Microsoft Word 刘珊珊-二校出2269

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1 6 6 Vol. 6 No Journal of Food Safety and Quality Jun., 2015 刘珊珊 1 1,, 陈季旺 2*, 陈露 1 1,, 丁文平 2, 吴永宁 (1., ; 2., ; 3., ) 1, 3 摘要 : 目的 (rice cadmium-binding protein, RCBP), RCBP 方法 Sephedex G-75 (Ethanol precipitation protein, EP) RCBP, SDS-PAGE RCBP, FTIR RCBP 结果 b( RCBP), b RCBP, 32 kda b 56.76% 19.41%, b N-H C=O, β- β- 结论 RCBP 关键词 : ; ; ; Purification and biochemical properties of cadmium binding protein from rice LIU Shan-Shan 1, CHEN Ji-Wang 1, 2*, CHEN Lu 1, DING Wen-Ping 1, 2, WU Yong-Ning 1, 3 (1. College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan , China; 2. Hubei Collaborative Innovation Center for Processing of Agricultural Products, Wuhan , China; 3. National Center for Food Safety Risk Assessment, Beijing , China) ABSTRACT: Objective To investigate the purification and biochemical properties of cadmium binding protein from rice and binding mechanism between cadmium and rice protein. Methods Rice cadmium-bining protein (RCBP) was purified from ethanol precipitation protein in rice using gel chromatography. The purity, molecular weight, amino acid composition, and secondary structure of RCBP were analyzed using SDS-PAGE, amino acid automatic analyzer, and FTIR. Results Fraction b (objective RCBP) with uniform purity and 32 kda of molecular weight was collected from elution buffer of ethanol precipitation protein in rice, and hydrophobic amino acids and aromatic amino acids in fraction b were accounted for 56.76% and 19.41%, respectivly. Moreover, β-sheet and β-turn were mainly secondary structures infraction b, and also existed N-H relative to C=O only in the form of trans-configuration. Conclusion The formation of RCBP was through binding between cadmium and characteristic group of hydrophobic or aromatic amino acids in rice protein. 基金项目 : ( ) (2011Z05) ( ) (2014CFB888) (2012cx012, 2013cx011) Fund: Supported by the Project to Special Scientific Research of Grain Public Welfare Profession ( ), Major Project to Special Cultivate of Wuhan Polytechnic University (2011Z05), International Cooperation Projects of Wuhan City ( ), National Natural Science Foundation of Hubei Province (2014CFB888) and Graduate Education Innovation Fund of Wuhan Polytechnic University (2012cx012, 2013cx011) * 通讯作者 :,,, jiwangchen1970@126.com *Corresponding author: CHEN Ji-Wang, Professor, College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan , China. jiwangchen1970@126.com

2 KEY WORDS: rice cadmium binding protein; purification; gel chromatography; amino acid composition 1 引言 [1] 1957, Margoshes [2],, [3] [4] [5] [6] [7], [8] 1977, Casterline [9] Kagi [10], 20, 61,,, Tanaka [11], 30%, 70 kda,,, ( ) Cohen [12] SDS-PAGE [13] SDS-PAGE, SDS-PAGE,, 85~45 kda, 85 kda (Fourier transform infrared spectrometer, FTIR) [14] FTIR [15] FTIR (15~95 ) [16] FTIR Sephedex G-75 RCBP (rice cadmium-binding protein, RCBP), SDS-PAGE RCBP, FTIR RCBP, RCBP, RCBP, 2 材料与方法 2.1 仪器与试剂 TAS-990 (, ); Ø cm ( ); HL-2S HD-3 BSZ-100 ( ); DDS-307 ( ); PerkinElmer Lambda 25 ( ); Bio-Rad Mini-PROTEAN-3 (, (Bio-Rad) ); NEXUS 670 ( ); L-8900 ( ) : X 9 (, 157 μg/kg, 6.86%, 14.55%, ), (Tris) (sodium dodecyl sulfonate, SDS)(, ); Sephedex G-75( ); SDS- (SDS-PAGE) (, ) 2.2 实验方法 RCBP 的纯化, g 1000 ml, 600 ml NaOH(1%, W:W),, 1 h, NaOH(1%, W:W) ph 11, 40 2 h( 120 r/min), 4000 r/min 10 min, (alkali extraction protein, AP) AP min, r/min 15 min,, (thermal denaturation protein, TP) TP 3, -20, r/ min 15 min,, 40

3 6, : 2271, (ethanol precipitation protein, EP), Sephedex G-75 EP, : 25 ml( 10 mg/ml), - (0.05 mol/l ) (ph9.8), 0.6 ml/min, 0.8 ml/min, 210 nm, 10 min 1, 210 nm A, A 0.3,,, RCBP SDS-PAGE 分析 10 mg/ml 80 μl 1.5 ml, 5 SDS 20 μl,, 10 min Tris- (ph8.3), 10 μl, 60 V, 40 min 110 V,, 0.1% R h, 10% -5%, 氨基酸组成分析 1.00 g, 6 mol/l 15 ml,,, h,,, 100 ml, 20 μl 40, 0.2 mol/l HCl 1 ml FTIR 分析, KBr, 1:100,,,,, KBr, 4 cm -1, 16, (4000~500 cm -1 ) 3 结果与分析 3.1 EP 的凝胶层析纯化及蛋白质与镉含量分析 Sephedex G-75 EP 1, X 9, a b, a b,, 1 Fig. 1 1 EP Elution curve of EP using gel chromatography 表 1 EP 组分 a 组分 b 中蛋白质和镉的质量 Table 1 The content of protein and Cd of EP, fraction a, and fraction b (g/300 g ) (ng/300 g ) Cd/Pro(ng/g) EP a b , EP Sephedex G-75, a b EP 90.60% 4.25%, EP 38% 25%; a b, ng ng, b a 13.7, b a, b, Sephedex G-75 RCBP 3.2 SDS-PAGE 分析 SDS-PAGE EP a b, 2 2, a EP, 32 kda 16 kda, a 32 kda 16 kda, a, ; b 32 kda, 32 kda, b, 32 kda Sephedex G-75 RCBP, 32 kda,

$2272 6 2 b SDS-PAGE Fig. 2 SDS-PAGE analysis of EP, fraction a and fraction b 3.3 氨基酸组成分析 b 2 2, b (Pro), 15.79%, (Leu) (Phe), (Cys) 0.13%; b 4.17%, 19.41%, 29.78%, 56.76%; EP ( ), b,, b EP (4.$

4 b SDS-PAGE Fig. 2 SDS-PAGE analysis of EP, fraction a and fraction b 3.3 氨基酸组成分析 b 2 2, b (Pro), 15.79%, (Leu) (Phe), (Cys) 0.13%; b 4.17%, 19.41%, 29.78%, 56.76%; EP ( ), b,, b EP (4.25%),, Thomas [17],, 13.5 kda 14 kda, (Cys) 5% 3%, b, RCBP 3.4 FTIR 分析 FTIR b, FTIR 3, b cm cm -1, b -CH 3 -CH 2 C-H -C=O ; b cm ~1520 cm -1, b -NO 2 C=N N=O NH C-N, 1550 cm cm -1, N-H C=O [18] ; Ⅰ cm -1 α cm -1 β-, III cm -1 α cm cm -1 β-, Omnic 8.0 FTIR, Peakfit, b N-H C=O, 3 3 3, b 1555 cm -1 表 2 组分 b 的氨基酸组成 Table 2 Amino acid composition of fraction b b(g/100 g ) b(g/100 g ) Asp 0.56 Leu Thr 0.60 Tyr 8.69 Ser 0.52 Phe Glu 4.99 His 6.88 Pro Lys 7.70 Gly 3.53 Arg 9.64 Ala 3.87 Trp / Cys Val Met Ile

5 6, : b Fig. 3 FTIR spectroscopy of fraction b Table 3 表 3 组分 b 的二级结构及 N-H 相对于 C=O 构型峰面积 Peak area of secondary structure and configuration of N-H relative to C=O of fraction b among FTIR spectra region by integration b /cm -1 α- β- 1650~1659 / 1290~1340 / 1615~ ~ β- 1660~ ~1288 / N-H C=O 1450 / , b N-H C=O b β , β , α-, b β- β-, b cm -1 b -NH 2 4 结论 (1) SephedexG-75 EP, b : NaHCO 3 - NaOH(pH 9.8), 0.8 ml/min 210 nm SDS-PAGE b, 32 kda (2) FTIR b N-H C=O, β- β-, α- b 56.76% 19.41%,, (Cys), 0.13%, RCBP

2274 6 参考文献 [1]. [M]. :, 2006. Zhao XM. Metallothionein[M]. Changsha: Central South University Press, 2006. [2] Margoshes M, Vallee B. A cadmium protein from equine renal cortex [J].

The dynamics of protein and metal metabolism in acclimated and Cd-exposed freshwater crabs (Potamonautes warreni) [J]. Comp Biochem Phys A, 2007, 145: 457 463.

6 参考文献 [1]. [M]. :, Zhao XM. Metallothionein[M]. Changsha: Central South University Press, [2] Margoshes M, Vallee B. A cadmium protein from equine renal cortex [J]. J Am Chem Soc, 1957, 79: [3] Pedersen SA, Kristiansen E, Andersen RA, et al. The dynamics of protein and metal metabolism in acclimated and Cd-exposed freshwater crabs (Potamonautes warreni) [J]. Comp Biochem Phys A, 2007, 145: [4] Hensbergen PJ, van Velzen MJM, Nugroho RA, et al. Metallothionein-bound cadmium in the gut of the insect Orchesella cincta (Collembola) in relation to dietary cadmium exposure [J]. Comp Biochem Phys A, 2000, 125: [5] Dennis RW, Rajagopalan KV. Purification and some properties of Cd-binding protein from rat liver [J]. Arch Biochem Biophys, 1972, 153: [6] Oomah BD, Berekoff B, Li-Chan ECY, et al. Cadmium-binding protein components of flaxseed: Influence of cultivar and location [J]. Food Chem, 2007, 100(1): [7] Shute T, Macfie SM. Cadmium and zinc accumulation in soybean: A threat to food safety [J]. Sci Total Environ, 2006, 371: [8] Hervé-Fernández P, Houlbrèque F, Boisson F, et al. Cadmium bioaccumulation and retention kinetics in the Chilean blue mussel Mytilus chilensis: Seawater and food exposure pathways [J]. Aquat Toxicol, 2010, 99(4): [9] Casterline I, Barnett N. Cadmium-binding components in soybean plants [J]. Plant Physiol, 1982, 69: [10] Kagi J, Nordberg M. Metallothionein and other low molecular weight metal- binding proteins [J]. Experientia Supp, 1979, 34: [11] Tanaka T, Hayashi Y, Ishizawa M. Subcellular distribution and binding of heavy metals in the untreated liver of the squid: Comparison with data from the livers of cadmium and silver-exposed rats [J]. Experientia Supp, 1983, 39: [12] Cohen AS, Karger BL. High-performance sodium dodecyl sulfate polyaeryl amide gel capillary eleetrophoresis of peptides and proteins [J]. J Chromatogr A. 1987, 397: [13],,. SDS-PAGE [J]., 2012, 28(18): Shang HL, Meng X, Zhang T. Extraction and SDS-PAGE analysis of proteins from Job S tears seed [J]. Chin Agric Sci Bull, 2012, 28(18): [14],,,. FTIR [J]., 2010, 30(8): Liu B, MA HL, Li SJ, et al. Study on the effect of ultrasound on the secondary structure of BSA by FTIR [J]. Spectrosc Spect Anal, 2010, 30(8): [15],,,. [J]., 2003, 20(2): Xiao HL, Sun SQ, Zhou Q, et al. Two-dimensional correlation analysis of FTIR spectrum for collagen during variable temperature denaturing process [J]. Chin J Atomic Mol Phys, 2003, 20(2): [16],. [J]., 2008, 29 (5): Yan J, Ma XJ. Effect of different desiccant manner on the secondary structure of soybean protein [J]. Food Sci Tech Ind, 2008, 29(5): [17] Thomas D, Solbe J, Kay J, et al. Environmental cadmium is not sequestered by metallothionein in rainbow trout [J]. Biochem Biophys, 1983, 110: [18],,. FTIR [J]., 2008, 28(1): Lu Y, Zhang WW, Wang GK. Progress in study of secondary structure of denaturized protein by FTIR [J]. Spectrosc Spect Anal, 2008, 28(1): 作者简介 ( 责任编辑 : 白洪健 ) 刘珊珊, 硕士研究生, 主要研究方向为粮食油脂及植物蛋白工程 ashleyss@163.com 陈季旺, 教授, 主要研究方向为粮食油脂及植物蛋白工程 jiwangchen1970@126.com

i

i i ii 1 2 3 4 % N) 5 50.0 AMINO ACID #64 [modified by Administrator] IntAmp_1 nc 40.0 30.0 20.0 10.0 0.0-10.0 1 - ala - 10.483 2 - thr - 11.107 3 - gly - 11.872 4 - val - 13.458 5 - ser - 14.903 6 - pro

Microsoft Word 刘珊珊-二校 出2269

Microsoft Word 刘珊珊-二校出2269