Simple polyacrylamide affinity gel electrophoresis using oleic acid for the isolation of chymotrypsin inhibitor

Journal of Bioscience and Bioengineering VOL. 110 No. 3, 276 – 280, 2010 www.elsevier.com/locate/jbiosc

Simple polyacrylamide affinity gel electrophoresis using oleic acid for the isolation of chymotrypsin inhibitor Jin Ik Lim,1 Kook-Jin Lim,1,2 Yun-Cheol Na,3 and Yong-Keun Lee4,⁎ National Core Research Center for Nanomedical Technology, Yonsei University, 262 Seongsanno, Seodaemun-gu, Seoul, 120-749, Republic of Korea 1 Binex Co., Ltd. 541 Dohwa-dong, Mapo-gu, Seoul 121-701, Republic of Korea 2 Joint Bioanalytical Research Team, Korea Basic Science Institute, 126-16 Anam-Dong, Sungbuk-Gu, Seoul, 136-713, Republic of Korea 3 and Denforus Co., 3001-2 Bangbae-dong, Seocho-Gu, Seoul, 137-974, Republic of Korea 4 Received 9 December 2009; accepted 12 March 2010 Available online 15 April 2010

Protease inhibitors have been usually isolated through a number of steps using various chromatographical methods, which are time consuming and tedious. In this report, an efficient and low-cost acrylamide affinity gel electrophoresis method for the detection and isolation of chymotrypsin inhibitor from a crude extract was studied. The affinity gel was obtained by immobilization of chymotrypsin on 5% (w/v) poly acrylamide-oleic acid gel, and the immobilized chymotrypsin showed high stability under varied concentrations of urea (0 to 8 M), pH (4 to 10) and temperature (30 to 80 °C). The affinity gel made of immobilized chymotrypsin was applied to polyacrylamide affinity gel electrophoresis and reverse electrode electro-elution using a modified commercial electrophoresis kit. Polyacrylamide affinity gel electrophoresis method showed higher isolation efficiency for chymotrypsin inhibitor from Ganoderma lucidum crude extract than a chromatographical method. Specific activity and yield of chymotrypsin inhibitor increased around 2.3-folds and 1.4-folds, respectively, compared with a chromatographical method. Also, two isomers of the inhibitor could be isolated by this method. Therefore, this method can be applied for the detection and isolation of bio-active molecules as a fast and economical method. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Bio-molecular detection technique; Affinity electrophoresis; Biosensor; Oleic acid; Immobilization]

Protease inhibitors are widely distributed in biological world (1–4), which are believed to be involved in the control of a variety of fundamental physiological proteolytic processes (5–7). Several purified exogenous protease inhibitors were proved to be beneficial in regulating certain diseases where proteolytic enzymes were related with pathological pathways (8–10). In general, protease inhibitors have been usually detected and purified through a number of steps using various chromatographical methods (11–13), which are generally time consuming and tedious. Therefore, varied novel methods have been developed to improve the isolation and detection efficiency (14,15). Affinity chromatography is one of the most efficient methods for the isolation of biological macromolecules (16–19). Compared with other techniques such as ion-exchange process (20), salting-out crystallization (21), organic reagent precipitation (22) and ultra filtration (23), the advantages of affinity chromatography are continuous online separation, specific selection and real time detection of the target molecules by immobilized bio-molecules (17,18). Immobilized enzymes are defined as the physically confined or localized enzymes with their catalytic activity, which can be used ⁎ Corresponding author. Denforus Co., Bangbae-dong, Seocho-Gu, Seoul 137-853, Republic of Korea. Tel.: +82 2 592 2870; fax: +82 2 592 2879. E-mail addresses: [email protected] (J.I. Lim), [email protected] (K.-J. Lim), [email protected] (Y.-C. Na), [email protected] (Y.-K. Lee).

repeatedly and continuously (24,25). There are varied methods by which enzymes can be immobilized, ranging from covalent chemical bonding to physical entrapment (24,26,27). The merit of immobilized enzymes from an analytical standpoint is primarily their reusability; therefore, cost saving, high efficiency and control of their catalytic activity are possible (25,26). Recently, capillary affinity gel electrophoresis method as a kind of affinity electrophoresis methods was introduced for the ingredient analysis of nucleotides or proteins (28,29). Compared with conventional methods including affinity chromatography, the merits of this method are applicability to small quantity of sample, and fast and quantitative analysis by dilution of limited amount of sample with electro-elution (30). However, this method can be applied only to the microanalysis of crude extracts, not to the recovery of bio-active molecules. Detection and isolation of bio-active molecules from natural products such as a curative plant are generally boring and difficult work; however, they were widely studied because of the importance of new medicine. Recently, protease inhibitor from medicinal mushrooms was reported as a multi-functional regulator in biological system, which could reveal anti HIV and anti cancer activity (31–33). Among these, Ganoderma lucidum is a popular medicinal mushroom in Asia because of numerous pharmacological effects including immunomodulatory and antitumor activities (34). Also, protease inhibitor from this mushroom was already reported (30,32). However, previous studies were on the confirmation of protease

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inhibition activity or partial purification with chromatographical methods. Therefore, novel method for an improved analytical study and simple isolation method should be developed (35). In this study, we studied a simple and fast polyacrylamide affinity gel electrophoresis (PAAGE) method involving reverse electrode electro-elution (REE) for the isolation of chymotrypsin inhibitor using immobilized chymotrypsin. The aims of this study were to (i) confirm the compatibility of oleic acid, which was used as a space arm, with acrylamide to immobilize chymotrypsin, (ii) evaluate the efficiency of immobilized chymotrypsin and (iii) determine the optimal condition for PAAGE-REE method for the isolation of the chymotrypsin inhibitor. MATERIALS AND METHODS Chemical reagents Electrophoresis kit (HSI SE 250 Mighty Small II; Hoefer Scientific Instruments, San Francisco, CA, USA) was used for the affinity electrophoresis and electro-elution. Chymotrypsin, acrylamide, lithium chloride, trifluoro ethanol, oleic acid, 1-ethyl–3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxyl succinimide (NHS) and synthetic substrates were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used without further purification except for trifluoro ethanol. Preparation of crude extract from G. lucidum Dried G. lucidum (Korean cultured, 6 years old) of 3 g was homogenized in 200 ml lysis buffer (0.05 M Tris–HCl buffer, pH 7.6, 0.1 M NaCl, 5 mM mercaptoethanol) containing 1 mM phenylmethylsulphonyl fluoride. The homogenate was centrifuged (6000×g for 30 min) and the supernatant was fractionated by ammonium sulfate saturation. Obtained precipitates were dissolved in 5 ml of 0.05 M Tris–HCl buffer (pH 7.6) (30). Preparation of affinity gel by immobilization of chymotrypsin Mixture of 5% (w/v) acrylamide for native polyacrylamide electrophoresis (PAGE) (36,37) and oleic acid with the final concentration of 0.05 M was poured in a sandwich type glass plate mold of the electrophoresis kit, and was polymerized for 15 min. Oleic acid as spacer arm was used between chymotrypsin. In Fig. 1, the schematic showed randomly addition polymerized oleic acid and acryl amide by free radical reaction. The gel was washed in 70% (v/v) ethanol for 12 h. After removal non-reacted oleic acid, the gel was washed for 5 h in distilled water (DW). Carboxyl group of the gel was activated by 0.1 M N-ethyl-N′-(dimethylaminopropyl) carbodiimide and 0.3 M N-hydroxysuccinimide) for 1 h. After washing in DW for 1 h, mixture of 0.2 M chymotrypsin (C7762; SigmaAldrich) and 0.3 M N-benzoyl-L-tyrosine ethyl ester was added. After 5 min at room temperature, chymotrypsin immobilized gel was washed in 4 °C phosphate buffered saline (PBS) for 3 h. Immobilized chymotrypsin stability assay Stability of immobilized chymotrypsin gel (10 × 10 × 4 mm) and free chymotrypsin was measured by the remaining activity of chymotrypsin. For the stability determination according to the concentration of urea as a denaturant, immobilized and free chymotrypsin were soaked in varied concentrations of urea (0 to 8 M) for 1 h at room temperature. In case of the reaction time, immobilized and free chymotrypsin were soaked up to 180 min in 5 M urea and were removed from urea every 20 min. For the stability determination according to pH, immobilized and free chymotrypsin were soaked for 4 h in varied pH buffer (4–10). All immobilized and free chymotrypsin from varied urea and pH conditions were washed in 0.05 M Tris–HCl buffer of pH 7.6 for 5 min, then the remaining activity was measured using the chymotrypsin activity assay method. Also, immobilized and free chymotrypsin were soaked for 1 h in varied temperatures (30, 40, 50, 60, 70 and 80 °C), and then the remaining activities were determined by the activity assay method. Reusability of the immobilized chymotrypsin gel was assessed by the remaining activity according to the number of reuse. For this, the gel was stored in 0.05 M Tris–HCl buffer of pH 7.6 at 4 °C. Polyacrylamide affinity gel electrophoresis (PAAGE) and reverse electrode electro-elution (REE) Electrophoresis kit (HSI SE 250 Mighty Small II; Hoefer Scientific Instruments, San Francisco, CA, USA) was used for the affinity electrophoresis and electro-elution. Four milliliters of 5% (w/v) polyacrylamide as supporting gel was

FIG. 1. A schematic representation of gel structure by acryl amide and oleic acid.

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made in a sandwich type glass plate, and the affinity gel was placed on top of the supporting gel. Then, 2 ml of 5% (w/v) polyacrylamide solution as stacking gel over the affinity gel was prepared (Fig. 2A). Crude extract from G. lucidum (2 ml) was loaded and was run at 10 mA for 2 h. Loaded crude extract was moved to the bottom of 5% (w/v) supporting gel. The affinity gel was removed from the kit. Seven milliliters of 5% (w/v) polyacrylamide supporting gel was made in a sandwich type glass plate. Then the removed affinity gel was placed on top of the supporting gel and was fixed by adding the small amount of 5% (w/v) polyacrylamide solution. The electrode solution (0.5 M Tris) was added on the top of the affinity gel (about 3.0 ml). Instead of the anodic electrode equipped with the commercial kit, a new platinum electrode was used. Then polarity of the power supply was reversely plugged to change the polarity of the electrodes. Electrophoresis was run at 30 mA for 1 h upward instead of the usual running down (Fig. 2B). Isolation of chymotrypsin inhibitor by chromatographical methods Crude extract of G. lucidum (2 ml) was loaded onto a Sephadex G-75 column of 1.6 × 80 cm (GradiFrac; Pharmacia Biotec, Uppsala, Sweden) and proteins were eluted using the equilibration buffer (0.05 M Tris-HCl buffer, pH 7.6). Fractions were collected at a flow rate of 0.4 ml/min. 250 μl of concentrated sample from the gel filtration chromatography was loaded onto a UNO-Q1 column (1 × 5 cm) of a fast protein liquid chromatography (Biologic HR, Bio-Rad, Hercules, CA, USA). Bound proteins were eluted by a linear gradient of ionic strength (0∼ 1 M NaCl in 0.05 M Tris–HCl buffer, pH 7.6). Fractions were collected at a flow rate of 0.3 ml/min and those corresponding to chymotrypsin inhibitory activity were collected and concentrated by ultrafiltration (38). Chymotrypsin and chymotrypsin inhibitor activity assay Chymotrypsin or isolated chymotrypsin inhibitor from G. lucidum extract activity was assayed according to the reported method (39). Standard assay composition contained 0.05 M Tris–HCl buffer of pH 7.6, 2 × 10−5 M synthetic peptide substrate (N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide) and chymotrypsin or chymotrypsin inhibitor in 0.5 ml total volume. The activity was measured by the absorbance (390 nm) of p-nitroaniline from the substrate dissociated by chymotrypsin using an UV–Vis spectrophotometer. Characterization of chymotrypsin inhibitor For molecular weight determination of chymotrypsin inhibitor, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 15% (w/v) separating gel was performed with molecular weight markers and product by PAAGE-REE, and compared with protein band position on native PAGE of the same condition using Coomassie blue dye. After another native PAGE, before dyeing, protein bands including some surrounding the gel by native PAGE were cut and applied to the REE method for recovery of protein on the cut gel, and then concentrated (30). Recovered proteins were soaked for 1 h in each temperature (40, 50, 60, 80 and 100 °C) and removed and then the remaining inhibition activities were determined by the chymotrypsin activity assay method.

RESULTS AND DISCUSSION PAAGE-REE process and immobilized chymotrypsin Immobilized chymotrypsin was applied to PAAGE and REE. This method was easily applicable after minimum modifications of commercial electrophoresis kit of a vertical type (Fig. 2). Therefore, expensive other devices were not necessary except for platinum wire as an electrode for REE. Following this method, it took about 21 h for the fabrication of affinity gel and the required time for PAAGE-REE method was only 3 h. In case of the conventional chromatographical methods, it usually takes

FIG. 2. A schematic representation of the (A) polyacrylamide affinity gel electrophoresis (PAAGE) and (B) reverse electrode electro-elution (REE) process; (A′) commercial platinum anodic electrode; (B, B′) electrode solution; (C) 5% stacking gel; (D, C′) affinity gel; (E, D′) 5% supporting gel; (A, E′) cathodic electrode. Arrows indicate the direction of migration.

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FIG. 3. Difference in the remaining activity of immobilized and free chymotrypsin (A: according to urea concentration, B: according to immersion time in 5 M urea, C: according to pH, D: according to temperature).

over 24 h for each chromatography steps including the preparation of column and operation. Immobilization efficiency of chymotrypsin was determined to be 30 ± 6% as compared with the amount of initial loaded free chymotrypsin for immobilization. After immobilization, unreacted free chymotrypsin was collected and concentrated to be used for the fabrication of another affinity gel. Stability of immobilized chymotrypsin Urea was used as a denaturant for the inactivation of the chymotrypsin. As to the difference in stability between immobilized and free chymotrypsin, urea was added and the remaining activity of immobilized and free chymotrypsin was measured according to the urea concentration and reaction time. The remaining activity decreased by increasing the urea concentration, and immobilized chymotrypsin showed high stability than free chymotrypsin (Fig. 3A). In case of immobilized chymotrypsin, it showed the remaining activity to be higher than 50% at 6 M urea; however, free chymotrypsin showed a low activity of below 5%. The remaining activity of immobilized and free chymotrypsin was measured according to time under the condition of 5 M urea. After 1 h, the remaining activity of immobilized chymotrypsin was higher than 75%; however, free chymotrypsin lost almost all activity which was lower than 20% (Fig. 3B).

Ionization trend of amino acids in an enzyme changed by pH condition (40), and the binding and active sites of almost all enzymes were affected by pH condition (41). Therefore, activity of immobilized chymotrypsin was measured under varied pH conditions and compared with that of free chymotrypsin. Immobilized chymotrypsin showed higher stability in acidic or basic conditions except for neutral pH condition. In case of pH 7 and pH 8 conditions, the activity of immobilized chymotrypsin was similar to that of free chymotrypsin. Therefore, we supposed that differences in the activity between immobilized and free chymotrypsin in these conditions were due to some measurement errors. The following values for pH, remaining activity (%) of immobilized chymotrypsin/remaining activity (%) of free chymotrypsin were obtained; 4, 72 ± 4 / 56± 2; 5, 80 ± 2 / 70 ± 1; 9, 83 ± 5/75 ± 4; 10, 71 ± 3/63 ± 3 (Fig. 3C). Also, heat stability increased by immobilization of chymotrypsin (Fig. 3D). In case of free chymotrypsin, the activity was completely lost at 60 °C, but immobilized chymotrypsin showed relatively stable activity of over 40%. Therefore, higher stability of immobilized chymotrypsin than free chymotrypsin was confirmed.

Table 2. Isolation efficiency by polyacrylamide affinity gel electrophoresis-reverse electrode electro-elution (PAAGE-REE) method and chromatographical method. Isolation method Table 1. Remaining activity of immobilized chymotrypsin by the number of reuse. Number of reuse Remaining activity (%)

1

2

3

5

7

10

15

20

30

100 ±3

99 ±3

97 ±7

98 ±5

99 ±2

97 ±3

99 ±3

94 ±5

90 ±3

Crude extract Chromatographical method PAAGE-REE method

Protein (mg)

Activity (Unit)

Specific activity (U/ mg)

Yield (%)

21.7 0.63

1423 238

65.6 377.8

100 16.7

0.38

325

855.3

22.8

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FIG. 4. Molecular weight and separated protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE (lane 1: separated chymotrypsin inhibitor-1 (CI-1) on native PAGE, land 2: separated CI-2 on native PAGE, lane 3: two bands on SDS-PAGE, lane 4: molecular weight markers).

Reusability is a common merit of immobilized enzymes (42). Therefore, reusability according to the number of reuse using the same immobilized chymotrypsin gel was evaluated. Table 1 shows the remaining activity of immobilized chymotrypsin according to the number of reuse. The result of one reaction per day using the same immobilized chymotrypsin gel showed high reusability at least over 90% up to 30 times (number of reuse 10: 97 ±3%, 20: 94±5% and 30: 90±3%). Isolation efficiency by PAAGE-REE method and chromatographical method Isolation efficiency by PAAGE-REE method was evaluated by the amount of recovered chymotrypsin inhibitor from G. lucidum. In Table 2, the purification details of the chymotrypsin inhibitor by a chromatography and also by the PAAGE-REE method are listed. Besides timesaving, purification by the PAAGE-REE enhanced the specific activity of purified inhibitor around 2.3-folds as compared to that by the chromatographical purification. Also, yield was increased around 1.4-fold. This high isolation efficiency by PAAGE method seemed to be due to affinity of immobilized chymotrypsin with chymotrypsin inhibitor and REE was economical method because the method is not need to additional equipment. Also, REE showed high recovery efficiency in as little as 1 h. Characterization of isolated chymotrypsin inhibitor from G. lucidum In Fig. 4, two bands [chymotrypsin inhibitor-1(CI-1) and CI-2 in lane 3] on SDS-PAGE were observed, which were separated to each protein by native PAGE and REE method. Lanes 1 and 2 on the native PAGE represented each separated protein band. In this study, molecular weights of chymotrypsin inhibitors were determined to be 17574.7 Da and 17546.5 Da, respectively, compared with the molecular weight marker (lane 4) on SDS-PAGE. General Kunitz type protease inhibitor showed molecular weight of around 18,000–20,000 Da and heat unstable activity (43). Also, Bowman-Birk type inhibitor showed around 8,000–10,000 Da molecular weight and heat stable activity due to a high cystine content (usually seven disulfides) (44), although a Bowman-Birk type protease inhibitor of 18,500 Da was reported (45). Therefore, molecular weights of CI-1 and CI-2 were within the range of both of the Kunitz type and the Bowman-Birk type protease inhibitor. But both inhibitors showed good heat stability higher than 70% at 100 °C (Fig. 5); therefore, heat stability of CI-1 and CI-2 was within the range of Bowman-Birk type. Therefore, we supposed that two proteins were Bowman-Birk type protease inhibitors. In this study, chymotrypsin was immobilized on polyacrylamide-oleic acid gel. Immobilized chymotrypsin showed improved stability and reusability than free chymotrypsin. For detection and isolation of chymotrypsin inhibitor from a crude extract, PAAGE-REE using the

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FIG. 5. Heat stability comparison of chymotrypsin inhibitor isomers (CI-1 and CI-2) under varied temperature condition.

immobilized chymotrypsin was performed. This method was confirmed as a fast and reliable method for the detection and isolation of chymotrypsin inhibitor from G. lucidum crude extract than chromatographical method. Therefore, the PAAGE-REE method can be widely applied for the detection and isolation of bio-active molecules from various natural products. ACKNOWLEDGMENTS This work was supported by a grant from the National Core Research Center for Nano Medical Technology, Yonsei University (Grant R15-2004-024-00000-0).

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