized MFP at both salt concentrations when treatedwith MTG was higher than the nonoxidized. Therefore, the oxidation of MFP at high saltconcentration was a good method to improve the quality of meat products when treated withMTG.In summary, the effect of oxidation on the MTG-catalyzed of MFP and myosincross-linking （degree, location, and site） was studied, and then verified by protein gelation. The mechanism of MTG catalysis influenced by protein oxidation was clarified, whichprovided a theoretical basis to explain the problems occurring in situ and to develop strategiesto over the problem when MTG is used as a meat processing aid.