The Distribution, Preparation And Interaction With Heavy Metals Of Thiol-containing Peptides In Soy Glycinin

Thiol-containing compounds are widely applied in health care and pharmaceutical products because of their specific bioactivities. However, most of them are synthetic. Natural thiol-containing compounds are gaining more and more attentions own to their many advantanges including safety and low health cost. The research object of this study is soy glycinin. First of all, the changes of the content of sulfhydryl groups（SH） and distributions of thiol-containing peptides（TCPs） in the process of hydrolysis were studied. And then TCPs in soy glycinin hydrolysates（SGHs） were prepared by the method of DTT reduction-covalent chromatography, which was highly specific and high throughput. At last, the interactions between thiol-containing peptides and heavy metal ions were studied. This study provides a certain theoretical basis for the preparation of novel functional peptides from food protein hydrolysates.1. The method of monobromobimane-high performance chromatography（m BBr–HPLC） method was established to detect TCPs, and its application in soy protein hydrolysate was further analyzed. The effects of the reaction conditions, including p H, temperature, time and the m BBr dosage, on the SH labelling by m BBr were studied with the reduced glutathione（GSH） as the standard. The optimized SH labelling condition was: p H being 7.5, temperature being 25°C, reaction time bing 90 min and the m BBr dosage being 10 times of the content of SH. Under this condition, m BBr which did not react with other side chain groups showed high specificity for SH and non-thiol-containing peptides in soy glycinin hydrolysates did not affect the labelling of SH. The fluorescence intensity of the reaction product showed no significant different avoid of light at 4°C in for 15 days, indicating a high stability. At concentration of SH within the scope of the 10-90 μM, fluorescent area and SH concentration showed good linear relationship, precision and accuracy were high, and the recovery of standard added was acceptable. Different soy TCPs showed no significant difference in fluorescence coefficient after labelling with m BBr. The labelling with m BBr showed a great effect on chromatographic retention behavior of TCPs. For RP-HPLC, the relationship between retention times of TCPs（t） and retention times of TCPs labelled with m BBr（t₀） was t=t₀+45.814×t₀^-0.863; and for SEC-HPLC, the relationship between them was t=0.7987+3.4713×t₀.2. Reduced soy glycinin was hydrolysated by three commercial protease: Alcalase, papain and pepsin. The changes of DH in the process was monitored by p H-Stat method. The results showed that 120 min gaven the maximum degree of hydrolysates for Alcalase, the hydrolysis rate of papain after 120 min was very slow and papain also showed a slow hydrolysis rate after 120 min. The reduction of disulfide bonds increased the hydrolysis rate of 11 S significantly. The changes of conent of SH was determined by m BBr-HPLC method. In the protection of N2 and ethylenediamine tetraacetic acid diamine（EDTA）, the content of SH showed no change during the intial 30 min, and then decreased with different degrees.The retention rate of thiol groups in Alcalase and papain hydrolysate was 75.2% and 83.2% after 2 h. The content of thiol groups showed no significant change during pepsin hydrolysis. The content of SH in SGHs with different hydrophobicity and molecular weight distribution was studied by the comparision of the fluorescence chromatograms of SGHs labelled with m BBr and ultravilate chromatograms of SGHs without labelling. For Alcalase and papain, the difference of the distribution of SH in hydrolysates with different structures became more and more small and tended to be more dispersed during the hydrolysis. For pepsin, the content of SH in the high molecular weight peptides was very low, and was high in the low molecular weight peptides. This distribution changed little during the pepsin hydrolysis.3. A highly selective procedure to extract TCPs from complicated SGHs was described. Firstly, the disulfide bonds of SGHs was reduced by DTT and he concentration of DTT was optimized in order to increase the content of TCPs in SGHs. And then the TCPs was prepared by Thiopropyl-Sephrose 6B covalent chromatography. The adsorption and desorption conditions of this method were studied. The results indicated that the optimezed concentration of DTT was 20 m M. The content of SH was 113.8 μmol/g after redution with being 1.8 μmol/g before reduction. The optimized conditions for the covalent chromatography was: the loading of the SGHs being 80% of the active site, bonding time being 30 min; the concentration of DTT for desorption being 20 m M, the desorption time being 20 min. In addition, C18 column could effectively remove DTT and 2-TP form TCPs. MALDI-TOF-MS showed that 36 of the 45 extracted peptides were TCPs, indicating that Thiolpropyl-Sepharose 6B showed high selectivity for TCPs.4. TCPs with different SH content and molecular weight distribution were prepared. The Pb²⁺, Cd²⁺ and Hg²⁺ binding capacities of TCPs were studied by the method of p Hpotentiometry with the stability constants as the indicator. It was indicated that TCPs from different SGHs with the same DH but different protease had different SH content as well as molecular weight distributions. The numbers of proton dissociable groups in TCPs decreased with the increase of DH. The major contribution of sulfhydryl groups was confirmed by the largest p H decrease between 8.0 and 8.5 of the p H titration curves. The complexation of TCPs with heavy metals was evaluated by stability constants（βn） of TCP-metal complexes whose stoichiometry was found to be 1:1（ML） and 1:2（ML₂）. TCPs from degree of hydrolysis of 25% hydrolysates gave high affinities towards Hg²⁺, Cd²⁺, and Pb²⁺. A significantly positive correlation was found between the logarithm of stability constants for ML₂（lgβ2） and the sulfhydryl group content. Molecular weight distribution of TCPs affected the complexation with Pb²⁺ more notably than Hg²⁺ and Cd²⁺.5. The preparation and and Hg²⁺ binding properties of of TCPs with different charges were studied. TCPs extracted by DTT reduction and covalent chromatography from glycinin hydrolysates were seperated by SP Sepharose C25 into two fractions: negative charge fraction（NCF） and positive charge fraction（PCF）. TCPs were characterized by amino acid analysis, molecular weight distributions and hydrophobic distributions. Hg²⁺-binding properties of TCPs were investigated by UV-Vis spectroscopy, CD spectroscopy and SEC-HPLC.The results indicated that TCPs was successfully seperated into two partes. UV-Vis spectroscopy and CD spectroscopy indicated that complexes with the ratio of Hg and sulhydryl groups being 1:2, 1:1 and 2:1 were formed with the increase of Hg²⁺. SEC-HPLC indicated that the forms of complexes were ML₂、M₂L₂、M₂L. Charge properties showed no effect on the forms of TCPs-Hg²⁺complexes, but influenced the stability constants of the complexes. For NCF, the stability constants of M₂L₂ and M2 L were 1.22×1017 and 1.82, respectively, with the stability constant of M₂L₂ and M2 L being 4.44×1018 and 2.12 for PCF. The Hg²⁺ binding capacity of NCF was stronger than that of PCF.