- Email: [email protected]
The use of papain inhibitor immobilized onto polyaniline for bioaffinity chromatography of cysteine proteases
Separation and Purification Technology 120 (2013) 467–472
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
The use of papain inhibitor immobilized onto polyaniline for bioaffinity chromatography of cysteine proteases Adriane G. Gamboa, Karla A. Batista, Flavio M. Lopes, Kátia F. Fernandes ⇑ Laboratório de Química de Proteínas, Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia-GO, Brazil
a r t i c l e
i n f o
Article history: Received 18 January 2013 Received in revised form 11 October 2013 Accepted 15 October 2013 Available online 25 October 2013 Keywords: Stationary phase Papain Bromelain Ficin Purification
a b s t r a c t In this work, a papain inhibitor extracted from seeds of Adenanthera pavonina was covalently immobilized onto glutaraldehyde modified polyaniline (PANIG). The immobilization was very efficient, presenting 54.24% inhibitor retention in the following conditions: immobilization time of 30 min, at 4 °C, pH 7.0, inhibitor concentration of 20 mg and 5 mg of PANIG. The resultant material (PANIG-I) was tested as stationary phase for the cysteine proteases papain, ficin and bromelain purification through bioaffinity chromatography. A commercial preparation of papain was efficiently purified, resulting in a single band with 25 kDa. SDS–PAGE of purified bromelain showed a characteristic band around 28 kDa and this procedure resulted in 0.89-fold purification and 32.4% yield. The purification process was more effective for ficin reaching 2.6-fold purification and a single band of 25 kDa in the SDS–PAGE. These results showed that the stationary phase containing A. pavonina inhibitors immobilized onto PANIG is a very promising material for a single step purification of cysteine proteases through bioaffinity chromatography. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The cysteine proteases comprise a group of enzymes with varied physical and biochemical properties. Papain extracted from Carica papaya latex is the representative enzyme for this family [1]. Cysteine-proteases are widely distributed in the nature, presenting several endogenous actions. In general, these enzymes present low selectivity through peptide linkage to be hydrolyzed and consequently they may have a diversity of applications [2]. However, the use of cysteine proteases is frequently restricted due to the difficulty inherent of proteolytic enzyme purification. The classic purification procedure consists of sequential chromatographic techniques including gel filtration and ion-exchange, hydrophobic or affinity chromatography [3,4]. The proteases purification is commonly accompanied by significant losses due to autolysis occurring during the sequence of chromatographic procedures. Therefore, the purification of proteases demands meticulous handling in a slow and high cost procedure [5]. An additional problem in the protease purification is the limited use of bioaffinity chromatography, since the stationary phase suffers the hydrolytic attack of the enzyme, compromising its integrity. In this sense, a stationary phase resistant to the proteolytic attack of the proteases could be helpful in a purification approach.
⇑ Corresponding author. Address: PB 131, Goiânia-GO, CEP 74001-970, Brazil. Tel.: +55 (62) 3521 1492; fax: +55 (62) 3521 1190. E-mail address: [email protected] (K.F. Fernandes). 1383-5866/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2013.10.027
In this study, a papain inhibitor was covalently immobilized in a glutaraldehyde modified polyaniline and the resultant material used as a stationary phase for bioaffinity chromatography. This stationary phase was tested as a single step procedure for purification of papain, ficin and bromelain. 2. Methodology 2.1. General experimental procedures The seeds from Adenanthera pavonina used in this study were collected in the farm of the Universidade Federal de Goiás (Goiânia-GO, Brazil). Papain was from Calbiochem. Bromelain was obtained from the common commercially cultivated pineapple (Ananas comosus). Ficin was obtained from the latex of Ficus carica cultivated in the farm of the Universidade Federal de Goiás. The electrophoresis reagents were from GE Healthcare Life Sciences. All other reagents were of analytical grade. 2.2. Polyaniline synthesis and activation (PANIG) HCl-doped polyaniline (PANI) was synthesized by oxidation of a 0.44 mol L 1 aniline solution with a 0.68 mol L 1 ammonium persulphate solution, as described by Fernandes et al. [6]. PANI was subsequently activated (PANIG) under reflux for 2 h, using a 2.5% (v/v) glutaraldehyde solution prepared in 0.1 mol L 1 phosphate buffer, pH 6.0 [7]. The PANIG was exhaustively washed to assure
468
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
glutaraldehyde was completely removed. Then, PANIG was dried in a desiccator and stored in opaque plastic flask, at room temperature. 2.3. Extraction of A. pavonina seeds inhibitors The extraction and ammonium sulfate precipitation of papain inhibitors from A. pavonina was carried out according to methodology described by Silva et al. [8]. Seeds of A. pavonina were broken in a blade mill and the cotyledon fragments were separated as much from the testa pieces. The fragments were ground in a Wiley mill and defatted with hexane. The papain inhibitors were extracted by using 0.3 mol L 1 NaCl in 0.1 phosphate buffer, pH 8.0 (1:10 w/v). The supernatant (crude extract) was collected by centrifugation and heating at 90 °C for 10 min. The soluble proteins were collected by centrifugation and submitted to ammonium sulfate fractionation at 30% and 60% saturation. The resulting fractions were collected by centrifugation, dialyzed and lyophilized. The fraction 30–60 (98 mg of protein) was purified in a column (2.3 6.0 cm) filled with Sephadex G-75, equilibrated and eluted with 0.1 mol L 1 sodium phosphate buffer, pH 7.0. The tubes containing inhibitory activity from the first eluted peak (F1) were pooled, dialyzed against distillated water and lyophilized. The protein content was measured by the Bradford method [9], using bovine serum albumin as standard. The purity of purified papain inhibitor was analyzed using 12% denaturating polyacrilamide gel electrophoresis (SDS–PAGE) according to the method described by Laemmli [10]. Electrophoresis was run at 25–40 mA for 4 h at room temperature. Gels were stained according to the methodology described by Blum et al. [11], using silver nitrate. 2.4. Immobilization of papain inhibitor in PANIG Attempting to obtain the maximum amount of immobilized inhibitor, several tests were performed. For such, the amount of inhibitor (5–30 mg) and the pH of reaction (5.0–8.0) were varied. The buffers used in the pH tests were 0.1 mol L 1 sodium acetate for pH 5.0 and 0.1 mol L 1 sodium phosphate for the other pH values. The efficiency on the immobilization was measured making the PANIG- inhibitor derivative (PANIG-I) to react with a papain solution (7.6 U). After optimization, the immobilization process was made by adding 5 mg of PANIG to a solution containing 20 mg of inhibitor in 0.05 mol L 1 sodium phosphate buffer, pH 7.0. The reaction proceeded for 30 min, at 4 °C under stirring. The mixture was centrifuged at 9000g for 5 min to separate PANIG-I from supernatant. The PANIG-I was washed with the same buffer until unbounded inhibitor was completely removed. 2.5. Determination of inhibitory activity The papain inhibitory activity was determined according to the method of Kunitz as modified by Arnon [12]. Inhibitory activity was determined by comparing proteolytic activity of mixtures of preparations containing PANIG-I and enzyme with the activity of pure enzyme. The inhibitory activity was expressed in units of inhibitor (UI), according to definition of Silva et al. [8]. 2.6. Bioaffinity chromatography 2.6.1. Preparation of crude extracts of proteases The crude extract of bromelain was obtained from the stem of pineapple (A. comosus). Briefly: the stems of ripe pineapple fruits were separated from the freshly fruits. The stems were cut in small pieces, crushed in a mortar and vacuum filtered. The clear solution
was precipitated with cold acetone (1:3 v/v), and the powder obtained after evaporation of acetone was suspended in 0.1 mol L 1 sodium phosphate buffer, pH 7.0. This material was submitted to enzyme activity assay and used on the purification process. The crude extract of ficin was prepared according to Sgarbieri [13]. The latex from peduncles of unripe fruits of F. carica was collected and centrifuged at 9000g for 20 min. The clear yellow supernatant containing the crude ficin was stored in freezer at 20 °C until its use for activity assay and affinity chromatography. 2.6.2. Chromatography tests In order to test the PANIG-I as a stationary phase for bioaffinity chromatography, a glass tube was filled with 20 mg of PANIG-I and the column was equilibrated with 0.05 mol L 1 Tris buffer, pH 8.0. 500 lL of a commercial grade papain solution (171.4 U) (Calbiochem, La Jolla, CA, USA) was applied to this column and the same Tris buffer was used as mobile phase for elution of the nonbounded proteins. The elution of the retained papain was carried out by applying 500 lL of 0.1 mol L 1 NaOH solution, followed by immediate neutralization with 0.5 mol L 1 HCl solution. This fraction was precipitated with cold acetone (1:3 v/v) and dried at room temperature. The purity of this material was evaluated by electrophoresis. The same procedure was used for purification of bromelain and ficin. In order to evaluate the stability of the stationary phase for bioaffinity chromatography, test of repeated use were conducted using papain as enzyme to be purified. After elution of the retained papain, PANIG-I was washed twice with 0.05 mol L 1 Tris buffer, pH 8.0 and after this a new papain solution was left to react as described above. 2.6.3. Electrophoresis in polyacrylamide gel (SDS–PAGE) The purified proteins were analyzed for purity by using 12% denaturating polyacrylamide gel electrophoresis (SDS–PAGE) according to methodology described by Laemmli [10]. The amount of protein applied were determined following the method described by Bradford [9]. Electrophoresis was run at 25–40 mA for 2 h at room temperature. Gels were stained according to methodology described by Blum et al. [11], using silver nitrate. Molecular weight markers (New England Biolabs, MA) ranging from 25 to 116 kDa were used as running standard. 3. Results and discussion 3.1. Polyaniline synthesis and activation The chemical synthesis of polyaniline resulted in a fine brilliantblack-green powder, which became black-opaque after the activation with glutaraldehyde (Fig. 1a). The scanning electron microscopy (SEM) showed that glutaraldehyde modified polyaniline (PANIG) has a granular morphology, the particles presenting a variable size (Fig. 1b). The presence of cavities in PANIG provides a microenvironment that protects the immobilized molecule and explains the improved properties of several immobilized proteins. The cavities also enlarge the surface area of this material, resulting in a high available area for protein linkage. This structure explains several properties observed in the different systems where polyaniline was used as support for immobilization [7,14–16]. 3.2. Purification of the papain inhibitor from A. pavonina seeds The results of papain inhibitory activity, protein content, specific inhibitory activity and purification factor obtained in the purification of the papain inhibitor from A. pavonina seeds are
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
469
Fig. 1. Photograph (a) and scanning electron micrograph (b) of the PANIG.
Table 1 Purification of the papain inhibitors from Adenanthera pavonina.
Protein content (mg mL 1) Activity (IU mL 1) Specific activity (IU mg 1 protein) Purification factor Yield (%)
Crude extract
Fraction 30–60
Purified inhibitor
167.9 64 0.38 1.0 100
4.63 4.9 1.06 2.79 7.7
0.78 2.5 3.18 8.37 3.9
shown in Table 1. As can be observed, the protein content present in the fraction 30–60 was about 36-hold lower than in the crude extract. This lower content of protein in the fraction 30–60 leads to an increase of the specific inhibitory activity, resulting in approximately 3-fold purification (Table 1). In addition, the chromatography through Sephadex G-75 resulted in about 8-fold purification and a yield of 3.9%. The purification of the papain inhibitor was confirmed by denaturizing polyacrylamide gel electrophoresis (Fig. 2). The presence of a single band at 60 kDa in opposition to the multitude of protein present in the crude extract is a confirmation of the effectiveness of the purification procedure. According to Xavier-Filho [17], the leguminous plant seeds, like A. pavonina contain at least three classes of papain inhibitor: (1) a low molecular weight class (5–12 kDa); (2) an intermediate size class (20–30 kDa) and a high molecular weight class (60–80 kDa). The purified papain inhibitor from A. pavonina was used for immobilization onto glutaraldehyde modified polyaniline (PANIG).
Fig. 2. SDS–PAGE of the papain inhibitors from A. pavonina in the different purification steps. R1: molecular weight marker; R2: crude extract; R3: fraction 30– 60; R4: purified papain inhibitor (Shepadex G-75).
3.3. Immobilization of the papain inhibitor onto PANIG The maximum inhibitor retention was reached when 20 mg of inhibitor was offered to 5 mg of PANIG (Fig. 3). Above this concentration, the retention remained unchanged probably because all reactive groups available for the inhibitor linkage were occupied. The influence of the reaction pH on the immobilization efficiency was also observed (Fig. 4). The amount of immobilized inhibitor varied by almost 3-fold over a narrow pH range, with optimum immobilization being achieved at pH 7.0. Such large variation probably reflects the state of ionization of the support and the enzyme since the coupling reaction depends on the appropriate ionization of the reactive groups in both materials [15,18,19]. It is possible that at pH 7.0 the basic residues present on the exposed surface of papain inhibitor perform an important role in the immobilization reaction. As reported by Silva et al. [8], the papain
inhibitor from A. pavonina seeds presents 4 histidine, 9 arginine and 4 lysine residues. Considering the highly conserved structure of the cystatin’s inhibitor family, it is possible that such basic aminoacids are exposed in the protein surface being the targets for the reaction with the carbonyl groups of glutaraldehyde (Fig. 5) [20]. The optimal immobilization was obtained after 30 min of reaction of 5 mg PANIG and 20 mg of papain inhibitor in 0.1 mol L 1 sodium phosphate buffer pH 7.0, at 4 °C. Under these conditions, 3.58 U of inhibitor were immobilized, corresponding to 54.24% yield. The efficiency of the immobilization process, expressed by the ratio between the specific activity of the PANIG-I and the inhibitor offered for immobilization, was 0.77. It means that 77% of the inhibitor molecules were properly immobilized and only 23% were inactive or inaccessible to react with enzyme.
470
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
Fig. 3. Optimal amount of offered papain inhibitor for immobilization onto PANIG. Reaction: 4 °C; pH 7.0; 30 min of immobilization; 5.0 mg of PANIG.
Fig. 4. Optimal pH for papain inhibitor immobilization onto PANIG. Reaction: 4 °C; 30 min of immobilization; 5.0 mg of PANIG; 20 mg of papain inhibitor.
3.4. Chromatography tests
Fig. 5. Scheme representing the mechanism of reaction between papain inhibitor and PANIG.
The evaluation of PANIG-I as a stationary phase for bioaffinity chromatography was initially conducted with a commercial grade papain (Table 2). The purity of the papain eluted from the PANIG-I column was analyzed in a 12% SDS–PAGE. As can be seen in Fig. 6(a), the purified papain consisted of a single band of about 25 kDa, described in the literature as the molecular weight of papain from C. papaya [21–23]. It is important to note that the commercial papain had an additional band around 22 kDa, which disappeared after the bioaffinity purification. Vasu et al. [24] reported the presence of several contaminant enzymes and proteins in commercial preparations of papain. The stability of the stationary phase was evidenced by the absence of a 60 kDa band in the SDS–PAGE, corresponding to the papain inhibitor. The absence of this band confirms that the inhibitor remained bounded to the PANIG even after the elution of papain with NaOH solution. The chemical integrity of the PANIG-I was confirmed in the repeated use tests. In these tests, PANIG-I preserved 100% of the initial retention capacity after four cycles of reuse.
The purification of bromelain and ficin was also successfully obtained with PANIG-I bioaffinity chromatography. In the case of bromelain, 0.89-fold purification and 32.4% yield was achieved in a single step procedure (Table 2). The purity of the bromelain was evidenced in the SDS–PAGE by the presence of a single band around 28 kDa (Fig. 6b). Devakate et al. [25] using a 5-step methodology involving ammonium sulfate precipitation (40–80% saturation followed by 40–70% saturation), dialysis and ion exchange chromatography reached 10-fold purification with 84.5% yield. In another approach Kumar et al. [26] obtained 9.38fold purification with 15% yield in a 2-step affinity based reverse micellar purification process. Regarding to the ficin purification, the electrophoresis revealed a single band at 25 kDa (Fig. 6c). The efficiency of this purification procedure is presented in Table 2. As can be seen, the eluted fraction showed a 2.6-fold purification and a yield of 26.7%. Englund et al. [27] observed a purification yield of 8.8% using 5-step
Table 2 Purification of cysteine proteases by using a PANIG-I bioaffinity chromatography. Papain
Activity (U mL 1) Protein content (mg mL 1) Specific activity (U mg 1 protein) Purification factor Yield (%)
Bromelain
Ficin
Commercial
Purified
Crude extract
Purified
Crude extract
Purified
171.4 0.10 1714 0.89 62.2
106.6 0.07 1522
68 2.61 26.1 0.89 32.4
22 0.94 23.4
72 13.6 5.3 2.60 26.7
19 1.38 13.8
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
471
Fig. 6. SDS–PAGE of enzyme extracts and purified proteins through PANIG-I bioaffinity chromatography. (a) papain: (R1) molecular weight marker (kDa), (R2) commercial papain, (R3) purified papain; (b) bromelain: (R1) molecular weight marker (kDa), (R2) crude extract from Ananas comosus stems, (R3) purified bromelain; (c) ficin: (R1) molecular weight marker (kDa), (R2) crude extract from Ficus carica peduncles, (R3) purified ficin.
methodology including salt precipitation, dialysis and gel filtration. The same author also reported a better yield around 16% when salt precipitation step was removed from the purification approach. Sigiura and Sasaki [28] reported a yield of 9.5% using 4-step purification procedure based on CM-cellulose chromatography. Anderson and Hall [29] using a bioaffinity column of mercurial modified sheparose reached 1.9-fold purification with 66.3% yield.
4. Conclusions The immobilization of the papain inhibitor from A. pavonina onto PANIG confirmed the high retention capacity of polyaniline and evidenced its ability to actuate as a support for protein immobilization. The methodology for the PANIG-I synthesis is fast, simple, low cost and demands non-toxic reagents. The stability of the linkage between the papain inhibitor and PANIG was successfully demonstrated through SDS–PAGE analysis. Furthermore, the effectiveness of the proposed bioaffinity purification methodology was showed by SDS–PAGE analysis evidencing a single band for bromelain and ficin. In this sense, the PANIG-I is a very promising material for use as a stationary bioaffinity phase for a single step purification of cysteine proteases. References [1] Y.Q. Ling, H.L. Nie, S.N. Su, C. Branford-White, L.M. Zhu, Optimization of affinity partitioning conditions of papain in aqueous two-phase system using response surface methodology, Separation and Purification Technology 73 (2010) 343– 348. [2] A.S. Fahmy, A.A. Ali, S.A. Mohamed, Characterization of a cysteine protease from wheat Triticum aestivum (Cv. Giza 164), Bioresource Technology 91 (2004) 297–304. [3] H.L. Nie, T.X. Chen, L.M. Zhu, Adsorption of papain on dye affinity membranes: isotherm, kinetic, and thermodynamic analysis, Separation and Purification Technology 57 (2007) 121–125. [4] D. Tombaccini, A. Mocali, E. Weber, F. Paoletti, A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts, Analytical Biochemistry 289 (2001) 231–238. [5] P.A.G. Soares, A.F.M. Vaz, M.T.S. Correia, A. Pessoa-Junior, M.G. Carneiro-daCunha, Purification of bromelain from pineapple wastes by ethanol precipitation, Separation and Purification Technology 98 (2012) 389–395. [6] K.F. Fernandes, C.S. Lima, H. Pinho, C.H. Collins, Immobilization of horseradish peroxidase onto polyaniline polymers, Process Biochemistry 38 (2003) 1379– 1384.
[7] K.F. Fernandes, C.S. Lima, F.M. Lopes, C.H. Collins, Properties of horseradish peroxidase immobilised onto polyaniline, Process Biochemistry 39 (2004) 957–962. [8] K.F.F. Silva, C.S. Lima, R.R. Val, J. Xavier-Filho, Cysteine proteinase inhibitors from seeds of Adenanthera pavonina L, Revista Brasileira de Botânica 18 (1995) 137–141. [9] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding, Analytical Biochemistry 72 (1976) 680–685. [10] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T 4, Nature 227 (1970) 680–685. [11] H. Blum, H. Beier, H.J. Gross, Improved silver staining of plant proteins, RNA and DNA in polyacrilamide gels, Electrophoresis 8 (1987) 93–99. [12] R. Arnon, Papain, in: G. Perlmann, D.L. Loran (Eds.), Methods in Enzymology, Academic Press, New York, 1970, pp. 226–244. [13] V.C. Sgarbieri, Enzimas proteolíticas do látex de diversas variedades de Ficus carica L, Bragantia 24 (1965) 109–124. [14] K.F. Fernandes, E.R. Asquieri, R.N. Silva, Immobilization of Aspergillus niger glucoamylase onto a polyaniline polymer, Process Biochemistry 40 (2005) 1155–1159. [15] L.L.A. Purcena, S.S. Caramori, S. Mitidieri, K.F. Fernandes, The immobilization of trypsin onto polyaniline for protein digestion, Material Science and Engineering C 29 (2009) 1077–1081. [16] A.M. Pascoal, S. Mitidieri, K.F. Fernandes, Immobilisation of a-amylase from Aspergillus niger onto polyaniline, Food and Bioproducts Processing 89 (2011) 300–306. [17] J. Xavier-Filho, The biological roles of serine and cysteine proteinase inhibitors in plants, Revista Brasileira de Fisiologia Vegetal 4 (1992) 1–6. [18] K. Kang, C. Kan, A. Yeung, D. Liu, The immobilization of trypsin on soap-free P(MMA-EA-AA) latex particles, Material Science and Engineering C 26 (2006) 664–669. [19] A.H.M. Cavalcante, L.B. Carvalho Jr., M.G. Carneiro-da-Cunha, Cellulosic exopolysaccharide produced by Zoogloea sp. as a film support for trypsin immobilisation, Biochemical Engineering Journal 29 (2006) 258–261. [20] F. Xi, J. Wu, Z. Jia, X. Lin, Preparation and characterization of trypsin immobilized on silica gel supported macroporous chitosan bead, Process Biochemistry 40 (2005) 2833–2840. [21] B.P. Ó-Hara, A.M. Hemming, D.J. Buttle, L.H. Pearl, Crystal structure of glycil endopeptidase from Carica papaya: a cysteine endopeptidase of unusual substrate specificity, Journal of the American Chemical Society 34 (1995) 13190–13195. [22] M. Azarkan, A. El-Moussaoui, D.V. Wuytswinkel, G. Dehon, Y. Looze, Fractionation and purification of enzymes stored in the latex of Carica papaya, Journal of Chromatography B 790 (2003) 229–238. [23] T. Galindo-Estrella, R. Hernández-Gutiérrez, J. Mateos-Díaz, G. SandovalFabián, L. Chel-Guerrero, I. Rodríguez-Buenfil, S. Gallegos-Tintoré, Proteolytic activity in enzymatic extracts from Carica papaya L. cv. Maradol harvest byproducts, Process Biochemistry 44 (2009) 77–82. [24] P. Vasu, B.J. Savary, R.G. Cameron, Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation, Food Chemistry 133 (2012) 366–372. [25] R.V. Devakate, V.V. Patil, S.S. Waje, B.N. Thorat, Purification and drying of bromelain, Separation and Purification Technology 64 (2009) 259–264.
472
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
[26] S. Kumar, A.B. Hemavathi, H.U. Hebbar, Affinity based reverse micellar extraction and purification of bromelain from pineapple (Ananas comosus L. Meryl) waste, Process Biochemistry 46 (2011) 1216–1220. [27] P.T. Englund, T.P. King, L.C. Craig, A. Walti, Studies on ficin. I. Its isolation and characterization, Biochemistry 7 (1968) 163–175.
[28] M. Sigiura, M. Sasaki, Studies on proteinases from Ficus carica var.Hôraishi. V. Purification and properties of a sugar-containing proteinase (Ficin S), Biochimica and Biophysica Acta 350 (1974) 38–47. [29] C.D. Anderson, P.L. Hall, Purification of ficin by affinity chromatography, Analytical Biochemistry 60 (1974) 417–423.
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
The use of papain inhibitor immobilized onto polyaniline for bioaffinity chromatography of cysteine proteases Adriane G. Gamboa, Karla A. Batista, Flavio M. Lopes, Kátia F. Fernandes ⇑ Laboratório de Química de Proteínas, Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia-GO, Brazil
a r t i c l e
i n f o
Article history: Received 18 January 2013 Received in revised form 11 October 2013 Accepted 15 October 2013 Available online 25 October 2013 Keywords: Stationary phase Papain Bromelain Ficin Purification
a b s t r a c t In this work, a papain inhibitor extracted from seeds of Adenanthera pavonina was covalently immobilized onto glutaraldehyde modified polyaniline (PANIG). The immobilization was very efficient, presenting 54.24% inhibitor retention in the following conditions: immobilization time of 30 min, at 4 °C, pH 7.0, inhibitor concentration of 20 mg and 5 mg of PANIG. The resultant material (PANIG-I) was tested as stationary phase for the cysteine proteases papain, ficin and bromelain purification through bioaffinity chromatography. A commercial preparation of papain was efficiently purified, resulting in a single band with 25 kDa. SDS–PAGE of purified bromelain showed a characteristic band around 28 kDa and this procedure resulted in 0.89-fold purification and 32.4% yield. The purification process was more effective for ficin reaching 2.6-fold purification and a single band of 25 kDa in the SDS–PAGE. These results showed that the stationary phase containing A. pavonina inhibitors immobilized onto PANIG is a very promising material for a single step purification of cysteine proteases through bioaffinity chromatography. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The cysteine proteases comprise a group of enzymes with varied physical and biochemical properties. Papain extracted from Carica papaya latex is the representative enzyme for this family [1]. Cysteine-proteases are widely distributed in the nature, presenting several endogenous actions. In general, these enzymes present low selectivity through peptide linkage to be hydrolyzed and consequently they may have a diversity of applications [2]. However, the use of cysteine proteases is frequently restricted due to the difficulty inherent of proteolytic enzyme purification. The classic purification procedure consists of sequential chromatographic techniques including gel filtration and ion-exchange, hydrophobic or affinity chromatography [3,4]. The proteases purification is commonly accompanied by significant losses due to autolysis occurring during the sequence of chromatographic procedures. Therefore, the purification of proteases demands meticulous handling in a slow and high cost procedure [5]. An additional problem in the protease purification is the limited use of bioaffinity chromatography, since the stationary phase suffers the hydrolytic attack of the enzyme, compromising its integrity. In this sense, a stationary phase resistant to the proteolytic attack of the proteases could be helpful in a purification approach.
⇑ Corresponding author. Address: PB 131, Goiânia-GO, CEP 74001-970, Brazil. Tel.: +55 (62) 3521 1492; fax: +55 (62) 3521 1190. E-mail address: [email protected] (K.F. Fernandes). 1383-5866/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.seppur.2013.10.027
In this study, a papain inhibitor was covalently immobilized in a glutaraldehyde modified polyaniline and the resultant material used as a stationary phase for bioaffinity chromatography. This stationary phase was tested as a single step procedure for purification of papain, ficin and bromelain. 2. Methodology 2.1. General experimental procedures The seeds from Adenanthera pavonina used in this study were collected in the farm of the Universidade Federal de Goiás (Goiânia-GO, Brazil). Papain was from Calbiochem. Bromelain was obtained from the common commercially cultivated pineapple (Ananas comosus). Ficin was obtained from the latex of Ficus carica cultivated in the farm of the Universidade Federal de Goiás. The electrophoresis reagents were from GE Healthcare Life Sciences. All other reagents were of analytical grade. 2.2. Polyaniline synthesis and activation (PANIG) HCl-doped polyaniline (PANI) was synthesized by oxidation of a 0.44 mol L 1 aniline solution with a 0.68 mol L 1 ammonium persulphate solution, as described by Fernandes et al. [6]. PANI was subsequently activated (PANIG) under reflux for 2 h, using a 2.5% (v/v) glutaraldehyde solution prepared in 0.1 mol L 1 phosphate buffer, pH 6.0 [7]. The PANIG was exhaustively washed to assure
468
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
glutaraldehyde was completely removed. Then, PANIG was dried in a desiccator and stored in opaque plastic flask, at room temperature. 2.3. Extraction of A. pavonina seeds inhibitors The extraction and ammonium sulfate precipitation of papain inhibitors from A. pavonina was carried out according to methodology described by Silva et al. [8]. Seeds of A. pavonina were broken in a blade mill and the cotyledon fragments were separated as much from the testa pieces. The fragments were ground in a Wiley mill and defatted with hexane. The papain inhibitors were extracted by using 0.3 mol L 1 NaCl in 0.1 phosphate buffer, pH 8.0 (1:10 w/v). The supernatant (crude extract) was collected by centrifugation and heating at 90 °C for 10 min. The soluble proteins were collected by centrifugation and submitted to ammonium sulfate fractionation at 30% and 60% saturation. The resulting fractions were collected by centrifugation, dialyzed and lyophilized. The fraction 30–60 (98 mg of protein) was purified in a column (2.3 6.0 cm) filled with Sephadex G-75, equilibrated and eluted with 0.1 mol L 1 sodium phosphate buffer, pH 7.0. The tubes containing inhibitory activity from the first eluted peak (F1) were pooled, dialyzed against distillated water and lyophilized. The protein content was measured by the Bradford method [9], using bovine serum albumin as standard. The purity of purified papain inhibitor was analyzed using 12% denaturating polyacrilamide gel electrophoresis (SDS–PAGE) according to the method described by Laemmli [10]. Electrophoresis was run at 25–40 mA for 4 h at room temperature. Gels were stained according to the methodology described by Blum et al. [11], using silver nitrate. 2.4. Immobilization of papain inhibitor in PANIG Attempting to obtain the maximum amount of immobilized inhibitor, several tests were performed. For such, the amount of inhibitor (5–30 mg) and the pH of reaction (5.0–8.0) were varied. The buffers used in the pH tests were 0.1 mol L 1 sodium acetate for pH 5.0 and 0.1 mol L 1 sodium phosphate for the other pH values. The efficiency on the immobilization was measured making the PANIG- inhibitor derivative (PANIG-I) to react with a papain solution (7.6 U). After optimization, the immobilization process was made by adding 5 mg of PANIG to a solution containing 20 mg of inhibitor in 0.05 mol L 1 sodium phosphate buffer, pH 7.0. The reaction proceeded for 30 min, at 4 °C under stirring. The mixture was centrifuged at 9000g for 5 min to separate PANIG-I from supernatant. The PANIG-I was washed with the same buffer until unbounded inhibitor was completely removed. 2.5. Determination of inhibitory activity The papain inhibitory activity was determined according to the method of Kunitz as modified by Arnon [12]. Inhibitory activity was determined by comparing proteolytic activity of mixtures of preparations containing PANIG-I and enzyme with the activity of pure enzyme. The inhibitory activity was expressed in units of inhibitor (UI), according to definition of Silva et al. [8]. 2.6. Bioaffinity chromatography 2.6.1. Preparation of crude extracts of proteases The crude extract of bromelain was obtained from the stem of pineapple (A. comosus). Briefly: the stems of ripe pineapple fruits were separated from the freshly fruits. The stems were cut in small pieces, crushed in a mortar and vacuum filtered. The clear solution
was precipitated with cold acetone (1:3 v/v), and the powder obtained after evaporation of acetone was suspended in 0.1 mol L 1 sodium phosphate buffer, pH 7.0. This material was submitted to enzyme activity assay and used on the purification process. The crude extract of ficin was prepared according to Sgarbieri [13]. The latex from peduncles of unripe fruits of F. carica was collected and centrifuged at 9000g for 20 min. The clear yellow supernatant containing the crude ficin was stored in freezer at 20 °C until its use for activity assay and affinity chromatography. 2.6.2. Chromatography tests In order to test the PANIG-I as a stationary phase for bioaffinity chromatography, a glass tube was filled with 20 mg of PANIG-I and the column was equilibrated with 0.05 mol L 1 Tris buffer, pH 8.0. 500 lL of a commercial grade papain solution (171.4 U) (Calbiochem, La Jolla, CA, USA) was applied to this column and the same Tris buffer was used as mobile phase for elution of the nonbounded proteins. The elution of the retained papain was carried out by applying 500 lL of 0.1 mol L 1 NaOH solution, followed by immediate neutralization with 0.5 mol L 1 HCl solution. This fraction was precipitated with cold acetone (1:3 v/v) and dried at room temperature. The purity of this material was evaluated by electrophoresis. The same procedure was used for purification of bromelain and ficin. In order to evaluate the stability of the stationary phase for bioaffinity chromatography, test of repeated use were conducted using papain as enzyme to be purified. After elution of the retained papain, PANIG-I was washed twice with 0.05 mol L 1 Tris buffer, pH 8.0 and after this a new papain solution was left to react as described above. 2.6.3. Electrophoresis in polyacrylamide gel (SDS–PAGE) The purified proteins were analyzed for purity by using 12% denaturating polyacrylamide gel electrophoresis (SDS–PAGE) according to methodology described by Laemmli [10]. The amount of protein applied were determined following the method described by Bradford [9]. Electrophoresis was run at 25–40 mA for 2 h at room temperature. Gels were stained according to methodology described by Blum et al. [11], using silver nitrate. Molecular weight markers (New England Biolabs, MA) ranging from 25 to 116 kDa were used as running standard. 3. Results and discussion 3.1. Polyaniline synthesis and activation The chemical synthesis of polyaniline resulted in a fine brilliantblack-green powder, which became black-opaque after the activation with glutaraldehyde (Fig. 1a). The scanning electron microscopy (SEM) showed that glutaraldehyde modified polyaniline (PANIG) has a granular morphology, the particles presenting a variable size (Fig. 1b). The presence of cavities in PANIG provides a microenvironment that protects the immobilized molecule and explains the improved properties of several immobilized proteins. The cavities also enlarge the surface area of this material, resulting in a high available area for protein linkage. This structure explains several properties observed in the different systems where polyaniline was used as support for immobilization [7,14–16]. 3.2. Purification of the papain inhibitor from A. pavonina seeds The results of papain inhibitory activity, protein content, specific inhibitory activity and purification factor obtained in the purification of the papain inhibitor from A. pavonina seeds are
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
469
Fig. 1. Photograph (a) and scanning electron micrograph (b) of the PANIG.
Table 1 Purification of the papain inhibitors from Adenanthera pavonina.
Protein content (mg mL 1) Activity (IU mL 1) Specific activity (IU mg 1 protein) Purification factor Yield (%)
Crude extract
Fraction 30–60
Purified inhibitor
167.9 64 0.38 1.0 100
4.63 4.9 1.06 2.79 7.7
0.78 2.5 3.18 8.37 3.9
shown in Table 1. As can be observed, the protein content present in the fraction 30–60 was about 36-hold lower than in the crude extract. This lower content of protein in the fraction 30–60 leads to an increase of the specific inhibitory activity, resulting in approximately 3-fold purification (Table 1). In addition, the chromatography through Sephadex G-75 resulted in about 8-fold purification and a yield of 3.9%. The purification of the papain inhibitor was confirmed by denaturizing polyacrylamide gel electrophoresis (Fig. 2). The presence of a single band at 60 kDa in opposition to the multitude of protein present in the crude extract is a confirmation of the effectiveness of the purification procedure. According to Xavier-Filho [17], the leguminous plant seeds, like A. pavonina contain at least three classes of papain inhibitor: (1) a low molecular weight class (5–12 kDa); (2) an intermediate size class (20–30 kDa) and a high molecular weight class (60–80 kDa). The purified papain inhibitor from A. pavonina was used for immobilization onto glutaraldehyde modified polyaniline (PANIG).
Fig. 2. SDS–PAGE of the papain inhibitors from A. pavonina in the different purification steps. R1: molecular weight marker; R2: crude extract; R3: fraction 30– 60; R4: purified papain inhibitor (Shepadex G-75).
3.3. Immobilization of the papain inhibitor onto PANIG The maximum inhibitor retention was reached when 20 mg of inhibitor was offered to 5 mg of PANIG (Fig. 3). Above this concentration, the retention remained unchanged probably because all reactive groups available for the inhibitor linkage were occupied. The influence of the reaction pH on the immobilization efficiency was also observed (Fig. 4). The amount of immobilized inhibitor varied by almost 3-fold over a narrow pH range, with optimum immobilization being achieved at pH 7.0. Such large variation probably reflects the state of ionization of the support and the enzyme since the coupling reaction depends on the appropriate ionization of the reactive groups in both materials [15,18,19]. It is possible that at pH 7.0 the basic residues present on the exposed surface of papain inhibitor perform an important role in the immobilization reaction. As reported by Silva et al. [8], the papain
inhibitor from A. pavonina seeds presents 4 histidine, 9 arginine and 4 lysine residues. Considering the highly conserved structure of the cystatin’s inhibitor family, it is possible that such basic aminoacids are exposed in the protein surface being the targets for the reaction with the carbonyl groups of glutaraldehyde (Fig. 5) [20]. The optimal immobilization was obtained after 30 min of reaction of 5 mg PANIG and 20 mg of papain inhibitor in 0.1 mol L 1 sodium phosphate buffer pH 7.0, at 4 °C. Under these conditions, 3.58 U of inhibitor were immobilized, corresponding to 54.24% yield. The efficiency of the immobilization process, expressed by the ratio between the specific activity of the PANIG-I and the inhibitor offered for immobilization, was 0.77. It means that 77% of the inhibitor molecules were properly immobilized and only 23% were inactive or inaccessible to react with enzyme.
470
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
Fig. 3. Optimal amount of offered papain inhibitor for immobilization onto PANIG. Reaction: 4 °C; pH 7.0; 30 min of immobilization; 5.0 mg of PANIG.
Fig. 4. Optimal pH for papain inhibitor immobilization onto PANIG. Reaction: 4 °C; 30 min of immobilization; 5.0 mg of PANIG; 20 mg of papain inhibitor.
3.4. Chromatography tests
Fig. 5. Scheme representing the mechanism of reaction between papain inhibitor and PANIG.
The evaluation of PANIG-I as a stationary phase for bioaffinity chromatography was initially conducted with a commercial grade papain (Table 2). The purity of the papain eluted from the PANIG-I column was analyzed in a 12% SDS–PAGE. As can be seen in Fig. 6(a), the purified papain consisted of a single band of about 25 kDa, described in the literature as the molecular weight of papain from C. papaya [21–23]. It is important to note that the commercial papain had an additional band around 22 kDa, which disappeared after the bioaffinity purification. Vasu et al. [24] reported the presence of several contaminant enzymes and proteins in commercial preparations of papain. The stability of the stationary phase was evidenced by the absence of a 60 kDa band in the SDS–PAGE, corresponding to the papain inhibitor. The absence of this band confirms that the inhibitor remained bounded to the PANIG even after the elution of papain with NaOH solution. The chemical integrity of the PANIG-I was confirmed in the repeated use tests. In these tests, PANIG-I preserved 100% of the initial retention capacity after four cycles of reuse.
The purification of bromelain and ficin was also successfully obtained with PANIG-I bioaffinity chromatography. In the case of bromelain, 0.89-fold purification and 32.4% yield was achieved in a single step procedure (Table 2). The purity of the bromelain was evidenced in the SDS–PAGE by the presence of a single band around 28 kDa (Fig. 6b). Devakate et al. [25] using a 5-step methodology involving ammonium sulfate precipitation (40–80% saturation followed by 40–70% saturation), dialysis and ion exchange chromatography reached 10-fold purification with 84.5% yield. In another approach Kumar et al. [26] obtained 9.38fold purification with 15% yield in a 2-step affinity based reverse micellar purification process. Regarding to the ficin purification, the electrophoresis revealed a single band at 25 kDa (Fig. 6c). The efficiency of this purification procedure is presented in Table 2. As can be seen, the eluted fraction showed a 2.6-fold purification and a yield of 26.7%. Englund et al. [27] observed a purification yield of 8.8% using 5-step
Table 2 Purification of cysteine proteases by using a PANIG-I bioaffinity chromatography. Papain
Activity (U mL 1) Protein content (mg mL 1) Specific activity (U mg 1 protein) Purification factor Yield (%)
Bromelain
Ficin
Commercial
Purified
Crude extract
Purified
Crude extract
Purified
171.4 0.10 1714 0.89 62.2
106.6 0.07 1522
68 2.61 26.1 0.89 32.4
22 0.94 23.4
72 13.6 5.3 2.60 26.7
19 1.38 13.8
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
471
Fig. 6. SDS–PAGE of enzyme extracts and purified proteins through PANIG-I bioaffinity chromatography. (a) papain: (R1) molecular weight marker (kDa), (R2) commercial papain, (R3) purified papain; (b) bromelain: (R1) molecular weight marker (kDa), (R2) crude extract from Ananas comosus stems, (R3) purified bromelain; (c) ficin: (R1) molecular weight marker (kDa), (R2) crude extract from Ficus carica peduncles, (R3) purified ficin.
methodology including salt precipitation, dialysis and gel filtration. The same author also reported a better yield around 16% when salt precipitation step was removed from the purification approach. Sigiura and Sasaki [28] reported a yield of 9.5% using 4-step purification procedure based on CM-cellulose chromatography. Anderson and Hall [29] using a bioaffinity column of mercurial modified sheparose reached 1.9-fold purification with 66.3% yield.
4. Conclusions The immobilization of the papain inhibitor from A. pavonina onto PANIG confirmed the high retention capacity of polyaniline and evidenced its ability to actuate as a support for protein immobilization. The methodology for the PANIG-I synthesis is fast, simple, low cost and demands non-toxic reagents. The stability of the linkage between the papain inhibitor and PANIG was successfully demonstrated through SDS–PAGE analysis. Furthermore, the effectiveness of the proposed bioaffinity purification methodology was showed by SDS–PAGE analysis evidencing a single band for bromelain and ficin. In this sense, the PANIG-I is a very promising material for use as a stationary bioaffinity phase for a single step purification of cysteine proteases. References [1] Y.Q. Ling, H.L. Nie, S.N. Su, C. Branford-White, L.M. Zhu, Optimization of affinity partitioning conditions of papain in aqueous two-phase system using response surface methodology, Separation and Purification Technology 73 (2010) 343– 348. [2] A.S. Fahmy, A.A. Ali, S.A. Mohamed, Characterization of a cysteine protease from wheat Triticum aestivum (Cv. Giza 164), Bioresource Technology 91 (2004) 297–304. [3] H.L. Nie, T.X. Chen, L.M. Zhu, Adsorption of papain on dye affinity membranes: isotherm, kinetic, and thermodynamic analysis, Separation and Purification Technology 57 (2007) 121–125. [4] D. Tombaccini, A. Mocali, E. Weber, F. Paoletti, A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts, Analytical Biochemistry 289 (2001) 231–238. [5] P.A.G. Soares, A.F.M. Vaz, M.T.S. Correia, A. Pessoa-Junior, M.G. Carneiro-daCunha, Purification of bromelain from pineapple wastes by ethanol precipitation, Separation and Purification Technology 98 (2012) 389–395. [6] K.F. Fernandes, C.S. Lima, H. Pinho, C.H. Collins, Immobilization of horseradish peroxidase onto polyaniline polymers, Process Biochemistry 38 (2003) 1379– 1384.
[7] K.F. Fernandes, C.S. Lima, F.M. Lopes, C.H. Collins, Properties of horseradish peroxidase immobilised onto polyaniline, Process Biochemistry 39 (2004) 957–962. [8] K.F.F. Silva, C.S. Lima, R.R. Val, J. Xavier-Filho, Cysteine proteinase inhibitors from seeds of Adenanthera pavonina L, Revista Brasileira de Botânica 18 (1995) 137–141. [9] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding, Analytical Biochemistry 72 (1976) 680–685. [10] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T 4, Nature 227 (1970) 680–685. [11] H. Blum, H. Beier, H.J. Gross, Improved silver staining of plant proteins, RNA and DNA in polyacrilamide gels, Electrophoresis 8 (1987) 93–99. [12] R. Arnon, Papain, in: G. Perlmann, D.L. Loran (Eds.), Methods in Enzymology, Academic Press, New York, 1970, pp. 226–244. [13] V.C. Sgarbieri, Enzimas proteolíticas do látex de diversas variedades de Ficus carica L, Bragantia 24 (1965) 109–124. [14] K.F. Fernandes, E.R. Asquieri, R.N. Silva, Immobilization of Aspergillus niger glucoamylase onto a polyaniline polymer, Process Biochemistry 40 (2005) 1155–1159. [15] L.L.A. Purcena, S.S. Caramori, S. Mitidieri, K.F. Fernandes, The immobilization of trypsin onto polyaniline for protein digestion, Material Science and Engineering C 29 (2009) 1077–1081. [16] A.M. Pascoal, S. Mitidieri, K.F. Fernandes, Immobilisation of a-amylase from Aspergillus niger onto polyaniline, Food and Bioproducts Processing 89 (2011) 300–306. [17] J. Xavier-Filho, The biological roles of serine and cysteine proteinase inhibitors in plants, Revista Brasileira de Fisiologia Vegetal 4 (1992) 1–6. [18] K. Kang, C. Kan, A. Yeung, D. Liu, The immobilization of trypsin on soap-free P(MMA-EA-AA) latex particles, Material Science and Engineering C 26 (2006) 664–669. [19] A.H.M. Cavalcante, L.B. Carvalho Jr., M.G. Carneiro-da-Cunha, Cellulosic exopolysaccharide produced by Zoogloea sp. as a film support for trypsin immobilisation, Biochemical Engineering Journal 29 (2006) 258–261. [20] F. Xi, J. Wu, Z. Jia, X. Lin, Preparation and characterization of trypsin immobilized on silica gel supported macroporous chitosan bead, Process Biochemistry 40 (2005) 2833–2840. [21] B.P. Ó-Hara, A.M. Hemming, D.J. Buttle, L.H. Pearl, Crystal structure of glycil endopeptidase from Carica papaya: a cysteine endopeptidase of unusual substrate specificity, Journal of the American Chemical Society 34 (1995) 13190–13195. [22] M. Azarkan, A. El-Moussaoui, D.V. Wuytswinkel, G. Dehon, Y. Looze, Fractionation and purification of enzymes stored in the latex of Carica papaya, Journal of Chromatography B 790 (2003) 229–238. [23] T. Galindo-Estrella, R. Hernández-Gutiérrez, J. Mateos-Díaz, G. SandovalFabián, L. Chel-Guerrero, I. Rodríguez-Buenfil, S. Gallegos-Tintoré, Proteolytic activity in enzymatic extracts from Carica papaya L. cv. Maradol harvest byproducts, Process Biochemistry 44 (2009) 77–82. [24] P. Vasu, B.J. Savary, R.G. Cameron, Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation, Food Chemistry 133 (2012) 366–372. [25] R.V. Devakate, V.V. Patil, S.S. Waje, B.N. Thorat, Purification and drying of bromelain, Separation and Purification Technology 64 (2009) 259–264.
472
A.G. Gamboa et al. / Separation and Purification Technology 120 (2013) 467–472
[26] S. Kumar, A.B. Hemavathi, H.U. Hebbar, Affinity based reverse micellar extraction and purification of bromelain from pineapple (Ananas comosus L. Meryl) waste, Process Biochemistry 46 (2011) 1216–1220. [27] P.T. Englund, T.P. King, L.C. Craig, A. Walti, Studies on ficin. I. Its isolation and characterization, Biochemistry 7 (1968) 163–175.
[28] M. Sigiura, M. Sasaki, Studies on proteinases from Ficus carica var.Hôraishi. V. Purification and properties of a sugar-containing proteinase (Ficin S), Biochimica and Biophysica Acta 350 (1974) 38–47. [29] C.D. Anderson, P.L. Hall, Purification of ficin by affinity chromatography, Analytical Biochemistry 60 (1974) 417–423.