Purification and Characterization of Trypsin Inhibitor from Chickpea Seeds

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ * DEAE- 52 Sephadex G-100 CPTI CPTI 80% 32% SDS-PAGE CPTI 25 7 kd CPTI min 78% Lineveaer-Burk CPTI Ki mol / L Q946 1 R284 2 Purification and Characterization of Trypsin Inhibitor from Chickpea Seeds ZHAO Xin 1 FU Xuan-He 1 ZHANG Zong-Shen 1 * LIU Tong-Xiang 2 WANG Ji-Feng 2 1 School of Biological EngineeringDalian Polytechnic UniversityDalian 2 Laboratory of Cell and BiochemistryBeijing University of Chinese MedicineBeijing China China Abstract Chickpea trypsin inhibitor CPTI was purified and characterized from the seeds of Cicer arietinum L using ammonium sulfate grade precipitationanion exchange chromatography DEAEcellulose 52 and gel filtration on Sephadex G-100 The extraction from Cicer arietinum L seeds strongly inhibit trypsin activity by 80% and inhibit chymotrypsin activity by 32% but exhibited no effects on three proteases of pepsinpapain and subtilisin The approximate molecular weight of CPTI was estimated to be 25 7 kd by SDS-PAGE CPTI was also heat-resistant and maintained 78% of the activity after heated in 100 for 60 min The kinetic analysis showed that CPTI inhibited trypsin following the competitive model with an inhibition constant K i of mol / L Key words Cicer arietinum L chickpea trypsin inhibitor isolation and purification characterization trypsin inhibitorti HIV 6 ~ 8 TI No * Tel zhangzs@ dlpu edu cn Received September Accepted January Supported by National Natural Science Foundation of China No ~ 4 TI * Corresponding author Tel zhangzs@ dlpu edu cn

2 CPTI 5 10 μg Cicer arietinum L 20 μg 3 CPTI 1 4 CPTI 9 10 SDS- PAGE 14 chickpea trypsin 15% 100 V 4 h inhibitorcpti CPTI CPTI 1 5 CPTI CPTI min Cicer arietinum L DEAE- 52 Sephadex G-100 Pharmacia -DL- Benzoyl-DL-arginine ρ-nitroanilide hydrochloridebapna μg 2 min 1 2 CPTI g 40 ph h r / min 15 min ph % ~ 75% NH 4 2 S CPTI mmol / L Tris-HCl ph DEAE-52 CPTI DEAE NaCl 5 ph mol / L Tris-Hcl Fig 1 DEAE cm 15 cm 25 mmol / L Tris-HCl ph8 0 NaCl 20 ~ 240 mmol / L Sephadex G-100 Sephadex G-100 Sephadex G cm 40 cm 25 mmol / L Tris-HCl ph8 0 CPTI 0 3 ml / min 1 3 CPTI 2 2 CPTI 12 SDS-PAGE 1 mg Fig 3 4 DTT 1 U DTT 13 CPTI 800 μg 20 μg 30 min CPTI min CPTI CPTI BAPNA / L 150 μg CPTI Lineweaver-Buck CPTI 1 7 Bradford 15 2 NaCl 60 mmol / L Ⅱ Fig 2 Ⅰ CPTI Table 1 Fig 4 CPTI 25 7 kd

3 289 Table 1 Purification of the trypsin inhibitor from Chickpea seeds Purification step Total protein / mg Total activity / U Specific activity / U mg - 1 Yield % Crude extuact 330 7 2 364 10 2 100

3 3 289 Table 1 Purification of the trypsin inhibitor from Chickpea seeds Purification step Total protein / mg Total activity / U Specific activity / U mg - 1 Yield % Crude extuact % ~ 75% NH 4 2 SO Fig 1 The discontinuous gradient elution curve of DEAE-cellulose 52 column 2 5 cm 15 cm The DEAE-cellulose 52 colume was pre-equilibrated with 25 mmol / L Tris-HCl ph8 0 and then was eluted by a gradient from 0 to 240 mmol / L NaCl solution in the same bufferand protein concentration was monitored by 280 nm absorption The flow rate was 0 75 ml / minand 2 ml / tube was collected CPTI inhibitory activity A NaCl gradient DEAEcellulose 52 Sephadex G Fig 2 A representative chromatography of Sephadex G- 100 column 2 5 cm 40 cm separation of CPTI The protein fractions peakⅡon the anion-exchange column with a positive inhibitory activityas detected by activity assayswere loaded onto a Sephadex G-100 gel 2 5 cm 40 cm that had been equilibrated and eluted with 25 mmol / L Tris-HCl ph8 0 This process was carried out at the constant flow rate of 0 3 ml / min The fraction marked with Ⅰ was the active component - A CPTI inhibitory activity Fig 3 SDS-PAGE analysis of CPTI The samples was analyzed on 7 5% ~ 15% gradient geland protein bands were stained with Coomasie blue SDS-PAGE of samples containing trypsin inhibitory activity at different purification stages M standard marker proteins Lane 1crude extracts of CPTI lane 2Active fraction of the second DEAE-52 lane 3Active fraction of the first Sephadex G-100 with DTT lane 4Active fraction of the first Sephadex G-100 without DTT M Mark Arrow indicates the band of CPTI Table 2 The controlled expriment of trypsin inhibitors * Standard soybean trypsin inhibitor 82 ± 4 7% 36 ± 5 1% CPTI Table μg Purified chickpea trypsin inhibitor 71 ± 2 4% 19 ± 3 8% μg 30 * The concentration of trypsin were 50 μg / ml Positive min experiment Hydrolysis of CPTI with subillisin Negative 25 7 experiment Hydrolysis of CPTI with subillisin 10 μg for 30 kd CPTI minutes Group Positive experiment relative activity % Negative experiment relative activity %

4 Fig 4 Determination of molecular weight of CPTI by SDS- PAGE The R f of the standards are marked on the x-axis and the y-axis shows the standard proteins displayed in a logarithmic fashion y = log 97 2 kd 66 4 kd 44 3 kd 29 kd 20 1 kd 14 3 kd The molecular weight of CPTI was measured as 25 7 kd by the Rf of CPTI according to the standard line 2 3 CPTI BAPNA CPTI Table 3 CPTI CPTI Lineweaver-Buck Fig 5 BAPNA CPTI Km mol / L Km = Km 1 + I / Ki b c d e Ki mol / L mol / L mol / L mol / L Km Ki mol / L CPTI 2 4 CPTI CPTI Fig 6 Fig Table 3 The effect of inhibitors from Chickpea seeds on various protease activities * Enzymes Trypsin Chymotrypsin Pepsin Papain Subtilisin Relative activity % 80 ± 5 6% 32 ± 8 4% No inhibition No inhibition No inhibition * The concentrations of enzymes were 20 μg / ml and CPTI were 10 μg / ml Fig 5 Linewearer-Burk polts of Trypsin activity- CPTI kinetics Inhibition activity was analyzed by a double reciprocal plot as a function of substrate concentration at different inhibitor levels The reaction mixture 100 μl contained 10 μmol / L Tris-HCl ph μg trypsincpti at 0 μg μg μg μg or μg The rate of inhibition was measured during 10 minutes at 37 And the Linewearer-Burk plots indicate a competitive mode of inhibition with a increase in V max Fig 6 CPTI CPTI Effect of temperature on the inhibitory activity of Effect of temperature on the inhibitory activity of 30 mincpti min 10% min 80% min CPTI 3

5 3 291 Fig 7 Temperature stability of CPTI CPTI was heattreated at 100 for the indicated times and residual inhibitory activity of CPTI was measured Control inhibitory activity prior to heat treatment was designed as 100% J Tong-XiangZhang Zong-Shenet al orthogonal design methodj J Liu Tong-Xiang proteinase- inhibiting ingredients J J CPTI cellsj Chin J Biochem Mol Biol DEAE- 52 Sephadex G-100 Chem CPTI L seeds J CPTI J CPTI Zhang Zong-Shen et al Ki BAPNA Km CPTI Med Res CPTI J Zhang ZhengWang Chuan-HuaLin Ru-Fa buckwheat seedsj Chin J Biochem Mol Biol References 1 HIV J Yang Qin-GangHe Xu-Chang Bai Dong-Lu Current status of HIV protease inhibitorsj Acta Pharm Sin J Zhang NingWang Feng-Shan Research status of trypsin inhibitorj Chin J Biochem Pharm Boateng J AViquez O MKonan K Net al Screening of a peanut Arachis hypogaea L cdna library to isolate a Bowman-Birk trypsin inhibitor clonej J Agric Food Chem Pouvreau L Gruppen H van Koningsveld G Aet al Tentative assignment of the potato serine protease inhibitor group as beta-Ⅱ proteins based on their spectroscopic characteristicsj J Agric Food Chem Schuler T HPoppy G MKerry B Ret al Insect-resistant transgenic plantsj Trends Biotechnol Du Xu-DongLiu Optimize the extraction procedure of trypsin inhibitor of Houttuynia cordata Thunb by Lishizheng Med Mater Med Res Niu Jian-Zhao Du Xu-Donget al Selection of Chinese herbs containing Med China J Chin Mater 8 Li FangLi Yu-YingBai Chong-Zhi et al Effect of recombinant buckwheat trypsin inhibitor on apoptosis and caspases activity in human hepatoma HepG Poltronieri FAreas J A GColli C Extrusion and iron bioavailability in chickpea Cicer arietinum L J Food 10Clemente AVioque JSanchez-Vioque R Effect of processing on water adsorption and softening kinetics in chickpea Cicer arierinum J Sci Food Agric Fu Xuan- HeLiu Tong-Xiang Study on the extraction procedure of trypsin inhibitor of chickpea seedsj Lishizheng Med Mater et al Purification and some properties of the trypsin inhibitor from tartary J Chen

292 27 ZhongYang Xiao-QuanPeng Zhi-Ying Hydrolization of soybean trypsin inhibitor by subtilisinj J South China Univ Technol Nat Sci Ed 200129 6 23-26 14 16 KUNTIZ J Kang Zhuang J Xie Wang JingDu

Kuntiz-type trypsin Res Dev 200517 2 143-146 15Bradford M M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingj Anal

6 ZhongYang Xiao-QuanPeng Zhi-Ying Hydrolization of soybean trypsin inhibitor by subtilisinj J South China Univ Technol Nat Sci Ed KUNTIZ J Kang Zhuang J Xie Wang JingDu Lin-Fang Purification and some properties of a Ke-FangDong Ai-WuCao Kai-Minget al A study of the trypsin inhibitor from Spinacia oleracea L seedsj Nat Prod stability and insect resistance of soybean Kuntiz-type trypsin Res Dev Bradford M M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingj Anal Biochem inhibitorj J Fudan Nat Sci Ed ISBN ISBN DNA John M Walker METHODS IN MOLECULAR BIOLOGY John M Walker ATP zhouwenyu@ mail sciencep com http / / shop sciencepress cn www lifescience com cn lifescience@ mail sciencep com

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