Microsoft Word - Chem100409-f - edited_tobefinalised.doc 19-29 SQU Journal For Science, 15 (2010) © 2010 Sultan Qaboos University 19 Screening and Purification of a Chymotrypsin Inhibitor from Entrolobium Saman Seeds Maher Ali. Maqtari* and A.B. Mohamed. Saad Department of Chemistry, Sanaá University Faculty of Sciences, Sultanate of Oman, *Email: proteinase_613@hotmail.com.. سامان انترولوبيوم بذور من وتنقيه مثبط الكا يموتربسين البروتينيفحص محمد سعدالباسط وعبد ألمقطري أحمد علي ماهر سامان آايموتربسين انهيبيتورقد تم فصله وتنقيته بالطرق التقليدية لتنقيه البروتينات مثل الترسيب انترولوبيوم :خالصة ٍ (وجل فلتريشن ) (DEAE-Celluloseالتبادل أاليوني الكروماتوجرافي م،بالحامضيه ،الحرارة، آبريتات االمونيو Sephadex G75 .( ألفصل الكهربائي النتائج النهائية وجدت بأنه متجانس من خالل استخدام) SDS-PAGE و( PAGE Gel, filtration و Isoelectric focusing . ون بواسطة دالت17978الوزن الجزئي لمثبط الكايموتربسين آان SDS-PAGE و Gel filtration .سامان آايموتربسين انترولوبيوم. . أالمينو اسيد لهذا المثبط وجدت بأنها سيرين ويظهر طيف امتصاصي في منطقه االشعه فوق البنفسجية مع قيمه امتصاص ,)SHٍ( انهيبيتور يفتقر إلى مجموعه الثايول يشير إلى عدم Circular dichroic (CD) .هر عدم وجود التربتوفانظمثبط يالطيف القاعدي لهذا ال. 2,99منخفضة هي درجه مئوية آما اظهر هذا المثبط ثبات 97وجود أي ترآيب ثنائي للبروتين مع ثبات عالي في مختلف درجات الحرارة حتى ) Samanea saman( مان التخصص لهذا المثبط من انترولوبيوم سا .12,0 إلى 2,0عالي عند المدى الهيدروجيني من آايموتربسين يثبط بقوه بواسطة آايموتربسين انهيبيتور، . التظهر أي نشاط ضد السيستين، الكاربوآسيل وأيضا ميتالوبروتينيز آايموتربسين انهبيتور يظهر معقد مترابط بثباته مع الكايموتربسين وهذا المعقد قد تم فصله ودراسته ونتائج هذه الدراسة دلت ثابت التفكك لمعقد . إلنزيم الكايموتربسين ) Single headed Inhibitor( جود موقع واحد نشط على هذا المثبط على و دراسة التحوير الكيميائي للمثبط آان نوع . مولر 8ـ9,05X 10الكايموتربسين والكايموتربسين انهبيتور قد تم تعينها وهي الكايموتربسين ،فان هذا المثبط يظهر نشاطيه ضد بعض أنواع البكتيريا والفطريات إلى جانب نشاط هذا المثبط تجاه . تايروسين .الممرضة ولكن لم يتم آتابتها في هذا البحث ABSTRACT: A chymotrypsin inhibitor was isolated and purified from the seeds of Enterolobium saman (Leguminaceae family) by extraction with 100 mM phosphate buffer, heat treatment, ammonium sulphate precipitation, ion-exchange chromatography on DEAE- cellulose and filtration through Sephadex G-75. The final preparation appeared to be homogeneous by both chromatographic and electrophoretic analyses. ESCI had a molecular weight of about 17,890 and an isoelectric point of 5.8. ESCI inhibited bovine chymotrypsin at an inhibitor-enzyme molar ratio of 1:2. The inhibition mode of chymotrypsin inhibitor was competitive on bovine chymotrypsin. Investigation has been carried out on the complex formed between chymotrypsin and chymotrypsin inhibitor by physico-chemical methods. An apparent dissociation constant (Ki) of 9.05 X 10-8 M has been calculated for the complex. This enzyme- MAHER ALI AL MAQTARI and A.B. MOHMED SAAD 20 inhibitor complex was isolated by gel filtration on Sephadex G-75 and a molecular weight of 43.000 was estimated for the complex. The inhibitor did not have any effect on other proteinases, such as papain, bromelin, elastase, α-amylase, trypsin and pepsin. The chemical modification of lysine residues indicated that –NH2 groups are not essential for the activity of ESCI toward chymotrypsin. The inhibitor was an acidic protein and was stable over a wide pH range of 2-12 and temperature range of 10oC-97oC. KEYWORDS: Samanea saman; Serine proteinase inhibitor; Chymotrypsin inhibitor; Single- headed inhibitor. Abbreviations: ESCI, Enterolobium saman chymotrypsin inhibitor; CIA, Chymotrypsin inhibitory activity; ATEE, N-Acetyl-L-tyrosine ethyl ester; NB, Nutrient broth; SDS-PAGE, Sodium dodecyl sulphste-polyacrylamide gel electrophoresis; CD, Circular dichroism; TPCK, L-1-chloro-3-(4-tosylamide)-4-phenyl-2-butanone HC; APNE, N-acetyl-D,L phenylalanyl-β- naphthyl ester; TNBS, Trinitrobenzene sulfonic acid; CHD, 1, 2 cyclohexane dion, PDA, potato dextrose agar; MIC, Minimal Inhibitory Concentration; CIU, Chymotrypsin Inhibitory Unit; PVDF, (polyvinylidenedifluoride membrane); CAPS, [3-(cyclohexylamino) propane-1- sulphonic acid; PI, proteinase inhibitor; BSA, bovine serum albumin; TCA, trichloroacetic acid 1. Introduction he protease inhibitor (PI) protein, the natural antagonists of protease, is a small protein which is quite common in nature and present in all life forms (Fritz, 2000). Plant protein inhibitors of proteases have been known for many years and their function is presently the subject of much interest. Serine proteinase inhibitors are universal throughout the plant kingdom and have been described in many plant species. Therefore, the number of known and partially characterized inhibitors of serine proteinases is enormous (Haq et al., 2004). Serine proteinase inhibitors have been reported from a variety of plant sources and are the most studied class of proteinase inhibitors (Mello et al., 2002; Haq and Khan, 2003). Although the biological role of protein proteinase inhibitors is not still sufficiently clear, it has been suggested that they may perform three main functions-serving as storage proteins, being regulators of activity of endogenous proteinases and acting as agents protecting plants against insects and pathogenic microflora. These inhibitors are probably important physiologically and even in molecular evolution. Furthermore, they serve as excellent models for research on protein-protein interaction. The PIs are important tools to achieve a better understanding of fundamental principles and can be used to design new substances for the control of diseases and pathologic processes. Plant protein inhibitors of proteases have been known for many years and their function is presently the subject of much interest. Enterolobium saman (Rain tree) is a member of the Leguminaceae family. The seeds and the pulp are edible and nutritious for livestock and make an excellent feed supplement (George and Craig, 2005). The present paper describes the extraction, purification and biochemical characterization of chymotrypsin inhibitor from Enterolobium saman seeds. 2. Material and Method Enterolobium saman (Samanea saman) seeds were procured from Bangalore (India), the local area. All biochemical's and enzymes used in this investigation were purchased from Sigma Chemical Co. (St. Louis, USA). All other chemicals used were of analytical grade. T SCREENING AND PURIFICATION OF A CHYMOTRYPSIN 21 2.1 Extraction of chymotrypsin inhibitor The whole seeds were ground to fine powder and defatted with chilled acetone at 4oC. The defatted flour was extracted with 100 mM phosphate buffer (pH 7.6) for 3 h. at 4oC (1:10 W: V ratio). The supernatant was separated by centrifugation (30 min at 10.000 rpm) and the clear supernatant was used as a crude extract for estimation of chymotrypsin inhibitory activity and protein. Protein was estimated by the method of Lowry et al. (1951). 2.2 Assay of chymotrypsin and chymotrypsin inhibitory activity The inhibition spectrum of ESCI was established by assay proteolytic or esterolytic activity of the enzyme on appropriate substrates. In principle, a fixed amount of the enzyme was incubated with various amounts of the inhibitor and the residual enzyme activity was assayed. The activity of chymotrypsin or its inhibition was routinely assayed by the method of Kakade et al., (1969) and the method suggested by Schwert and Takenaka (1955) using casein and ATEE as substrate, respectively. 2.3 Purification of chymotrypsin inhibitor Enterolobium Saman chymotrypsin inhibitor was isolated in an apparently homogeneous form by the following procedure. Acetone defatted Enterolobium Saman flour was stirred with 100 mM phosphste buffer (pH 7.6) for 3 hr at 4oC. The extract was centrifuged at 10.000 rpm for 30 min. The pH of the supernatant was adjusted to 5.0 with 0.2 M acetic acid and the suspension was heated to 70oC with stirring. After 10 min at 70oC the supernatant was rapidly chilled in an ice-bath and centrifuged at 10.000 rpm for 10 min to remove the precipitate. The pH of the supernatant from the previous step was adjusted to 7.0 with diluted ammonium hydroxide. Solid ammonium sulphate was added to 80% saturation with constant stirring at 0oC. The suspension was centrifuged at 10.000 rpm for 15 min .The precipitate was dissolved in 10 mM Tris-HCl buffer (pH 8.0) and dialyzed. The ammonium sulphate fraction (80 mg protein) was applied to the DEAE-Cellulose column and eluted with a starting buffer. Thereafter, a gradient elution with increasing strength (0.2M NaCl) was performed. The fraction containing CIA was pooled, dialyzed and lyophilized. Protein (20 mg) from DEAE-preparation in 1.0 ml phosphate buffer (pH 7.0) was applied to a sephadex G-75 column and chromatographed using 100 mM phosphate buffer (pH 7.0) as the effluent. The active fractions were pooled, dialyzed and lyopholilized. The final preparation showed a single protein band in PAGE and this band was shown to possess CIA by a specific staining method. 2.4 PAGE analysis of inhibitors Polyacrylamide gel electrophoresis was performed according to the method of Davis (1964). For visualization of the inhibitor in acrylamide gels, after electrophoresis the gels were first incubated with trypsin solution (100 µg /ml) and then with the substrate (N-acetyl-D,L phenylalanyl-β-naphthyl ester)-dye (Diazoblue- B mixture by the method of Filho and Moreira, (1978). The CIA appeared as a clear zone against a pink background. 2.5 Determination of molecular weight For estimation of M.W. the sample and the marker proteins were incubated at 50oC for 1 hr in 0.1 ml phosphate buffer, with 10 µl 20% SDS and 10 µl β-mercaptoethanol by the method of Weber and Osborn (1969). The MW marker proteins used were BSA (66 KDa), Ovalbumin (43KDa), Carbonicanhydrase (29 KDa), SoybeanTI (20.1 KDa) and Lysozyme (14.3 KDa) for SDS-PAGE. 2.6 Isoelectric point Isoelectro focusing was done by the method of Wringley (1969) in 7.5 % PAGE tube gels with 2% ampholyte gradient between pH 3 and pH 10. The upper and lower reservoirs were 0.2 % sulphuric acid and 0.4 % MAHER ALI AL MAQTARI and A.B. MOHMED SAAD 22 ethanolamine, respectively. The run was performed in room temperature. After the run, the gels were stained for CIA and protein. 2.7 N-terminal sequencing The purified (ESCI) was separated by SDS-PAGE in 12% gel and stained with Coomassie Brilliant Blue , then electro blotted on to a PVDF in 10 mM CAPS as described by Matsudaira, ( 1987) . The protein band was cut out and loaded on to the sequencer. 2.8 Stability of the inhibitor The stability of inhibitors at different pH (2-12) values was assayed by incubating chymotrypsin inhibitor solution at different pH values at 5oC overnight. Then the pH of the mixture was brought to 7.0 and its inhibitor activity towards chymotrypsin was assayed as described above. To study the thermostability, the inhibitor prepared was incubated at different temperatures (10-1210 C) by 10oC interval in a water bath for 10 min. 2.9 Circular dichroism Spectra (CD-Spectra) The CD spectra were recorded on a Jasco-810 CD spectropolarimeter with a cell length of 0.1 cm at 10oC. The inhibitor (2 mg/ml) in distilled water was placed in a 2 mm Quartz cuvette and spectra were recorded from 200-300 nm at intervals of 1 nm and a well time of 2 seconds. 2.10 Chemical modification 2.10. 1 Modification of the amino groups The free amino groups were modified according to the method of Haynes et al., (1967). 500 µg of ESCI was incubated in different test tubes with 0.1 % TNBS in 2 ml of 4 % Na2 CO3, (pH 8.5) at 40 oC for different time intervals. The reaction was stopped by the addition of 1 ml of 10 % SDS followed by 0.5 ml of 1 N HCl. The extent of modification of each sample was determined by measuring the absorbance of the solution at 344 nm. The residual CIA was determined during the course of modification by removing aliquots from each reaction mixture at pre determined time intervals. The control inhibitory activity was estimated from an inhibitor solution incubated in the same manner without TNBS. 2.10.2 Modification of Arginine group Arginine groups were modified by the method of Liu et al., (1968). 10 mg CHD was allowed to react with 5.0 mg ESCI in 5.0 ml 200 mM triethanolamine buffer, (pH 7.8) in the dark for 12 h. The residual CIA was determined in relation to an inhibitor control without CHD after dialysis in cold condition for 36 hours. 2.11 Isolation of the chymotrypsin-ESCI complex The enzyme-inhibitor complex was isolated by gel filtration on sephadex G-75. A mixture of inhibitor (2mg) and chymotrypsin (1mg) in 2 ml, 100 mM phosphate buffer (pH 7.6) was allowed to stand at room temperature for 15 min. This solution was then chromatographed on a sephadex G-75 column equilibrated with 100 mM phosphate buffer (pH 7.0) and the column was then developed with the same buffer solution. The A280 nm of the fractions was measured. CIA and chymotrypsin activity in the fraction was determined. Estimation of the apparent dissociation constant (Ki) of the ESCI-Chymotrypsin complex was performed according to the method of Grob (1950). Fixed concentrations were calculated assuming of a M.W of 17890 for ESCI and 25000 for bovine chymotrypsin. 2.12 Kinetic measurement The nature of inhibition of chymotrypsin by ESCI was studied by incubating chymotrypsin with varying concentrations of substrate (Casein) in the presence and absence of the inhibitor. The substrate (1-5 mg) was SCREENING AND PURIFICATION OF A CHYMOTRYPSIN 23 incubated with 40 µg chymotrypsin for 10 min at 37 oC .After the incubation period, the reaction was stopped by adding 6 ml 5% TCA. A 280 nm of the solution was read against a blank incubation sample, but without enzyme. The assay was repeated in the presence of 10 and 20 µg of the inhibitor in the reaction mixture. 2.13 Specificity of ESCI The inhibitory activity of ESCI towards α-chymotrypsin was determined using casein (Kakade and Liener, 1970) or ATEE (Schwert and Takenaka, 1955) as substrate. The casein digestion method (Murachi, 1970) was used to determine the activity or inhibition of papain and bromelin. Elastolyatic activity of elastase was determined by the method of (Naughton and sanger, 1961) using elastin-Congo red as substrate. The activity of pepsin was assayed by the method of (Anson, 1938). The esterolytic activity of subtilisin-BPN' was assayed using ATEE as substrate in a manner similar to the α-chymotrypsin assay; α-Amylase (Pancreatic) activity or its inhibition was measured by the method of (Saunders and Lang, 1973). 3. Result and Discussion ESCI was purified from mature seeds based on chymotrypsin inhibitory activity. A procedure has now been evolved for the purification of chymotrypsin inhibitor from Enterolobium saman seeds as summarized in Table 1. The CIA was eluted by a linear gradient of increasing ionic strength. The chymotrypsin inhibitor was desorbed from the DEAE-Cellulose column at an ionic strength of 0.063 M NaCl (Figure 1), and then was finally separated by Sephadex G-75 column (Figure 2). Table 1. Summary of purification of Enterolobium saman Chymotrypsin inhibitors (ESCI) (from 100gm of defatted flour) *Yield and fold purification are calculated on the basis of CIU using Casein as substrate. Purification steps Total protein (mg) Total activity units CIU X104 Specific activity CIU/mg Fold purification* CIU Yield %* CIU Crude extract 50400 215.88 42.83 1.0 100 pH&heated treated extract 28820 197.31 68.47 1.59 91.4 80% (NH4)2SO4 5320 138.26 259.90 6.06 64.0 DEAE-Cellulose 1220 103.32 846.80 19.77 47.86 Sephadex G-75 760 64.86 853.49 20.0 25.6 MAHER ALI AL MAQTARI and A.B. MOHMED SAAD 24 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 1 8 15 22 29 36 43 50 57 64 71 78 85 Fraction Number A 2 80 n m 0 50 100 150 200 250 300 350 400 (C IU ) X 10 Figure 1. Ion-exchange chromatography on DEAE-Cellulose Column of the ESCI. □□ Absorbance at 280 nm;●●Chymotrypsin inhibitory activity. 0 0.2 0.4 0.6 0.8 1 1 9 17 25 33 41 49 57 65 73 81 Fraction Numbe r A 2 80 n m 0 100 200 300 400 500 C IU /f ra ct io n Figure 2. Gel filtration on sephadex G-75 of ESCI. □□ Absorbance at 28n nm, ●●Chymotrypsin inhibitory activity 0 0.1 0.2 0.3 1 6 11 16 21 26 31 36 41 46 51 56 61 Fraction Number A 2 80 n m Figure 4. Gel filtration on Sephadex G-75 of a mixture of bovine chymoptrypsin and ESCI. Protein was monitored at 280 nm ● ● SCREENING AND PURIFICATION OF A CHYMOTRYPSIN 25 Table 2. Effect of pH and Temperature on ESCI. * Inhibitory activity at 10oC was taken as 100%. † Boiling water. ‡ Autoclaving at 1.04 Kg/cm2 pressure A B C D Figure 3. Electrophoretic analysis of ESCI. (a) PAGE of purified ESCI in 10% gel stained with coomassie brilliant blue (b) Visualization of the chymotrypsin inhibitory band on the gel. (c) Isoelectric point in a 7.5% tube gel. (d) Molecular weight determination of purified ESCI by SDS- PAGE in 12% gel The chymotrypsin inhibitor isolated from Enterolobium saman by the procedures described above appeared to be homogeneous by the criteria of PAGE (Figure 3a), SDS-PAGE (Figure 3d), gel filtration on Sephadex G- 75 (Figure 4) and isoelectric-focussing (Figure 3c). The final preparation showed one protein band in PAGE and this band was shown to possess CIA by a specific staining method (Figure 3b). The studies with pure preparation have shown that the CIA has a broad pH stability and thermo stability (Table 2). Temp.oC CIU% pH CIU/mg 10* 100 2.6 1673.3 25 100 3.2 1666.6 30 100 5.0 1670.0 40 99.3 6.0 1753.0 50 98.2 7.0 1703.5 60 97.9 7.6 1780.0 70 95.5 8.6 1640.0 80 95.31 9.6 1620.0 97† 92.62 10.6 1680.0 > 121‡ 0.0 12.6 1576.6 KDa 66 43 29 20.1 14.3 MAHER ALI AL MAQTARI and A.B. MOHMED SAAD 26 The Mr of ESCI was determined as 17890 ± 100 SDS-PAGE and gel filtration on sephadex G-75. Many legume inhibitors have their molecular weight in the range of 15,000-20,000 Da. Single headed inhibitors have been isolated from barley bean (Prakash et al., 1996), Crotalaria paulina seeds ((Luiza et al., 1999), and Caesalpinia echinata (Pau-brasil) seeds (Cruz-Silva .et al., 2004)????. The Kunitz-type inhibitor found in E.saman supports the notion that there is a relationship between the types of inhibitors found in leguminous seeds and the evolution of the leguminous plants, since only Kunitz-type inhibitors are present in the relatively primitive plants of Caesalpinoideae. This conclusion was reached by Norioka et al. (1988) who investigated the presence of both Kunitz and Bowman-Birk inhibitors in seeds of 34 legumes by gel filtration. The results were compared to the morphological classification of Leguminosae: the seeds of the more primitive Leguminosae (Caesalpinoideae and Mimosoideae) contain mainly Kunitz-type inhibitors, whereas those of a more advanced family (Papilinoideae) contain only Bowman-Birk-type inhibitors. 0 10 20 30 40 50 60 70 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 Figure 5. Lineweaver-Burk plot of the inhibition of caseinolytic activity of chymotrypsin by ESCI. ▲▲ without inhibitor, ■ ■ with 10 µg of ESCI, ●● with 20 µg of ESCI. 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 ESCI] ([M] X 10-7) Figure 6. Inhibition of bovine chymotrypsin by ESCI. C on ce nt ra tio n of c hy m ot ry ps in in hi bi te d ([ M ] X 1 0- 7 ) SCREENING AND PURIFICATION OF A CHYMOTRYPSIN 27 Figure 7. CD-spectra of the purified chymotrypsin inhibitor (ESCI) (2.0 mg/ml) in distilled water. 0 20 40 60 80 100 0 20 40 60 80 100 120 Incubation Time (min) (% ) F re e am in o gr ou ps & (% ) R es id ua l C hy m ot ry ps in in hi bi to ry ac tiv ity i nh ib ito ry Figure 8. Time course of modification of free amino groups with TNBS % free amino groups ■■% residual CIA ▲▲. Figure 9. N-terminal sequences of ES. Inhibitor N-terminal sequences ESCI S-N-L-L-L MAHER ALI AL MAQTARI and A.B. MOHMED SAAD 28 Chymotrypsin activity in the presence (10 and 20 µg) and absence of ESCI was measured at different substrate concentrations. The double-reciprocal plot of the kinetic data is shown in (Figure 5). It can be seen that ESCI inhibited trypsin by a competitive mechanism involving competition of the substrate and inhibitor for the site on the enzymes. Most naturally occurring proteinase inhibitions inhibit the respective enzymes competitively. The dissociation constants (Ki) of the ESCI-chymotrypsin was calculated to be 9.05 X 10-8 M. The low Ki indicates a high affinity between the inhibitor and chymotrypsin. The extreme stability of the ESCI in acidic pH and higher temperatures suggests that the ESCI has little or no ordered structure. The CD-spectra of the ESCI (Figure 7) in distilled water shows the complete absence of α- helical content. The changes in the conformation of the ESCI in different pH and temperatures could not be carried as the proteins exhibited random coil secondary structures in CD-spectra. The modification of arginine residues by a group specific reagent does not have any significant effect on the trypsin inhibitory activity of ESCI. The modification of lysine residues showed the participation of fast acting NH2 groups in the reactive site of ESCI (Figure 8). This suggests that lysine is perhaps at the active site of the ESCI. ESCI most probably belong to the class of tyrosine type inhibitor. Specificity of the inhibition of different classes of proteinases by ESCI was tested against the enzymes from various sources (Table 3). ESCI inhibited only bovine chymotrypsin and showed no activity against other proteolytic enzymes tested. This establishes that the inhibitory activity of ESCI against bovine chymotrypsin with ATEE as substrate (Figure 6) is stoichiometric. N-terminal groups of the Enterolobium saman inhibitors have been detected by precise protein sequencing system IISc, Bangalor (Figure 9). The N-terminal sequence up to 5-residues was S-N-L-L-L. 4. Conclusions In this work we describe the purification and characterization of a new chymotrypsin inhibitor from Enterolobium saman (Samanea saman) which belongs to the class of tyrosine inhibitor. The inhibitor existed as a monomer with molecular weight of 17,890 Da. Judging from its molecular mass and the inhibitory activity against chymotrypsin, chymotrypsin can be considered as a Kunitz type inhibitor. This inhibitor highly inhibited bovine chymotrypsin, but did not show any activity against other proteolytic enzymes tested. 5. Acknowledgments We gratefully thank the Department of Chemistry, Faculty of Sciences, Sana'a University and Bangalore University, India, for a Research Assistantship and the College of Agricultural Sciences, Sana'a, for technical assistance. 6. 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