Purification And Binding Mechanism Of Rice Cadmium Binding Protein

Cadmium is known as a kind of environmental pollutants because of the high toxicity and mutagenicity. Moreover, cadmium is easy to accumulate and has a half-life of ten to thirty years in the body. Therefore, cadmium has been listed the sixth toxic substances endangering human health by American poison Management Committee. Rice is an important staple food for Chinese people. However, rice sold in Chinese market is polluted heavily with cadmium.It has been found that the toxicity of cadmium is closely related to its chemical morphology. Therefore, it is a significant research to investigate the existing form of cadmium in rice.The detectable limit, precision and recovery rate of the graphite furnace atomic absorption spectrometry to investigate the feasibility of the method for determining the cadmium content in rice. Firstly, to choose a kind of high cadmium content rice as raw material, four kinds of protein in rice including albumin, globulin, gliadin and glutelin were extracted according to Osborne sequential extraction method. Secondly, the cadmium binding protein in rice were separated and purified by using ulfiltration and ion exchange chromatography from a high cadmium content of rice protein. Thirdly, the purity of rice cadmium binding protein(RCBP) was analyzed by using SDS-PAGE and MALDITOF-MS. Moreover, the UV absorption peak,amino acid composition, and N- terminal sequence of RCBP were analyzed by using UV spectrophotometer, amino acid automatic analyzer, and protein sequence automatic analyzer, and also analyzed and determined primary and secondary structure of RCBP using MALDI-TOF-MS/MS and FTIR. This work will establish a theoretical basis for reducing the absorption and transformation of cadmium in rice and human body injury caused by cadmium.(1) The method of Graphite Furnace Atomic Absorption Spectrometry(GFAAS) for determining the cadmium content in rice was investigated in this work. Rice was digested by using wet digestion for sample pretreatment and the detection limits, precision, repeatability, and recovery rate of GFAAS method were analyzed at preferred conditions. The detection limit of GFAAS method to determine the cadmium content in rice was 0.124μg/kg and the recovery rate was 98.85% under the preferred sample pretreatment conditions, including 1 g of rice, 10 m L nitric acid digestion reagent, 2m L hydrogen peroxide, and 12 h of digestion time. The results indicated that GFAAS method for the determination of cadmium content in rice was accurate and had lower test error, good repeatability at the preferred sample pretreatment conditions, and it can be utilized to determine the cadmium content in rice.(2) GFAAS method was used to determine the cadmium content in rice with different varieties and milling level. The results showed that the cadmium content in indica rice was higher than those in glutinous rice and japonica rice, and the cadmium content in rice decreased with the increase of milling level. All of cadmium content in brown rice, three-level rice, two-level rice, and one-level rice decreased, and there is a significant difference between brown rice and one-level rice. Four kinds of proteins in rice were extracted according to Osborne sequential extraction method, and also determined their cadmium content. There were remarkable differences for the cadmium content in four kinds of proteins, and the cadmium concentrations were 0.66, 0.31, 0.63, 0.23μg/g, respectively. Moreover, the cadmium concentration in albumin is the highest, followed by the alcohol soluble protein, globulin, and glutenin. Rice ultrafiltration albumin(RUA) was separated from rice albumin(RA) using ultrafiltration, and then RUA was purified from by using ion-exchange chromatography. Three kinds of rice protein components including component a, component b, and component c was collected, and the cadmium content of component c was higher than component a and component b. Therefore, component c was defined as the target cadmium binding protein in rice( RCBP).(3) SDS-PAGE and MALDI-TOF-MS were used to identify and determine the purity and molecular mass of RCBP. The results showed that RCBP had uniform purity, and also molecular mass of 13514 Da. The UV absorption peak of RCBP was 230 nm and 250 nm. Glutamate content of RCBP accounting for 18.31% was the highest, followed by 11.45% of cysteine, and tyrosine of 0.52% was the lowest. Moreover, aspartic acid and arginine content accounted for more than 9%. Compared to RUA, 10% of cysteine content in RCBP increased. It indicated that binding pattern between cadmium and albumin was through binding between cadmium and cysteine, and it is an untypical metallothionein. N-terminal amino acid sequence of RCBP was AEDEQPGI, and also identified primary structure of RCBP using MALDI-TOF-MS/MS. The results showed that RCBP was a new rice albumin sequence. 39.49% of α-helix, 23.48% of random coil, 19.54% of β-sheet, and 17.58% of β-turn was presented in RCBP, and 58.94% of ordered structures(α-helix and β-sheet) content increased 11.6% compared to RUA. It indicated RCBP through binding between cadmium and rice albumin had more stable structure.