| Interaction mode, nanoparticle formation and influencing factors of bovine serum albumin (BSA) with quercetin (QUE) and sulforaphane (SFN) will help us understand the interaction mechanisms, nanoparticle formation and functional changes of bioactive small molecules and biomacromolecules. Previous study indicated that S=O group of10%dimethyl sulfoxide (DMSO) might play an important role in the formation of BSA-QUE nanoparticles. This study investigated interaction mechanisms and nanoparticle formation of BSA and QUE in three solution systems of deionized water (dH2O),10%dimethyl sulfoxide (DMSO) and10%ethanol (EtOH) in order to verify the role of S=O group in the nanoparticle formation. Furthermore, a S=O containing bioactive small molecules, sulforaphane was selected study on influence of S=O group on BSA nanoparticle fonnation and the interaction mechanisms of BSA and SFN in three solution systems of dH2O,10%DMSO and10%EtOH. Furthermore, the binding capacity and stability of QUE and SFN bound by BSA nanoparticle in dH2O,10%DMSO and10%EtOH were evaluated. All the results are as follow:QUE had a great ability to quench BSA’s fluorescence in both static and dynamic modes, and that hydrophobic interaction and hydrogen bonds played a dominant role for BSA and QUE interaction in dH2O,10%DMSO and10%EtOH. QUE interacted with BSA at both tyrosine (Tyr) and tryptophan (Tip) residues at site I corresponding to the pocket of subdomain IIA in three solution systems. The binding constant values and binding site numbers between BSA and QUE were in the order of dH2O>10%DMSO>10%EtOH. The binding distances were in the order of10%EtOH>10%DMSO> dH2O. Referred to the binding distance, the binding forces were in the order of dH2O>10%DMSO>10%EtOH. The particles diameter were in the order of10%EtOH (50-55nm)> dH2O (40-45nm)>10%DMSO (10-15nm). The binding capacities of1mole of BSA for QUE were in the order of17mole (in dH2O)>15mole (in10%EtOH)>6mole (in10%DMSO).10%DMSO contains a S=O group, which competed for the amide’s hydrogens with the C=O groups of BSA and resulted in the disappearance of the α-helix, therefore, the partially unfolding of the polypeptide chain exposed the internal hydrophobic groups and thus promoted BSA aggregation and formed dense spherical particles by hydrophobic effects. Additionally, BSA aggregation reduced binding area and binding capacity with QUE. The combination of QUE and BSA only decreased the content of a-helical structure of BSA in dH2O and10%EtOH. BSA exposed a little hydrophobic group, and increased the particles diameter and binding capacity. Furthermore, the combination of BSA and QUE changed the disulfide bond configuration from ggg to ggt in dH2O and10%EtOH, but did not change in10%DMSO. Two methyl groups of10%DMSO can interact with BSA avoiding changes of two disulfide bond configuration, thus the conformational of BSA changed little and resulted in binding less QUE. Therefore, methyl group and S=O group of DMSO were favorable to forming the smaller size of BSA-QUE nanoparticles.SFN had ability to quench BSA’s fluorescence in static modes, and to interact with BSA at both Tyr and Trp residues. Hydrophobic forces, hydrogen bonds and van der Waals interactions were all involved in BSA and SFN interaction, which were not significantly changed by three solutions. The binding constant values and binding site numbers were in a descending order of dH2O>10%DMSO>10%EtOH. The values of free energy change were in a descending order of dH2O>10%DMSO>10%EtOH, which indicated that the binding forces were in a descending order of dH2O>10%DMSO>10%EtOH. BSA interaction with SFN can form dense, good dispersibility and spherical nanoparticles with diameter less than30nm in three solution systems. The binding capacities of1mole of BSA for SFN were in the order of32mole (in dH2O)>28mole (in10%EtOH)>19mole (in10%DMSO). Two methyl groups of10%DMSO and S=O, N=C=S groups of SFN have an important role for stabilizing the two disulfide bond configuration of BSA that is better for water-soluble SFN binding with BSA and no significant difference in particles diameter of BSA-SFN in three solution systems. However S=O group of10%DMSO competed for the amide’s hydrogens with the C=O groups of BSA and resulted in the disappearance of the a-helix, therefore, the partially unfolding of the polypeptide chain exposed the internal hydrophobic groups that will go against BSA binding with QUE, and decreased binding capacity of BSA. Therefore, S=O group of DMSO and S=O, N=C=S groups of SFN have an important role in the formation of BSA-SFN nanoparticles.No significant difference in DPPH radical scavenging rates between QUE and BSA-QUE was observed. While, the ABTS radical scavenging rate of QUE was significantly lower than the unbound QUE. No significant difference in antioxidant activity between QUE and BSA-QUE was observed in three solution systems. The-OH moieties of the QUE were very important for antioxidant activity, such as5,7-dihydroxylation at the A-ring and3’,4’-dihydroxylation at the B-ring. The5-OH at the A-ring formed an intennolecular hydrogen bond with BSA, and thus the antioxidant activity decreased. Nevertheless, the DPPH radical scavenging rate of QUE was weak and accordingly the DPPH radical scavenging rate was not decreased. There was no significant difference in antioxidant activity between SFN and BSA-SFN. Moreover, three solutions had not significant influence on antioxidant activity of SFN and BSA-SFN. BSA nanocarrier had protection effect on the antioxidant activity and oxidation stability of QUE and SFN in dH2O,10%DMSO and10%EtOH, and the protection effect on the activity of QUE and BSA were in the order of dH2O>10%EtOH>10%DMSO. It inferred that this difference resulted from the binding capacity.In conclusion, both methyl, S=O groups of DMSO and S=O, N=C=S groups of SFN had an important role in the formation of BSA-QUE and BSA-SFN nanoparticles, while, the S=O group of DMSO and S=O group of SFN performed different functions, one destroyed a-helix, another generated new a-helix. |
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