Study Of The Molecular Binding Mechanism And Dissociation Discipline Between Rice Proteins And Cadmium

In 2012,China Center for Disease Control and Prevention?CCDCP?released a report indicating that the monthly intake of Cd for 2-6-and 7-14-year-old children had exceeded the Provisional Tolerable Monthly Intake?PTMI?regulated by FAO/WHO and Joint Expert Committee on Food Additives?JECFA?.Diet Cd from grains,especially from rice,contributed to half of the intake.The pollution of rice by Cd is becoming an urgent problem in cereal food safety in China.Compared to such modifications of soil and cultivations anti-Cd rice species that are financially and periodically costly,the removal of Cd from harvested rice is becoming a practical approach to address the concerns of such problems.Due to the fact there is a lack of such studies originated from thermodynamics,molecular dynamics and coordinating chemistry,current studies are yet to provide sufficient basis for the choices,optimizations and practical applications of proper Cd removal techniques.Based on the above concerns,this study aimed to investigate the binding state of Cd in rice,and from a thermodynamics perspective,to screen out the most stable protein composition binding with Cd.By means of molecular dynamics simulation and experimental verification,the typical binding mechanism between rice proteins?RPs?and Cd was investigated.Lastly,from a perspective of coordinating chemistry,the effects of acids and competing agents on the stability of protein-Cd complexes were studied.The study was additionally verified by practices of removing Cd from RPs by acids and chelating agents.Based on the above findings,this study is to lay the theoretical foundation for the future design of effective and green techniques to remove Cd from rice.Principle findings:?1?The binding state and thermodynamics attributes of Cd in rice.Cd is unevenly distributed in rice grains.For brown rice containing 0.32 mg/kg Cd,by investigating the Cd content in each component of rice from bran to endosperm,it was found that the Cd content was on the decrease from the outmost layer as 0.52 mg/kg to the innermost layer as 0.29 mg/kg,in the same trend as the content of RPs.Alkali-extracted proteins?ARPs?and enzyme-extracted proteins?ERPs?had Cd content of 2.70 and 1.85 mg/kg,respectively,which were significantly higher than that in rice powder?0.30 mg/kg?,indicating that proteins were the dominating components binding with Cd.Using modified Osborn to extract different proteins and analyze their respective Cd content,it was found that prolamin and albumin had larger Cd amounts?L?,being 2.46 mg/kg and 2.09 mg/kg,respectively,than those of glutelin and globulin.This result implied that Cd mainly bound with prolamin and albumin.The bindings of Cd to ERPs,globulin,prolamin and glutelin were studied.The binding amount q was found to be varied in line with time,temperature and initial Cd concentration.The bindings of Cd to the above proteins were quite rapid.The equilibrium was reached within 30 min,and respective q for 5-min bindings were 89.56%,59.06%,70.9%,and 90%that of equilibrium binding,respevtively.The q for prolamin was 23.78 mg/g,the largest among all the protein components.The SEM images showed that ERPs and prolamin presented compact,lamellar structures,inferring that most of the Cd was bound to the surface of the proteins.All the four proteins binding with Cd showed qusi-second order adsorption behaviors,indicating that the bindings were due to chemical adsorption.Upon different reaction temperatures,the bindings of Cd to the proteins can be applied by a Langmuir model,meaning that the adsorption was a mono-layer adsorption.Thermodynamics studies showed that the bindings had a?G°<0 and a?H°>0,suggested that the bindings were endothermic and spontaneous.The prolamin had the largest?H°of58.75kJ/mol,evidencing a multidentate binding.By correlation analysis,it was found that the maximum q(q_max)significantly correlated?R²=0.967?with the contents of Glu,Asp,indicating that Cd was more prone to bind with these two amino acids in proteins.?2?Typical molecular mechanism underlying the bindings of Cd to RPs.Using a modified Osborn technique,13 kDa prolamins that present mono-band in SDS-PAGE and mono-peak in HPSEC were obtained.The prolamins were then subjected to enzymatic hydrolysis and upon Total Ion Current?TIC?analysis by mass spectrum?MS?,unique peptides were collected.The analysis of the peptides was performed by comparison of the existing data in Uniprot by MaxQuant software?v1.5.6.0?,and 3 major subunits of the same molecular weight were screened out and their peptide sequences were identified.Among those subunits,one with the PDB ID of Q0D840 had the highest iBAQ in TIC and its structure was characterized.The subunit Q0D840 had 10 Glu and 9 Asp residues,accounting for 11.17%and 9.10%of the subunit weight,respectively.There were also 2 His in the sequence located at 12 and 112 amino acid sites.In addition,the structures of Q0D840 was characterized by?-helix secondary structures.Using molecular dynamics simulation,it was found that Glu75,Asp34 and His12were apt to bind with free Cd,and the structures of Q0D840 underwent helix-sheet transitions.As a result,the intramolecular interactions were intensified and the compactness of the protein molecule was improved.Eventually,the bindings of Cd to the whole 13 kDa prolamins were studied by fluorescence spectrum,circular dichroism spectrum and X-ray photoelectron spectroscopy,the results were in good agreements with the findings of molecular dynamics simulations.?3?Dissociation behaviors of RP-Cd complexes.Acid had a remarkable effect on the bindings of Cd to prolamins and ERPS,and the q for respective proteins decreased with reducing pH in a sigmoidal style.For example,at a pH of 7.5,the q for prolamins and ERPS were 5.75 and 3.76 mg/g,respectively,which decreased to 0.51 and 0.21 mg/g,respectively,when the pH was reduced to 3.0.The existence of Na⁺had a negligible effect on the RP-Cd stability that was even slightly improved at high Na⁺concentrations.In terms of the resistance of the bindings of Cd to RPs,the inhibition followed an order of EDTA-2Na>sodium pyrophosphate>sodium citrate.At an EDTA concentration of 10^–4 mol/L,the q values were readily reduced to 2.16 and 0.66 mg/g for prolamins and ERPS,respectively.However,natrium aceticum slightly improved the bindings of Cd to RPs.In this study,the metal ions were found to resist the bindings of Cd to RPs.Due to the differences in hydrolysis of metal ions,the inhibition was in an order of Zn²⁺>Cu²⁺>Ca²⁺>Fe³⁺.The most potent competition agent Zn²⁺reduced the q to 0.68 and 2.06 mg/g for prolamins and ERPS,respectively,at a concentration of 10^–3 mol/L.?4?Effects of the removal of Cd on the physicochemical properties of RPs.The removal of Cd by acid was rapid and the Cd removal rate(R_Cd)reached 86.7%within 5-min reaction.The increase of temperature had no significant effects on the R_Cd.Besides,the diameter of RPs slightly affected R_Cd.For example,the R_Cd increased from 88.6%to 92.3%when the average diameter?D50?decreased from 169?m to 20?m,indicating that Cd was bound to the surface areas of RPs.The post-washing of RPs by acid or water could also effectively improve the R_Cd.A one-time wash of RPs by water boosted the R_Cd from 75.2%to 92.3%,and repeated water-wash elicited limited further improvements of R_Cd.The increase of the concentration of Cd in the initial acid solution reduced the R_Cd that was kept above 85%at a Cd concentration of 0.4mg/L,indicating that acid can be recycled for repeated uses.pH had the most significant effect on R_Cd,and the increase of RP concentration significantly reduced R_Cd,which was due to that the increased addition of RPs as well improved the buffering ability of the reaction solution.At neutral conditions,R_Cd reached 92.0%when EDTA-2Na was supplemented at a concentration of 40 mg/L,indicating that stable complexes were formed between EDTA-2Na and Cd.In addition,coordination conjugates were formed when EDTA was supplemented in acid condition,which lead to a rapid reduction of R_Cd.Both Cd removal methods had no effects on the amino acid compositions of RPs.However,coordination had more specificity of Cd removal,and the physicochemical properties of RPs were less affected than acid method.Acid method,by contrast,had significantly stronger effects on the minerals,functional properties,and in vitro digestibility of RPs.