Subunit Level Study On Heat Induced Disulfide Linked Products And Sulfhydryl Change Of Soy Protein

Sulfhydryl group (SH) and disulfide bond (SS) are two important functional groups ofsoy protein. Heating is an essential process in the manufacture of soy protein. SH/SSexchange and SH oxidation are two important reactions when soy protein are heated, in whichsoy proteins are dissociated and rearranged to some new SS linked products contributes to themodification of nutritional and structural properties. The subunit composition and functionalproperties of the SS linked products has been studied in previous studies. However, the SHchange on the subunits/polypeptides of SS linked products has been rarely studied and therelationship of SH/SS exchange and SH oxidation have not been explained clearly. In thisstudy, the subunit/polypeptides of SS linked products was firstly identified; the SH contentchange of different subunit/polypeptides was determined and the evidence for the present ofSH/SS exchange and SH oxidation was provided; their relationship in the formation of SSlinked products was explain quantitatively; whether formation of intermolecule SS was thedriving force in soy protein aggregation was investigated to supplying a strategy to controlaggregation in soy protein processing.An accurate SH content is crucial to understanding the SH reaction. The SH contents ofunheated and heated (90°C,5min) soy protein were detected under different conditions (pH,reagent addition order, SDS/GuHCl concentration, EDTA) using two aromatic disulfidereagents:5,5′-dithiobis-(2-nitrobenzoic acid)(DTNB) and4,4′-dithiodipyridine (DPS). Twofluorescent alkylating reagents, monobromobimane (mBBr) and N-(1-pyrenyl)-maleimide(NPM) were also used. Amino acid analysis was used to detect the SH (cysteine) contents ofunheated (7.51±0.45μmol SH/g soy protein) and heated (1.47±0.10μmol SH/g soy protein)soy protein, and similar results were obtained using enzymatic hydrolysis-assisted DPS. TheSH content detected by DTNB was affected by pH, denaturant species, and denaturantconcentration, and the best results were obtained at pH7.0when6M GuHCl was added afterDTNB. These results were lower than that of the amino acid analysis, however. The SHdetected by DPS was not as affected as that of DTNB by pH, denaturant species, anddenaturant concentration. Additionally, the results of the amino acid analysis were similar tothat of DPS at pH7.0in2%SDS and4to6M GuHCl when SDS and GuHCl were addedafter DPS. EDTA did not have a significant effect on SH detection when DTNB and DPSwere added before SDS and GuHCl. Finally, although mBBr and NPM can detect SH in lowprotein concentrations (1/10of that required for DTNB and DPS), mBBr and NPMoverestimated the SH content of soy protein. Therefore, using DPS at pH7.0when it is addedbefore SDS and GuHCl is the most reliable method for detecting the SH content of soyprotein.The SS-linked components of the unheated and heated oy protein were studied by diagonal SDS-PAGE and MALDI–TOF MS/MS. The glycinin subunit (AB) was themajor SS linked components of the unheated soy protein, in which the basic (B) and acidic (A)polypeptides was linked by SS. In addition, a littlie SS-linked dimer of α’ and/or α andSS-linked dimer of AB exist in unheated soy protein. By heating, the SS-linked polymerscomprised of the B and A of glycinin and a small amount of and of-conglycinin wasthe major SS liked products, in which the B and A was identidied from four glycinin subunit(G1-G4). The SS-linked dimers of B and monomers of A were the minor products by heatingsoy protein. The SH of B in AB (Cys298of G1, Cys289of G2, Cys440of G4) was found tobe converted to SS in polymer, indicating that they was prone to involved in SH/SS exchangeand SH oxidation.By determine the SH change of different subunit/polypeptides by heating, the evidencefor the occurrence of SH oxidation and SH/SS exchange was provided. The SH content ofpolymer was lower than that of AB and α’/α, suggesting the presence of SH oxidation formingpolymer. The SH content of B in polymer was lower than that in AB, and both of themdecreased after heating. The SH content of Ain polymer was increased and higher than that ofB in polymer and Ain AB, indicating the occurrence of SH/SS exchanges resulting in transferof SH from B to A. The SH/SS exchanges was more important than SH oxidation to formpolymer; the content of polymer formed by SH oxidation only account for~30%of totalpolymer production.Soy proteins were reduced by0.1-10mM DTT to obtain samples with increasing SHcontents (7.5-75.9mol/g soy protein). Through heating, larger amounts and sizes of proteinaggregates were formed, and aggregates dispersions with higher viscosity were obtained withincreasing reduction degrees. However, the SS content decreased and the less SS linkedpolymer was formed with increasing reduction degrees, revealing that intermolecular SS arenot necessary for protein aggregation and polymer has somewhat inhibiting effect on proteinaggregates formation. This study was meaningful in supplying a strategy to controlaggregation in soy protein processing.