Enzyme inhibitors from plants. Isolation and characterization of a ... - IAS

J. Biosci., Vol.5, Number 1, March 1983, pp. 21—33

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Enzyme inhibitors from plants. Isolation and characterization of a protease inhibitor from arrow root (Maranta arundinaceae) tuber N. MALLIKARJUNA RAO, H. NAYANA RAO and T. N. PATTABIRAMAN Department of Biochemistry, Kasturba Medical College, Manipal 576 119 MS received 11 November 1982; revised 9 February 1983 Abstract. A protease inhibitor from arrow root (Maranta arundinaceae) tuber has been isolated in a homogeneous form. The inhibitor has a Mr of 11,000-12,000; it inhibited bovine trypsin, bovine enterokinase, bovine α-chymotrypsin and the proteolytic activity of human and bovine pancreatic preparations. The inhibitor is resistant to pepsin, and elastase. It could withstand heat treatment at 100°C for 60 min and exposure to a wide range of pH (1.0-12.5) for 72 h at 4°C without loss of activity. Arginyl groups are essential for the action of the inhibitor. Preincubation of the inhibitor at pH 3.7 with trypsin or chymotrypsin caused nearly a two-fold increase in inhibitor potency. Keywords.

Arrow root tuber; protease inhibitor; isolation; properties.

Introduction Protease inhibitors from plant tubers like potato (Ryan, 1973; Bryant et al.,1976; Pearce et al., 1982; Richardson, 1977; Melville and Ryan, 1972), sweet potato (Sugiura et al., 1973), Alocasia macrorhiza (Sumathi and Pattabiraman, 1977) and Colocasia antiquorum (Sumathi and Pattabiraman, 1979) have been studied extensively. Sumathi (1979) screened several plant tubers and bulbs for antiprotease activity and found that arrow root tuber comes under the group of plant materials with high antiprotease activity. Recently, Bhat et al. (1981) reported enterokinase inhibitory activity in this tuber. In view of limited information available on enterokinase inhibitory activity of plant protease inhibitors, it was considered worthwhile to study the inhibitory factors in arrow root tubers in detail. In this communication we report, the isolation and properties of a low molecular weight inhibitor from this tuber which is capable of inhibiting trypsin, enterokinase and α-chymotrypsin.

Abbreviations used: BAEE, α-N-benzoyl L-arginine ethyl ester; ΒΑΡΝΑ, α-N-benzoyl DL-arginine p-nitro anilide; TNBS, trinitro benzene sulphonate; OMI, O-methvl isourea. CHD, cyclohexane dione.

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Materials and methods Arrow root rubers were procured from local sources. Bovine trypsin (salt-free, twice crystallized), bovine α-chymotrypsin (salt free, thrice crystallized) and porcine elastase (twice crystallized) were the products of Worthington Biochemical Corporation, USA. Peroxidase, cytochrome c, myoglobin, α-Νbenzoyl L-arginine ethyl ester (BAEE). α-N-benzoyl DL-arginine p-nitroanilide (ΒΑΡΝΑ), sodium trinitrobenzene sulphonate (TNBS), O-methyl isourea (OMI) and Dalton markVI were purchased from Sigma Chemical Company, St. Louis, Missouri, USA. Porcine pepsin (thrice crystallized) was obtained from Calbiochem, USA. Affigel-10 was the product of Bio-Rad Laboratories, USA. 1,2Cyclohexanedione (CHD) was obtained from Aldrich Chemicals, USA. Subtilisin BPN was purchased from Nagase Company, Osaka, Japan. Bovine enterokinase was purified up to the ammonium sulphate stage (Liepnicks and Light, 1979). Bovine pancreas was collected from local slaughter house. Human pancreas was collected during autopsy at the Kasturba Medical College Hospital, Manipal. The tissues were kept in frozen condition until they were processed. Acetone powders of human and bovine pancreas were prepared and used as sources of proenzymes. The preparations were activated using bovine enterokinase (Chandrasekher and Pattabiraman, 1982). Trypsin-Affigel was prepared as follows. Bovine trypsin (350 mg) in 50 ml of 0.1 Μ phosphate buffer pH 7.0 was added to 50 ml (wet volume) of Affigel-10 at 4°C and gently stirred for 6 h. After standing overnight at 4°C, the suspension was filtered and washed extensively with 1.0 Μ ethanolamine-HCl in 0.1 Μ phosphate buffer pH 7.0 at a flow rate of 50 ml h–l followed by the buffer containing 1.0 Μ NaCl till there was no absorbance at 260 nm. The immobilized enzyme preparation was stored at 4°C in 50 mM HCl until use. Caseinolytic assay of the proteolytic enzymes were performed (Sumathi and Pattabiraman,1975). Ten µg ofbovine trypsin, 12.5 μg of bovine α-chymotrypsin, 0.2 mg protein of activated human pancreatic preparation, 0.11 mg protein of activated bovine pancreatic preparation, 12.5 µg of elastase and 20 µg of subtilisin BPN were used in the assay systems to get comparable activities in the linear range. One unit of caseinolytic activity is defined as the amount of enzyme that liberated one mg of trichloroacetic acid - soluble peptides. Amidolytic activity of trypsin was measured, using ΒΑΡΝΑ as substrate according to the method of Erlanger et al. (1961). Esterase activity of bovine enterokinase was determined using BAEE as substrate (Bhat et al. 1981). Protein content was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard. The purified inhibitor was analyzed for carbohydrate content by the method of Dubois et al (1956) using galactose as standard. Active site titration of bovine trypsin was performed according to Sumathi (1979). The trypsin preparation was 65% active.

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The inhibitor activities were determined by including suitable amounts of inhibitor in the assay system. One inhibitor unit is defined as the amount of inhibitor required to suppress the enzyme activity by one unit. Controls without inhibitor were run simultaneously. Unless mentioned otherwise, the inhibitory activities discussed are those obtained by the caseinolytic method. Isolation of the major inhibitor Arrow root tuber was homogenized with 3 volumes (wt/vol) of 0.02 Μ phosphate buffer pH, 7.6 and the mixture was stirred in the cold (5°C) for 30 min and set aside for 90 min. The suspension was filtered under suction. To the clear filtrate, five volumes of cold (—5°C) acetone was added with stirring. After 20 min stirring, the precipitate was collected by suction filtration, washed with cold acetone and finally with diethyl ether. The precipitate was air dried and stored at 4°C under desiccation. One g of acetone powder (obtained from 30 g of tubers) was suspended in 70 ml of 0.02 Μ phosphate buffer, pH 7.6. This was designated as the crude extract. The crude extract was subjected to heat treatment at 90°C for 5 min, cooled to room temperature and centrifuged at 10,000 g for 20 min. The supernatant was designated as the heat treated fraction. To this fraction (vol. 60 ml) solid ammonium sulphate (23.4 g) was added to 60% saturation at 4°C and the suspension was left overnight. The precipitate was collected by centrifugation at 10,000 g for 20 min, dissolved in 0.02 Μ phosphate buffer, pH 7.6, dialyzed against 100 volumes of 2×10-3 Μ phosphate buffer, pH 7.6 for 16h and centrifuged at 10,000 g for 20 min. The clear supernatant (8.0 ml) was designated as the ammonium sulphate fraction. The next three stages of purification were carried out in batches. DEAE-cellulose chromatography: Four ml of the ammonium sulphate fraction was loaded onto a column of DEAE-cellulose (1.6 × 12.4 cm, bed vol. 25 ml) equilibrated with 2×10–3 Μ phosphate buffer, pH 7.6 at room temperature. The column was washed with 140 ml of the equilibration buffer at a flow rate of 20 ml h-1 and the washings containing the inactive proteins were discarded. The column was then eluted with a linear gradient system of NaCl formed between 95 ml of 0.02 Μ phosphate buffer pH 7.6 in the mixing chamber and 95 ml of the buffer containing 0.8 Μ NaCl in the reservoir. Ten ml fractions were collected at a flow rate of 20 ml h–1 . The active fractions (tube numbers 5 and 6, figure 1) were pooled and designated as DEAE-cellulose fraction. Affinity chromatography: The above fraction (20 ml) was allowed to flow through a column of trypsin-Affigel (1.6×12.4 cm, bed vol. 25.0 ml) equilibrated with 0.02 Μ phosphate buffer, pH 7.6 containing 0.3 Μ NaCl at 4°C. The column was washed with 70 ml of this solution at a flow rate of 10.0 ml h–l and the washings were discarded. The column was eluted with 50 mM HCl and ten ml fractions were collected. The active fractions (tube numbers 10 and 11, figure 2) were pooled and designated as trypsin Affigel fraction.

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Figure 1. Fractionation of ammonium sulphate fraction on DEAE-cellulose. (Details are given under materials and methods).

•

( ) Protein content; (O) Antitryptic activity.

Figure 2. Affinity chromatography of DEAE-cellulose fraction on trypsin-Affigel 10. (ExperirnentaLdetails are given under materials and methods).

•

( ) Protein content; (O) Antitryptic activity.

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Chromatography on Sephacryl S-200: The above fraction was dialysed against 0.04 Μ phosphate buffer, pH 7.6 at 4°C and concentrated to 4.0 ml by lyophilization. To this solution 70 mg of NaCl was added to give a final concentration of 0.3 Μ NaCl. This solution was applied to a column of Sephacryl S-200 (1.6 × 62.0 cm, bed vol. 120.0 ml) equilibrated with 0.2 Μ phosphate buffer, pH 7.6 containing 0.3 Μ NaCl. The column was eluted at room temperature with the same solution at a flow rate of 8 ml h–1 and 2.0 ml fractions were collected. The major active fractions (tube numbers 67-71, figure 3) were pooled and dialyzed against 100 volumes of distilled water at 4°C. This was designated as the Sephacryl fraction and was used for further studies.

Figure 3. Gel chromatography of trypsin-Affigel 10 fraction on Sephacryl S-200. (Experimental details are given under materials and methods).

•

( ) Absorbance at 280 nm; (O) Trypsin inhibitory activity.

Electrophoresis: Cellulose acetate membrane electrophoresis of the purified inhibitor was performed at pH 8.6 (0.05 Μ barbitone buffer) and at pH 5.0 (0.5 Μ acetate buffer) for 75 min at 200V. The strips were stained with 0.1% Coomassie Brilliant Blue R-250 in 7% acetic acid. Polyacrylamide gel electrophoresis was performed according to the method of Davis et al (1964) at pH 8.3 using 7.0% Polyacrylamide. The electrophoresis was performed for 60 min at 3 mA current per tube. Coomassie Brilliant Blue R-250 (0.1% in methanol-acetic acid-water 5:1:5 vol/vol) was used to stain the protein. SDS-Polyacrylamide gel electrophoresis was performed at pH 7.2 according to the method of Weber et al (1972) using 10.0% Polyacrylamide. The inhibitor was treated with 1% SDS, 1% mercaptoethanol and 36% urea in 0.01Μ phosphate buffer, pH 7.2 at 37°C for 2 h. The electrophoresis was performed for 5 h at 7 mA current per tube. Staining was done as described above.

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Molecular weight determination: This was determined by SDS-polyacrylamide gel electrophoresis using Dalton mark VI for standardization. Molecular weight was also determined by gel permeation chromatography on Sephadex G-100 column (0.9 × 56.5 cm, bed volume 36 ml). The column was equilibrated with 0.02 Μ phosphate buffer pH 7.6 containing 0.1Μ NaCl and the same system was used as eluant. The purified inhibitor (150 µg protein) in a volume of 1.0 ml was applied to the column and eluted at a flow rate of 8.0 ml h-1 at room temperature. One ml fractions were collected and assayed for protein and antitryptic activity. Cytochrome c, myoglobin, peroxidase, bovine serum albumin and bovine trypsin were used as marker proteins. Effect of pH on the stability of the inhibitor: The inhibitor (2.9 µg protein) in 0.1 ml water was incubated with 0.1 ml of different solutions or buffers for 72 h at 4°C (pH 1.0) 0.1MHCl; (pH 2.0) 0.1 ΜHCl/KCl; (pH 4.5) 0.1 Μ acetate; (pH 6.0 and 7.0) 0.1 Μ sodium phosphate; (pH 8.0) 0.1 Μ Tris-HCl; (pH 9.0) 0.1 Μ borate; (pH 10.0) 0.1 Μ sodium bicarbonate and (pH 12.5) 0.2 Μ NaOH. After this treatment, 0.4 ml of 0.2 Μ phosphate buffer, pH 7.6 was added and aliquots were assayed for antitryptic activity. Effect of temperature on the stability of the inhibition: The inhibitor (1.45 µg protein) in 20μl of water was subjected to heat treatment at 10°C for different time intervals, cooled and assayed for residual inhibitor activity against bovine trypsin and α-chymotrypsin. The inhibitor was also subjected to pressure cooking (15 lb/ in2) for 20 min. Chemical modification: For amino group modification (Haynes et al., 1967),14.5µg protein of the inhibitor was treated with 200μg of TNBS at pH 7.6 (0.18 mM phosphate) in a volume of 2.0 ml at room temperature for 24 h. The solution was dialysed against one litre of water for 8h at 4°C and aliquots of the dialysed solution were used for the determination of antitryptic and antichymotryptic activities. OMI also was employed to study the role of amino groups (Plapp et al., 1971). The inhibitor (7.3μg protein) was allowed to react with 1.0 mg of OMI in a volume of 1.0 ml containing 8.0μmοl of sodium hydroxide (final pH 11.5) for 24 h room temperature. The solution was dialysed and processed as described above. Guanidino groups were modified by treatment with ninhydrin(Chaplin,1976) and with CHD (Abe et al.,1978). The inhibitor (29.2 µg protein) was treated with 4.0 mg of ninhydrin at 37°C in a volume of 4.0 ml containing 640 μmοl of Na2HPO4 (final pH 9.2). Aliquots were withdrawn after definite time intervals and dialyzed against 0.1 Μ acetate buffer pH 4.0 for 5 h at 4°C. The dialyzed solutions were assayed for residual antitryptic and antichymotryptic activities. For CHD treatment, 36 μg of the inhibitor was incubated with either 500 µg 2.0 mg of the reagent in a volume of 5.0 ml containing 900 µmol of borate buffer pH 9.0 at 37°C for definite periods of time. Aliquots were dialysed against one litre of distilled water for 8 h at 4°C and assayed for residual inhibitory activities. The inhibitor was also subjected to heat treatment at 100°C for 10 min in an experiment prior to treatment with CHD. Controls without the chemical modifiers were run in all cases.

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Effect of protease on the inhibitor: The inhibitor (2.9 µg protein) was incubated with 3.0 µg of pepsin at 37oC for 6 h at pH 2.0 (0.1 ΜHCl/KCl buffer) in a volume of 0.25 ml. To this solution, 0.4 ml of 0.2 Μ phosphate buffer pH 7.6 was added and the solution was directly used for the determination of residual antitryptic activity. For studying the effect of elastase, 5.8 µg of the inhibitor was treated with 12.5 µg of elastase for one h at 37°C in a volume of 0.4ml in the presence of 80 µmol of phosphate buffer pH 7.6.Aliquots were directly used for antitryptic activity measurement using ΒΑΡΝΑ as substrate. Controls without the enzymes were run simultaneously. Preincubation studies with trypsin and α-chymotrypsin: The inhibitor (2.1 µg protein) was incubated with 10 µg of bovine trypsin for definite time intervals either at 4°C, 25°C, or at 37°C in the presence of 4.0 μmol of acetate buffer pH 3.7 in a volume of 0.5 ml. The pH was adjusted to 7.6 by the addition of 0.5 ml of 0.2 Μ phosphate buffer and assayed for antitryptic activity by the addition of 1.0 ml of 2%casein solution. Similarly, preincubation was done with 12.5 µg of bovine αchymotrypsin and the antichymotryptic activity was assayed. Preincubation studies were also done at pH 7.6 using 0.2 Μ phosphate buffer. Controls without the inhibitor were run simultaneously. Studies on complex formation. The inhibitor (150 µg protein) was incubated with 250 μg of bovine trypsin for one h at 37°C in the presence of 10 µmol phosphate buffer pH 7.6 and 50 μmol of NaCl in a volume of 0.5 ml. The solution was subjected to gel chromatography on Sephadex G-100 (see molecular weight determination) and the fractions were analyzed for protein, antitryptic and tryptic activites. Trypsin and the inhibitor were also subjected separately to gel chromatography. Results A crude extract of arrow root tuber was found to inhibit both bovine trypsin and αchymotrypsin. While the two activities could not be separated, the inhibitory activity in the tuber could be resolved into two fractions during gel filtration on Sephacryl S-200. Both the fractions showed anti-tryptic and antichymotryptic activities. The low molecular weight, major inhibitor (figure 3) accounted for about 80% of the recovered antitryptic activity. The major inhibitor was purified about 40 fold and the data on purification are summarized in table l. The inhibitor was homogeneous by several criteria. On cellulose acetate electrophoresis at pH 9·8 and at pH 5.0, it moved as a single protein band, 26.0; mm and 16.0 mm respectively, towards the anode (data not shown). During SDS-polyacrylamide gel electrophoresis at pH 7.2 and during gel electrophoresis in the absence of SDS at pH8.3the inhibitor movedas a single band (figure 4). During gel permeation chromatography on Sephadex G-100, the activity and protein levels corresponded in the individual fractions (figure 5). The molecular weight of the inhibitor was found to be 12,000 based on SDSpolyacrylamide gel electrophoretic data (figure 6a) and the value of 11,000 based on gel chromatography on Sephadex G-100 agreed fairly well with this value (figure 6b).

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Table 1. Purification of the major protease inhibitor from arrow root tuber.

The inhibitor units were calculated using bovine trypsin.

Figure 4.

Polyacrylamide gel electrophoresis of purified inhibitor.

A: In the presence of SDS at pH 7.2; B. In the absence of SDS at pH 8.3. (Experimental details are described under materials and methods).

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Figure 5. Studies on complex formation between trypsin and purified inhibitor by chromatography on Sephadex G-100. (Experimental details are described under materials and methods). (•) Inhibitor, protein; (Δ) Inhibitory (antitryptic) activity; (O) Trypsin, protein; (▲) Trypsininhibitor complex, protein.

Figure 6.

A. Molecular weight determination of purified inhibitor by SDS-PAGE.

(Experimental details are given under materials and methods). B. Molecular weight determination of purified inhibitor and trypsin-inhibitor complex by gel chromatography of Sephadex G-100. (Experimental details given under materials and methods).

The action of the inhibitor was linear with respect to protein concentration up to 60% inhibition of trypsin and 80% inhibition of α-chymotrypsin. Beyond these levels the inhibition was progressive but non-linear. The inhibitor affected both the

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caseinolytic and amidolytic activities of bovine trypsin to equal extents. The inhibitor was 1.6 times more active on trypsin than on α-chymotrypsin. This ratio was slightly less than the value of 1.8 obtained with crude extract of arrow root tuber. The purified inhibitor had no action on subtilisin BPN and porcine elastase even when used at 10 times higher concentration of the inhibitor that caused complete trypsin inhibition. The caseinolytic activities of bovine and human pancreatic preparations were equally diminished by the purified inhibitor. However, in both cases the inhibition was not linear beyond 25 % and the relative inhibition in terms of caseinolytic activity was much less compared to the action on bovine crystalline trypsin and α-chymotrypsin. The data are summarized in table 2 The purified inhibitor also inhibited bovine enterokinase. The inhibition was linear up to 60% activity. In terms of inhibition of trypsinogen activation, it can be calculated that 1.45 µg of the inhibitor blocked an amount of enterokinase that can activate 4.5 μg of trypsinogen under the assay conditions. Table 2. Action of the inhibitor on different proteases.

«

ΒΑΡΝΑ was used as substrate.

«« Expressed

as amount of trypsin generated under assay conditions.

The purified inhibitor was highly heat stable. No loss of antitryptic activity was noticed on subjecting it to heat treatment at 100°C for 60 min. However, pressure cooking for 20 min, resulted in the complete inactivation of the inhibitor. The inhibitor was also fully stable to exposure to a wide range of pH from 1.0 to 12.5 for 72 h at 4°C. Treatment with pepsin or elastase also did not cause any loss of the inhibitory potency. Based on the data on the linear range of inhibition it was found that 1.1 mol of the inhibitor could inactivate 1.0 mol of active bovine trypsin. The calculated Ki value for the inhibitor with respect to trypsin was 0.91 × 10–1M. Complex formation between bovine trypsin and the inhibitor was studied by gel chromatography on Sephadex G-100. The elution profiles of the inhibitor, trypsin and the enzyme inhibitor mixture are shown in figure 5. The single peak observed with the mixture did not display any caseinolytic activity or antitryptic activity, indicating that it

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represents the inactive complex between trypsin and the inhibitor. The molecular weight of the complex based on its Ve/Vo value was 34,000 which is close to the expected value of 35,000. The complex did not also display any antichymotryptic activity. The data on the effect of chemical modification of the inhibitor on its antiprotease activity are presented in table 3. Treatment with TNBS or OMI did not Table 3. Chemical modification of the inhibitor.

Details are given under materials and methods.

affect the antitryptic activity. However, these amino group modifiers caused a partial loss of the antichymotryptic activity. Treatment with CHD resulted in about 50% loss of the antitryptic and antichymotryptic activity. The magnitude of inactivation could not be increased further by increasing the concentration of CHD. Similarly, prior heat treatment of the inhibitor did not make it more susceptible to modification by CHD. Treatment with ninhydrin, another arginyl group modifier resulted in the rapid loss of inhibitory potency. The antichymotryptic activity was slightly more susceptible to ninhydrin than the antitryptic activity. Table 4 represents the data on the effect of preincubation of the inhibitor with trypsin and α-chymotrypsin. Preincubation at pH 3.7 at 37°C or 25°C with trypsin was found to nearly double its antitryptic activity. The time of preincubation in the range 15-60 min did not have any effect on this enhancement. The reaction was temperature dependent, since the magnitude of increase at 4°C was much less. Effect of preincubation at pH 7.6 at 37°C could not be assessed due to a gradual loss