ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2011 3 27 3 287 ~ 292 1 1 1 * 2 2 1 116034 2 100029 DEAE- 52 Sephadex G-100 CPTI CPTI 80% 32% SDS-PAGE CPTI 25 7 kd CPTI 100 60 min 78% Lineveaer-Burk CPTI Ki 3 99 10-7 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 116034 China 100029 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 3 99 10-7 mol / L Key words Cicer arietinum L chickpea trypsin inhibitor isolation and purification characterization trypsin inhibitorti HIV 6 ~ 8 TI - 1 2010-09-09 2011-01-22 No 30371775 * Tel 13898489972 E-mail zhangzs@ dlpu edu cn Received September 9 2010 Accepted January 22 2011 Supported by National Natural Science Foundation of China No 30371775 2 ~ 4 TI * Corresponding author Tel 13898489972 5 E-mail zhangzs@ dlpu edu cn
288 27 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 20 40 60 80 1 1 1 20 40 60 min Cicer arietinum L DEAE- 52 Sephadex G-100 Pharmacia -DL- Benzoyl-DL-arginine ρ-nitroanilide hydrochloridebapna 0 0 135 0 312 0 467 0 815 μg 2 min 1 2 CPTI 11 30 g 40 ph4 050 1 5 h 4 10 000 r / min 15 min ph 8 0 35% ~ 75% NH 4 2 S0 4 25 2 1 CPTI mmol / L Tris-HCl ph8 0 2 1 1 DEAE-52 CPTI DEAE-52 20 000 NaCl 5 ph8 00 025 mol / L Tris-Hcl Fig 1 DEAE- 52 2 5 cm 15 cm 25 mmol / L Tris-HCl ph8 0 NaCl 20 ~ 240 mmol / L 2 1 2 Sephadex G-100 Sephadex G-100 Sephadex G-100 2 2 5 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 100 30 min CPTI 100 1 6 CPTI BAPNA 0 4 0 6 0 8 1 0 / 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 35% ~ 75% NH 4 2 SO 4 40 2 1 320 32 8 55 8 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 280 - --NaCl gradient DEAEcellulose 52 Sephadex G- 100 8 6 714 83 0 30 2 3 3 322 97 6 13 6 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 280 - - -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 2 800 μg Purified chickpea trypsin inhibitor 71 ± 2 4% 19 ± 3 8% 30 20 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 %
290 27 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 1 2 4 5 -BAPNA CPTI Table 3 CPTI CPTI Lineweaver-Buck Fig 5 BAPNA CPTI Km 3 56 10-3 mol / L Km = Km 1 + I / Ki b c d e Ki 3 75 10-7 mol / L4 40 10-7 mol / L3 82 10-7 mol / L4 01 10-7 mol / L Km Ki 3 99 10-7 mol / L CPTI 2 4 CPTI CPTI Fig 6 Fig 7 20 40 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 8 0 100 μg trypsincpti at 0 μg 0 135 μg 0 312 μg 0 467 μg or 0 815 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 60 30 min 10% 100 30 min 80% 100 60 min CPTI 3
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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 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 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 Biochem197672 7 248-254 inhibitorj J Fudan Nat Sci Ed 20026 631-634 2011-1 2 ISBN 978-7-03-029789-1 108 00 16 2011 1 1 2 30 ISBN 978-7-03-030122-2 58 00 16 2011 2 - DNA John M Walker METHODS IN MOLECULAR BIOLOGY John M Walker ATP 1997 010-64031535email zhouwenyu@ mail sciencep com http / / shop sciencepress cn www lifescience com cn 010-64012501 email lifescience@ mail sciencep com