ISOLATION AND CHARACTERIZATION OF TWO TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM LENTIL SEEDS (LENS CULINARIS MEDIK.)

ISOLATION AND CHARACTERIZATION OF TWO TRYPSIN- CHYMOTRYPSIN INHIBITORS FROM LENTIL SEEDS (LENS

CULINARIS MEDIK.)’

RUDI MUELLERZ and JUERGEN K. P. WEDER3

Institute of Food Chemistry, Technical University of Munich, Garching, Federal Republic of Germany

Received for Publication March 7, 1988 Accepted for Publication August 21, 1988

ABSTRACT

The two major trypsin-chymotrypsin inhibitors of Lens culinaris Medik., LCI- I and LCI-4, were purijied to homogeneity from Italian red lentils by ammonium sulfate fractionation, gel and ion-exchange chromatography. At least two more trypsin-chymotrypsin inhibitors of minor importance were present. LCI-I and LCI-4 inhibited human trypsin less (68-74%) but human chymotrypsin better (268-279%) than the respective bovine enzymes. The two inhibitors contained no carbohydrates, no methionine and no tryptophan and high contents of cystine but no free suljhydryl groups (seven and eight disuljide bridges for LCI-I and LCI-4, respectively). In addition, LCI-4 contained no isoleucine. The isoelectric points were 5.35 and 7.70 and the average molecular weights 10,600 and 9,900 daltons for LCI-I and LCI-4, respectively. Inhibitor extracts, as well as purified inhibitors in solution, were relatively stable against heating and treatment with human gastric juice, while soaking the whole seeds overnight and subsequent boiling for two hours totally destroyed inhibitor activity.

INTRODUCTION

The inhibition of human pancreatic trypsin and chymotrypsin by naturally occurring plant inhibitors, among them those from legumes, has been studied by several authors. The work done with legume extracts and inhibitor preparations was reviewed recently (Weder 1986). In comparative studies it has frequently been demonstrated that some inhibitors displayed considerable differences in the

I Dedicated to Professor Friedrich Kiermeier on the occasion of his 80th birthday. Present address: Maizena GmbH, Postfach 2760, D-7100 Heilbronn, FRG Correspondence to: Dr. J. K. P. Weder, Institut fuer Lebensmittelchemie, Technische Universitaet Muenchen, LichtenbergstraDe 4, D-8046 Garching, FRG. To whom reprint requests should be directed.

Journal of Food Biochemistry 13 (1989) 39-63. AfZRighrs Reserved. 0 Copyright 1989 by Food & Nutrition Press, Inc. , Trumbull, Connecticut. 39

40 R. MUELLER and JUERGEN K. P. WEDER

inhibition of human and bovine trypsin and chymotrypsin. Among the econom- ically important grain legumes studied at our Institute (Belitz et al. 1982), bush beans, carob beans, chickpeas, lentils, lima beans and peas exhibited a characteristic feature of inhibition: they all inhibited human trypsin to nearly the same extent as the bovine enzyme but the human chymotrypsin was inhibited to a much higher extent (2 to 4-fold) than the bovine counterpart.

In view of these results, we chose lentils to isolate the inhibitors responsible for this difference in action and to clarify the mechanism causing this difference. Detailed information on proteinase inhibitors from this species was lacking in spite of its nutritional importance and wide consumption in Asia, northern Africa, Latin America and southern and eastern Europe (Duke 1981).

Jaffk (1950) was the first to demonstrate trypsin inhibitor activity in lentils. Some years later, the inhibition of chymotrypsin by an inhibitor preparation from lentil seeds was reported by Mansfeld et al. (1959). Very limited advances were made in the following years to improve the knowledge on the nature of these inhibitors. In 1981, Chavan and Hejgaard demonstrated the presence of seven trypsin inhibitors in lentils by isoelectric focusing in polyacrylamide gel rods, six of which inhibited also chymotrypsin. Weder et al. (1983) detected four inhibitors by disc electrophoresis, all of which inhibited both bovine trypsin and chymotrypsin. These authors reported on the isolation and initial characterization of the most potent inhibitor, designated LCI-4.

Thirty eight lentil accessions from different parts of the world were screened for both inhibitor activities and electrophoretic inhibitor patterns in order to choose the best suited sample for the isolation of all four inhibitors (Weder et al. 1985). All of the eight accessions tested also with the human enzymes exhibited the common feature of inhibiting human chymotrypsin much more than the bovine enzyme while the two trypsins were inhibited to nearly the same extent. Disc gel electrophoresis of seed extracts and negative staining for trypsin inhibitors showed the presence of four inhibitors in all samples. They were denoted LCI-1 to LCI-4 in order of decreasing mobility in the anodic system. We selected a red lentil from Italy (Lens culinaris Medik., ssp. microsperma) with a relatively high inhibitor activity and a well-balanced inhibitor pattern for this study. The purification of the two main trypsin-chymotrypsin inhibitors from this lentil seed sample, LCI-1 and LCI-4, their composition and some physical and biochemical properties are discussed in this paper. In addition, stability experiments with extracts and whole seeds were carried out using a variety of the same subspecies (Syrian local small).

MATERIALS AND METHODS

Reagents Enzymes. The juice from human duodena (HDJ) was taken from the duodenum

using a Dreiling tube after the stimulation of pancreas secretion with secretin

PROTEINASE INHIBITORS OF LENTILS 41

and cerulein. Human gastric juice was taken by oral probe. Both were obtained from a local hospital.

Bovine trypsin (BT), 3.5 U/mg, cryst., for biochemical use (24579) was from Merck; bovine alpha-chymotrypsin (BCT), 45 U/mg, 3x cryst., lyophil., purest (17160) from Serva.

Substrates. N"-benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPA), p.a, (pro analysi; Serva 14634); glutaryl-L-phenylalanine-4-nitroanilide (GLU- PHEPA; Merck 4244); N-acetyl-DL-phenylalanine beta-naphthylester (Schwarz/ Mann 90063 1); 4-nitrophenyl 4'-guanidinobenzoate hydrochloride (Merck 10562); 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone (Merck 10559).

Other Reagents. Acrylamide p.a. (LKB 1820-101); Bio-Gel P-100, 100-200 mesh (Bio-Rad); Blue Dextran (Sigma D 5751); bromophenol blue (Riedel de Haen); CM-52 cellulose cation exchanger, microgranular (Whatman); Coomassie Brilliant Blue (3-250 (Serva Blue G, C.I. 42655), purest (Serva 35050); Coom- assie Brilliant Blue R-250 (Serva Blue R, C.I. 42660) purest (Serva 17525); DE-52 cellulose anion exchanger, microgranular (Whatman); 5 , s ' -dithiobis-(2- nitrobenzoic acid) (DTNB, Ellman's reagent), purest (Serva 20735); Fast blue B salt, C.I. 37235, pure (Serva 21270); gel bond sheets, 0.2 mm (FMC 54727); N,N-methylenebisacrylamide, p.a. (LKB 1820-102); ODS-Hypersil RP-18, 5 pm (Shandon); Polyol = Silo0 and Polyol= Si200, 5 pm, prepacked GPLC- columns (Serva 4231 1 and 42439); Sephadex G-50 superfine (Pharmacia), N,N,N' ,N'-tetramethylethylenediamine, purest (Serva 35925).

Marker Proteins. Aprotinin, proteinase inhibitor (Sigma A 4529); bovine serum albumin, l x cryst., purest (Serva 11920); carbonic anhydrase B, from bovine erythrocytes (Sigma C 2522); conalbumin Type I, from chicken egg white, iron-free (Sigma C 0755); cytochrome C Type VI, from horse heart (Sigma C 7752); gamma-globulins, bovine, Cohn Fraction 11,111 (Sigma G 5009); Kunitz pancreatic trypsin inhibitor (PTI, Worthington 3212); lysozyme, purest (Serva 28260); myoglobin Type I, from horse skeletal muscle (Sigma M 0630); ovalbumin, lx cryst., pure (Serva 11850); Phaseolus coccineus proteinase inhibitor PCI-2 and Phaseolus vulgaris inhibitor PVI-3,, both prepared in our laboratory (Weder et al. 1975; Gerstenberg et al. 1980); phosphorylase a, from rabbit muscle (Sigma P 1261); ribonuclease (Boehringer Mannheim 109134); soybean trypsin inhibitor, Kunitz (SBTI), purest (Serva 37328).

All other chemicals used were of analytical grade from various suppliers.

Isolation of the Crude Inhibitor Preparation LCI-C

Commercial red lentils from Italy (Lens culinaris Medik., ssp. microsperma; from Lebascha, health food, Munich, FRG) were ground, sieved and extracted with 0.25 N H,SO, following the procedure of Weder et al. (1983). Ammonium sulfate precipitation (30-60% saturation), subsequent dialysis against distilled water and lyophilization gave the crude inhibitor preparation LCI-C.

42 R . MUELLER and JUERGEN K. P. WEDER

Gel Chromatographic Purification of LCI-C

In a typical experiment, 300 mg of LCI-C dissolved in 0.1 N acetic acid were applied onto a Bio-Gel column (P-100, 100-200 mesh; 50 x 400 mm, Pharmacia) equilibrated with 0.1 N acetic acid. Elution was performed with 0.1 N acetic acid at 48 mUh; 8 mL fractions were collected. Protein was detected in the eluate by recording the absorbance at 228 nm (Uvicord S 2, LKB). Inhibition of human and bovine trypsin and chymotrypsin were estimated in 0.1 mL samples of each fraction as described below with BT (15 pg), human trypsin (HT; 0.15 mL of HDJ), BCT (20 pg), and human chymotrypsin (HCT; 0.2 mL of HDJ), respectively. The fractions showing inhibitor activity were pooled as indicated in Fig. 1 and lyophilized to yield the purified lentil inhibitor preparation LCI-P.

Ion-Exchange Chromatographic Separation of the Isoinhibitors

LCI-P (25 mg, dissolved in 1 mL of starting buffer, 0. I M ammonium acetate buffer, pH 4.5) was placed onto a column (9 X 250 mm, Pharmacia) filled with carboxymethyl cellulose cation exchanger CM-52 (Whatman) and equilibrated with the starting buffer. Separation of the inhibitors was achieved at a flow rate of 24 mL/h (6 mL fractions were collected) using a non-linear pH-gradient, pH 4.5-6.5, prepared by mixing the starting buffer and 0.1 M ammonium acetate buffer, pH 6.5 (final buffer), as indicated in Fig. 2. Proteins and inhibitors were detected as outlined above. Fractions corresponding to LCI- 1, LCI-2 and -3 and LCI-4 (Fig. 2) were collected separately and lyophilized to yield the enriched isoinhibitor preparations.

Purification of LCI-1

A 6 mg sample of enriched LCI-1 was applied to a column (9 X 170 mm, Pharmacia) of diethylaminoethyl cellulose exchanger DE-52 (Whatman). The protein was eluted with 0.1 M ammonium acetate buffer, pH 8.5, at a flow rate of 24 mL/h and collected in 5 mL fractions. Proteins and inhibition of BT were detected as outlined above. The fractions showing inhibitor activity and electrophoretic homogeneity were lyophilized to yield the pure LCI-1 (cf. Fig. 3 ) .

Purification of LCI-4

A 7 mg sample of enriched LCI-4 was applied to a column (9 X 250 mm, Pharmacia) of carboxymethyl cellulose exchanger CM-52 (Whatman). Elution with 0.1 M ammonium acetate buffer (pH 5.5) and evaluation was carried out as for LCI-1 to yield pure LCI-4 (Fig. 4).


Trypsin Inhibitor Determinations

Trypsin inhibitor activities were determined with BAPA, a trypsin-specific synthetic substrate, by a modification of the procedures of Erlanger et al. (1961) and Nagel ef al. (1965).

The BT calibration curve was obtained by mixing 0.05-0.25 mL of the BT solution (10 mg of BT dissolved in 100 mL of 1 mM HCl) in test tubes with 0.2 M Tris/HCl buffer (pH 7.6, 66 mM CaCl,) to a total of 2.90 mL. After temperature equilibration to 30"C, the reaction was started by addition of 0.10 mL of BAPA solution (100 mg of BAPA dissolved in 10 mL of dimethyl formamide). After 10 min at 30"C, the reaction was stopped by mixing with 1.00 mL of 30% acetic acid. Blanks were obtained accordingly by adding the enzyme last. Absorbance at 405 nm was plotted against the amount of enzyme.

For the HT calibration curve 0.05-0.25 mL of HDJ (thawed immediately before the experiment) were used following the same procedure as given for BT. Comparison of the two calibration curves gave the amount of BT which produced the same absorbance change as a given amount of HDJ.

Inhibitor activities were determined by mixing 0.15 mL of BT solution or 0.15 mL of HDJ with 0.025-0.15 mL of inhibitor solution (inhibitor extract or preparations dissolved in distilled water) and bringing up to 2.90 mL with 0.2 M Tris/HCl buffer (pH 7.6, 66 mM CaCl,). After pre-incubation at 30°C for 15 min, the reaction was started by adding 0.10 mL BAPA solution and stopped after 10 min at 30°C by mixing with 1 mL of 30% acetic acid. The amounts of free enzyme present were read from the calibration curve using the sample absorbances at 405 nm after correction for blanks. Percent inhibition (related to the total amount of enzyme present in each test tube) was calculated and plotted against inhibitor content. The specific inhibitor activity (expressed as mg of active enzyme totally inhibited by 1 mg of inhibitor preparation) was calculated from the initial slope of this inhibition curve, taking into consideration the content of active trypsin determined by active-site titration with 4-nitrophenyl 4'-guanidinobenzoate (Chase and Shaw 1967; Klockow 1972). The batch used contained 73% active trypsin.

Chymotrypsin Inhibitor Determinations

Determinations were carried out using GLUPHEPA, a chymotrypsin-specific synthetic substrate, by a modification of the procedure of Erlanger et al. (1966).

The BCT calibration curve was obtained by making 0.05-0.30 mL of the BCT solution (10 mg of BCT/100 mL of 1 mM HCI) up to 2.90 mL with 0.5 M Tris/HCl buffer (pH 7.6, 66 mM CaCI,). The reaction was started by adding 0.10 mL of GLUPHEPA solution (100 mgllO mL of dimethylformamide) and stopped after 60 min at 30°C by addition of 1.00 mL of 30% acetic acid.


Absorbance at 405 nm (corrected for blanks) was plotted against amount of enzyme.

The HCT calibration curve was obtained accordingly using 0.05-0.30 mL of HDJ (thawed immediately before use). Comparison of the two calibration curves gave the amount of BCT which produced the same absorbance change as a given amount of HDJ.

Inhibitor activities were determined as described for the trypsins but using 0.20 mL of BCT solution or 0.20 mL of HDJ, respectively, 0.5 M TnslHCl buffer (pH 7.6, 66 mM CaCl,), and 0.10 mL of GLUPHEPA solution and incubating for 60 min at 30°C. Content of active chymotrypsin was determined with 2-hydroxy-5-nitro-aIpha-toluenesulfonic acid sultone (Heidema and Kaiser 1968; Klockow 1972). The batch used contained 77% active chymotrypsin.

SDS-Polyacrylamide Gel Electrophoresis in a Pore-Size Gradient

This was carried out on ultra-thin (0.5 mm) slab gels of 5-22.5% polyacrylamide prepared according to Goerg et al. (1982). Runs were performed with 0.4 M Tridglycine buffer (pH 8.6) in a Multiphor chamber (LKB). Electropho- retic conditions were 700 V, 25 W and 35 mA at 6°C. Molecular weight markers used were aprotinin, cytochrome C, myoglobin, carbonic anhydrase B, ovalbumin, gamma-globulin, bovine serum albumin, conalbumin, and phosphorylase a. Samples and markers (5-10 kg of protein per slot) were boiled for 5 min in 0.1 M Tris/HCI buffer (pH 6.8, 0.4% SDS, 2% 2-mercaptoethanol).

Proteins were stained according to Westenneier (1981) with Coomassie Bril- liant Blue G-250 and copper sulfate.

SDS-Polyacrylamide Gel Electrophoresis

Electrophoresis in vertical gel rods (4 X 70 mm) with continuous gel of 10% polyacrylamide was performed according to Weber and Osborn (1969) in a Tris/ glycine buffer (pH 8.2). Proteins were incubated overnight at 37°C ,in 0.1 M Tris/HCl buffer (pH 7.2, 0.4% SDS, 2% 2-mercaptoethanol) before sample application (100 pL). Conditions: voltage 100 V, which is equal to a current of 5 mA on each gel. Proteins were stained with 0.5% Coomassie Brilliant Blue R-250 in rnethano1:acetic acid:water = 90:5:90. Protein markers used were myoglobin, cytochrome C, BCT, PVI-3,.

Polyacrylamide Gel Electrophoresis in a Pore-Size Gradient

Electrophoresis of the native proteins was performed in a pore-size gradient of 5-22.5% polyacrylamide as described above for SDS electrophoresis but without the addition of SDS. Samples were dissolved in 0.1 M Tris/HCl buffer (pH 7.6) at a concentration of 1 mg/mL and 5-10 KL of the solution were applied


per slot. Electrophoretic conditions and staining for proteins were the same as for SDS-electrophoresis. Inhibitors were detected with N-acetyl-DL-phenylalanine beta-naphthylester and Fast blue B salt after incubation with BT or BCT according to Uriel and Berges ( 1968).

Gel Isoelectric Focusing

The isoelectric points of the inhibitors were determined with ultra-thin (0.5 mm) Ampholine-Ultro-PAG-Plates, pH 3.5-9.5 (LKB), according to the manufacturer’s instruction in a Multiphor chamber (LKB). Focusing conditions: prefocusing 10 min, focusing 50 min at 2000 V, 25 W at 8°C. The pH-gradient was measured directly on the gel surface with a surface electrode (Ingold LoT- 405-M 5 ) .

Reversed-Phase HPLC

Chromatography was performed on an ODs-Hypersil RP-18 column (4.6 X 240 mm) with 10 mM triethylammonium formate buffer (pH 6.0) and an acetonitrile gradient (LCI- 1 : 0.8 mg dissolved in 0.1 mL of the buffer, linear gradient from 10% to 30% of acetonitrile; LCI-4: 1 .O mg dissolved in 0.1 mL of the buffer, linear gradient from 4% to 40% of acetonitrile). Running conditions: 60 min at a flow rate of 2 mumin, a temperature of 60”C, and a pressure of 70 bars; the protein concentrations were measured at 220 nm.

Amino Acids, Protein and Carbohydrate Determination

Protein hydrolysates (about 350 pg of protein hydrolyzed in 2 mL of 6 M HCl for 24 h at 110°C in a vacuum desiccator) were analyzed on a Multichrom B amino acid analyzer (Beckman) following the manufacturer’s instruction. Tryptophan was determined by HPLC after alkaline hydrolysis (unpublished results).

Protein content was calculated from the contents of the individual amino acids. Determination of free sulfhydryl groups was carried out with Ellman’s reagent (5,5’-dithiobis(2-nitrobenzoic acid); Ellman 1959) in 100 pg portions of the purified lentil isoinhibitors. Carbohydrates were determined according to Dubois et al. (1956) with the phenol-sulfuric acid reagent.

Reduction of Lentil Isoinhibitors

Samples of 100 pg of LCI-1 or LCI-4, dissolved in 1 mL of 0.1 M sodium potassium phosphate buffer (pH 7.6), were treated with 2-mercaptoethanol (15 FL) at room temperature. Aliquots of 100 pL were taken after 60, 90, and 180 min and tested for inhibitor activity against BT.

46 K MUELLER and JUERGEN K. P. WEDER

Analytical Gel Chromatography

Molecular weights were also estimated by gel filtration on Sephadex G-50 superfine (column size 10 x 500 mm). The column was equilibrated with 0.1 N acetic acid (pH 3.0) at a flow rate of 8.0 mL/h. Sample load was 1.0 mL (0.5 mg protein). Molecular weight markers used were Kunitz soybean trypsin inhibitor, ribonuclease, lysozyme and myoglobin; the void volume was determined with Blue Dextran.

Gel Permeation Liquid Chromatography (GPLC)

GPLC was performed on prepacked HPLC-columns Polyol = Silo0 and Po- lyol = Si200 from Serva. After equilibration with the eluant, 10 p,L of sample solution (1 mg proteidml eluant) were applied to the column by a sample loop. The column was eluted with 0.2 M phosphate buffer (pH 7.2, 0.17 M NaCl) at a flow rate of 6.0 mL/h. Protein markers used were cytochrome C, PTI, PVI- 3 , , BT, BCT, carbonic anhydrase B, ovalbumin, and bovine serum albumin.

Exposure to Gastric Juice

The stability of the two lentil isoinhibitors was estimated by dissolving 300 kg of LCI-1 or LCI-4 in 2 mL of freshly thawed human gastric juice and keeping at 37°C for 240 min. Inhibitor activities left were determined after 60 and 240 min. In parallel, LCI-C ( I mg) was incubated in 1 mL of 0.1 N HCL.

The acidic extract from Syrian local small lentils (ILL 4401 from ICARDA, Aleppo/Syria) was brought to pH 1.5 (the pH of the human gastic juice used in the experiment) with 2 N NaOH. It was mixed with the same amount of human gastric juice (thawed immediately before use) and kept at 37°C for 4 h. Inhibitor activity determinations were performed after neutralization and dilution with 0.2 M Tris/HCl buffer (pH 7.6, 66 mM CaC1,).

Heating Experiments

The stability of pure lentil isoinhibitors against heating was estimated by holding 300 k g of LCI- 1 or LCI-4, dissolved in 3 mL of distilled.water, in small closed plastic tubes at 95°C for 90 min. The residual activities against human and bovine trypsin and chymotrypsin were compared with those of unheated samples. LCI-C (4 mg in 4 mL distilled water) was similarly treated.

The heat stability of the inhibitor extract from Syrian local small lentils was estimated by keeping 10 mL of the neutralized extract (70 mL of the 0.25 M sulfuric acid extracts, adjusted to pH 7.6 and brought up to 100 mL with 0.2 M Tris/HCl buffer, pH 7.6) in closed ground-glass test tubes at 95°C for 60 and 120 min and then testing for inhibitor activities.


Syrian local small lentil seeds (5 g) were soaked overnight in 45 mL of distilled water and boiled for 2 h at atmospheric pressure. After readjusting the volume, the sample was homogenized and extracted by addition of 50 mL of 0.5 M sulfuric acid following the usual procedure.

RESULTS AND DISCUSSION

Purification of the Two Main Lentil Isoinhibitors

Crude Inhibitor Preparation. Preliminary experiments with commercial red lentils from Italy, a variety of Lens culinaris Medik., ssp. microsperma, showed that the addition of ammonium sulfate in steps of 10% saturation to the neutralized extract (pH 7.5) precipitated most of the inhibitor activity between 30% and 60%. In a typical experiment, 250 g seed meal yielded 2.75 g of the crude inhibitor preparation (LCI-C) after dialysis and lyophilization of the supernatant. Some material without inhibitor activity precipitated during dialysis. The inhibitory activity of LCI-C against human and bovine proteinases are given in Table 1; data on yield and purification are included in Table 2. The specific inhibitor

TABLE 1.

TILS (LENS CULJNARIS MEDIK., SSP. MZCROSPERMA) SPECIFIC INHIBITOR ACTIVITIES OF PREPARATIONS FROM ITALIAN RED LEN-

I n h i b i t o r p r e p a r a t i o n

S p e c i f i c I n h i b i t o r A c t i v i t y a a q a i n s t BTD HT BCT HCT

LCI-c

LCI-P

0.083 0.077 0.062 0.168

0.228 0.227 0.154 0.387

LCI- I ( e n r i c h e d ) 0 .626 0.580 0.275 0.643

( p u r e 1 1.58 1.08 0.68 1.90

L C I - 4 ( e n r i c h e d ) 0.995 0.980 0.467 1.227

( p u r e ) 1.78 1.31 0.75 2.01

a Values are mg of active enzyme totally inhibited by 1 mg of inhibitor preparation; means of three determinations BT: bovine trypsin, HT: human trypsin, BCT: bovine chymotrypsin, HCT human chymotrypsin (concentrations in the assay: 3.65, about 4, 5.13, and about 5 Fg/mL, respectively)


TABLE 2. YIELDS AND PURIFICATION FACTORS OF PREPARATIONS FROM ITALIAN RED

LENTILS (LENS CUUNARIS SSP. MICROSPERMA)

I n h i b i t o r Y i e l d s a Recoveryb Puri f i ca t i o n C p r e p a r a t i o n ( $ 1 ( f o l d )

L C I - c

LCI -P

2.15 g 30

0.80 g 22

28

6 9

L C I - 1 ( e n r i c h e d ) 25.50 mg 1 . 4 139

( p u r e ) 6.50 mg 0.9 350

LCI-2/3 4 . 5 0 m g - -

LCI-4 ( e n r i c h e d ) 11.25 mg 1 . 0 242

( p u r e 1 5 . 0 0 mg 0.8 381

a Yields of a typical experiment from 250 g of lentil seed meal

c Average purification, related to the activity of the seed meal

Recovery of inhibitor activity (average against all the four enzymes) related to the activity of 250 g seed meal

activities clearly demonstrate that the typical differences in the inhibition of human and bovine proteinases are maintained after ammonium sulfate fractionation. The total activity of this preparation was 30% of the original activity present in the extract; the specific activity was increased about 30-fold. Electro- phoresis and staining for trypsin inhibitors showed that all the four isoinhibitors were present in LCI-C.

Purified Inhibitor Preparation. LCI-C was then subjected to gel chromatography on Bio-Gel, a procedure that had been found to be very effective in separating the inhibitors from the other protein material in studies with other legumes (Weder et al. 1975; Gerstenberg et al. 1980). Figure 1 shows the elution profile of a typical run. In contrast to the results obtained with a different lentil variety (Weder et al. 1983), measurement of absorbance at 228 nm resulted in one distinct protein peak preceded by two minor peaks. As found before, the

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Separation of Isoinhibitors. Since chromatography of the purified inhibitor preparations on an anion exchanger (DEAE-Sephadex) could only be used to isolate the most basic inhibitor LCI-4, while the other inhibitors are eluted very late and with poor yields from the column (Weder et al. 1983), cation exchangers were then tested. In preliminary experiments, the best results were obtained with carboxymethyl cellulose from Whatman (CM-52). A typical elution profile for LCI-P subjected to this cation-exchanger using volatile buffers and an increasing pH-gradient is presented in Fig. 2 . Protein absorbance and the inhibitor activity against bovine trypsin (BT) showed that some proteins without inhibitor activity were eluted first, followed by the inhibitors and finally by more proteins without inhibitor activity. Comparison with electrophoresis confirmed that the inhibitors were eluted in the order of decreasing acidity.

For preparative runs, the pH gradient was chosen so as to achieve a maximal separation of the two main inhibitors LC1-1 and LCI-4 from proteins without inhibitor activity and from the minor components, LCI-2 and LCI-3. The fractions were pooled as indicated by the bars. Because of their low concentration and poor separation, LCI-2 and LCI-3 were pooled together. Electrophoresis of the lyophilized enriched preparations showed the presence of small amounts of impurities in each of these samples. Inhibitor activities and yields of these products, denoted LCI-1 (enriched), LCI-4 (enriched) and LCI-2/3, are included in Tables 1 and 2. As expected from the elution profile, a further enrichment of the two main inhibitors was achieved in this step (2 and 3.5-fold for LCI-1 and LCI-4, respectively) together with poor yields. The presence of accompanying proteins with both similar molecular weights and ion exchange behaviour rendered nec- essary a sharp fractionation to obtain maximum purity. Thus, only 2.4% of the inhibitor activity present in the seeds were recovered after three purification steps (ammonium sulfate fractionation, gel and ion-exchange chromatography).

Examination of the inhibitory activities against all four enzymes in another run (data not shown) demonstrated that the inhibition of the other enzymes coincided with that of BT (Fig. 2). Further, it was also found that the maximum activity against all enzymes occurred at the same eluate fraction of the LCI-1 and LCI-4 peaks. As expected from previous experiments, BT was inhibited

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somewhat better than human trypsin (HT)and human chymotrypsin (HCT) was considerably more inhibited than bovine chymotrypsin (BCT). LCI-2 and LCI- 3 seemed to inhibit HCT less than BCT, but further experiments are needed to draw final conclusions.

Purification of LCI-1. Anion-exchange chromatography of the enriched LCI- 1 resulted in two protein peaks, the first of which showed no inhibitor activity and was eluted in the void volume of the column (Fig. 3). The second peak, which exhibited inhibitor activity, was shown by electrophoresis of its individual fractions to contain, besides LCI- 1 , an additional protein with smaller electrophoretic mobility, which corresponded to the fractions of the ascending slope of this peak. Therefore, only the fractions demonstrating 100% inhibition of BT and no impurities were pooled as indicated by the bar in Fig. 3 . A total of 6.5 mg of pure LCI-1 were obtained from 250 g lentil meal with a further 2.5-fold purification (Table 2).

Purification of LCI-4. The enriched LCI-4 was further purified by rechromatography at a constant pH of 5.5 on the cation exchanger used for isoinhibitor

L C I - 1

FIG. 3 . DEAE-CELLULOSE CHROMATOGRAPHY OF THE LENTIL ISOINHIBITOR LCI-I A sample o f 6 mg of enriched LCI-I was applied to a 9 x 170 mm column of Whatman DE-52 and eluted with 0.1 M ammonium acetate buffer (pH 8.5) at 24 mL/h (fraction volume: 5 mL).

Protein and inhibilion of bovine trypsin were detected as in Fig. 2.


separation. A protein without inhibitor activity and one inhibiting BT were separated by this procedure (Fig. 4). The inhibitor itself was eluted as a sym- metrical peak; electrophoresis showed that all fractions contained only LCI-4. The fractions pooled as indicated yielded a total of 5.0 mg of pure LCI-4 from 250 g lentils with a 1.6-fold purification in this step (Table 2).

As can be seen from the data presented in Table 1, the two isoinhibitors isolated and purified by these procedures, LCI-1 and LCI-4, displayed the same characteristic features as do the lentil extracts and LCI-C: They inhibit HT to a lesser extent than BT (68-74%) and on the contrary HCT is inhibited to a considerably higher extent than BCT (2.7 to 2.8-fold). From the data presented here it can be concluded that the isolated proteins are the main components responsible for the inhibition properties of this lentil variety. LCI-1 and LCI-4 are homogeneous proteins, as proven by PAGE in a pore-size gradient, SDS- PAGE in a pore-size gradient and reversed-phase HPLC.

0.2

E C s) h( @i

8 C < 0.1 0 v)

2 I I I

LC I - 4 -

10 Fraction no. 20 FIG. 4. RECHROMATOGRAPHY OF THE LENTIL ISOINHIBITOR LCI-4 ON

CM-CELLULOSE A sample of 7 mg of enriched LCI-4 was applied to a 9 X 250 mm column of Whatman CM-52 and eluted with 0.1 M ammonium acetate buffer (pH 5.5) at 24 mWh (fraction volume: 5 mL).

Protein and inhibition of bovine trypsin were detected as in Fig. 2.


Biochemical Characterization

Composition. The two lentil isoinhibitors, LCI-I and LCI-4, are similar in their amino acid composition and characterized by a high content of acidic amino acids (21 and 17 residues per mole LCI-1 or LCI-4, respectively) and 1/2-cystine (14 and 16 residues, Table 3). Both inhibitors lack methionine and tryptophan and LCI-4 additionally isoleucine. The calculation of the number of amino acid residues per mole leads to 89 residues with a molecular weight of 9800 Da for LCI-1 and 85 residues (9400 Da) for LCI-4.

No free sulfhydryl groups and no carbohydrate could be detected with Ellman’s reagent and with the phenol-sulfuric acid reagent, respectively. Hence, it can be concluded that all cysteine residues in the native inhibitors are involved in disulfide bridges and that the inhibitors are not glycoproteins. Reduction of LCI- 1 and LCI-4 with 2-mercaptoethanol resulted in a complete loss of inhibitor activity against BT.

Because of the high cystine content and the calculated number of 7 and 8 disulfide bonds for LCI-I and LCI-4, respectively, the two proteins belong to the Bowman-Birk soybean proteinase inhibitor family. The prototype of this inhibitor family is the so-called Bowman-Birk inhibitor from soybeans (BBI) with 71 amino acid residues per mole (Odani and Ikenaka 1972; data included in Table 3 for comparison). With respect to the number of amino acid residues, lentil inhibitors are near to those from Phaseolus lunatus (lima beans, LBI-IV and -1V’; Stevens et al. 1974), from Vignu angularis (adzuki beans, ABI-I; Ishikawa et al. 1979; and API-11; Yoshikawa el al. 1979) and from Vigna radiatu (mung beans, MBI-F; Wilson and Chen 1983). All Bowman-Birk inhibitors sequenced so far contain two homologous domains with a total of 7 disulfide bonds at the same positions of the molecule. They exhibit two independent reactive sites in the molecule each one able to inhibit one molecule of a serine proteinase like trypsin, chymotrypsin or elastase (see, e.g., Weder 1985).

Isoelectric Points. The data were obtained by isoelectric focusing of LCI-C, LCI-1, LCI-213, and LCI-4 in ampholine gels (average of 3 determinations each). The values determined by this method are all in agreement with the elution properties on the cation exchanger column and with the mobilities in an anodic electrophoresis system. LCI-1 is an acidic protein with a PI = 5.35. Assuming that all dissociable amino acid residues participate in charge differences this would accord with the determined number of 21 acidic and 13 basic amino acid residues if 8 of the acidic residues were amidized. From the figure presented by Chavan and Hejgaard (1981) a similar PI (5.2) can be taken for their most acidic lentil inhibitor. The two minor inhibitors LCI-2 and LCI-3 seem to be neutral proteins with a PI = 6.00 and 6.75, respectively. The second main lentil proteinase inhibitor, LCI-4, contains also a higher proportion of acidic than basic amino acid residues (i.e,, 17:14), but showed a basic pl = 7.70. This suggests


TABLE 3. AMINO ACID COMPOSITION OF THE TWO LENTIL ISOINHIBITORS LCI-I

AND LCI-4

B B I a - Amino acid LCI-1 LCI-4

Mole %b c d Mole %b c d d

Aspartic acide Threonine Serine

Gluatmic acidf Proline G3 ycine

Alanine 1/2-Cystine Valine

Methione Isoleucine Leucine

Tyrosine Phenyla lanine Histidine

Lysine Arginine Tryptophan

Total

12.4 5 . 8 9 .4

1 1 . 8 5.2 3.3

6.7 15.6 5.5

0 . 0 3 1 . 8 2.4

3.2 2 . 4 4.1

5.7 4.6 n.d.9

99 .9

1 0 . 8 5.0 8 . 2

10.2 4.6 2 . 9

5 . 9 13.5

4.8

0.03 1.5 2.1

2 .8 2.1 3.6

5.0 4.0 0

11 5 8

10 5 3

6 14 5

0 2 2

3 2 4

5 4 0

89

13.1 6.5 8 . 4

7.3 5.5 2.7

6 . 9 1 9 . 4 6.1

0.05 0.10 2.0

3.6 2.3 4.8

6.5 4 .7 n . d .

9 9 r 9

11.3 11 11 5.6 6 2 7.3 7 9

6.3 6 7 4 . 8 5 6 2.3 2 0

5.9 6 4 1 6 . 8 16 1 4

5 . 3 5 1

0.04 0 1 0 .08 0 2 1 . 7 2 2

3.1 3 2 2,o 2 2 4.1 4 1

5.6 6 5 4.1 4 2 0 0 0

85 71

8 BBI: Bowman-Birk inhibitor from soybeans (Glycine m a (L.) Merr.), data from Odani and Ikenaka ( 1972)

b Means of four determinations from two hydrolysates Residues per mole, calculated (based on minimum molecular weight (Edsall 1953))

d Residues per mole, nearest integer; for 1/2-cystine nearest even number = Includes asparagine

Includes glutamine g Not detectable

that a considerable part (7 residues) of aspartic and glutamic acid residues must be amidized. Weder et al. (1983) found a PI of 7 .4 for their LCI-4 preparation. This slight variation in PI may be due to the different methods used.

Molecular Weights. Results obtained by different techniques are compiled in Table 4. Molecular weights of 9,800 and 9,400 Da were calculated from the amino acid composition for LCI-1 and LCI-4, respectively. Slightly higher molecular weights were found using two SDS-electrophoretic methods. In a horizontal ultra-thin slab gel system under reducing conditions we obtained 10,500 and 9,500 Da for LCI-1 and LCI-4, respectively. In a vertical system using gel rods nearly the same value was found for LCI-1 but a somewhat higher value of 12,500 Da for LCI-4.

56 R. MUELLER and JUERGEN K . P. WEDER

With gel filtration on Sephadex we obtained higher values than by GPLC. From the retention properties of molar 1 : 1 LC1:BCT complexes on the GPLC column we calculated molecular weights of 11,400 and 8,400 Da for LCI-1 and LCI-4, respectively. Much lower values (not included in Table 4) were obtained with LC1:BT complexes (LCI-1: 5,700 Da, LCI-4: 4,300 Da). Chromatographic methods differed much more than electrophoretic ones. This may be due to different space volumes of the inhibitors under different separation conditions and/or to different interactions of the inhibitors with the column material.

Molecular weight calculations derived from the specific inhibitor activities cannot be used, because one of the prerequisites, the fully stoichiometric 1:l reaction between enzymes and inhibitors, is not fulfilled here (Weder and Mueller 1989).

As an average value calculated from all methods, we obtained 10,600 and 9,900 Da for LCI-I and LCI-4, respectively. These values are on the upper level of the range from 8,000 to 10,000 Da reported for most Bowman-Birk inhibitors (Kassell and Williams 1976). Higher molecular weights have already been reported earlier for some representatives of this family, e.g. 10,000-11,900 Da for Pisurn sativurn inhibitors (Weder and Hory 1972), 10,300-11,600 Da for

TABLE 4. MOLECULAR WEIGHTS OF THE TWO LENTIL ISOINHIBITORS LCI-1 AND LCI-4

Method Molecular weights (Dalton)a LCI-1 LCI-4

9800 9400 Aniino acid analysis

SDS/PAGE 11000 12500

SDS/pore-size gradient-PAGE 10500 9500

b

Gel chromatography

GPLC

GPLC (LCI : BCT complex)

12500 12000

8500 7500

11400 8400

Average 10600 9900

~~

* Means of two determinations b Calculated minimum molecular weight from amino acid composition (Edsall 1953)


some of the inhibitors from Phaseolus coccineus (Weder and Hory 1976) and 12,000 Da for the Cicer arietinum inhibitor (Birk 1974).

Inhibitor Stability

From the results of inhibitor activity determinations (Table l), it is clear that these inhibitors would affect protein digestion if they reached the small intestine unaltered. For example, 100 g of lentils would inactivate about one third of the daily production of trypsin and chymotrypsin in man which is about one g of each. Other legumes, e.g., kidney beans, chickpeas and soybeans, would inhibit even higher proportions (Belitz et al. 1982). From experiments with bovine enzymes it is known that the inhibitors are destroyed by cooking (Liener 1980; Sathe and Salunkhe 1984). The use of commercially available bovine enzymes is more convenient for testing the effect of processing. Therefore, we were interested to find out if there are differences in heat stability between the action on human and bovine proteinases. Another question is whether these inhibitors, because they are proteins, would survive the passage through the stomach. The present knowledge on this aspect is still limited, in particular with respect to human pepsin and human pancreatic proteinases (Weder 1986).

Influence of Pepsin. Exposure of the purified isoinhibitors to human gastric juice (pH 1.5) in vitro demonstrated a high stability against proteolysis with pepsin (Table 5 ) . Residual activities were between 70 and 96% after 1 h at 37°C and exceeded 82% after 4 h. Since similar alterations in activities were found after incubating LCI-C in 0.1 N HCI at 37°C for 4 h, it may be concluded that the slight effect to the inhibitors by gastric juice is actually due to the acidic

An interesting result was obtained when the acidic lentil extract was treated with human gastric juice (Table 6). While the inhibitor activity against the two chymotrypsins remained practically unaltered, trypsin inhibitor activities increased by 45%. At least two explanations are possible: that the inhibitor is present as a pre-inhibitor which requires hydrolysis for activation, or that the inhibitor is associated with a factor that is destroyed by the digestive enzyme. The inhibitor responsible for the increase in activity may be one of the known inhibitors or an additional one. Further experiments are needed to clarify this effect.

Influence of Heating. Heating the purified isoinhibitors in distilled water caused an intermediate reduction in the activity against the two trypsins and the two chymotrypsins (Table 5) . The decrease against the two human enzymes was about the same as that against the bovine ones. The considerable amounts of inhibitor (between 50 and 75%) that remained active are thought to be due to the compact structure of the Bowman-Dirk inhibitors, which are stabilized by disulfide bridges. Exposure of the crude inhibitor preparation, LCI-C, to the

PH.


TABLE 5 . STABILITY OF THE TWO LENTIL ISOlNHIBlTORS LCI-1 AND LCI-4

Treatment Inhibitory activitya aqainst H T ~ BT HCT BCT

LCI-1 - treated with HGJC for 1 h 92 79 70 78

for 4 h 118 89 83 85

- heated at 95°C for 1.5 h 53 49 70 62

LCI-4 - treated with HGJ for 1 h 91 96 86 89

for 4 h 120 97 89 103

- heated at 95OC for 1.5 h 68 70 75 71

a Percent activity related to untreated inhibitor; means of three measurements HT: human trypsin, B T bovine trypsin, HCT human chymotrypsin, BCT bovine chymotrypsin HGJ: human gastric juice

same heating conditions caused a somewhat smaller decrease in inhibitor activity (Table 6). This is in accordance with the well-known fact that lability of proteins increases with purification. Another reason may be the higher dilution with the purified isoinhibitors (0.1 vs. 1 .O mg per mL).

Heat treatment of the neutralized lentil extracts caused only moderate reduction of inhibitor activities (Table 6). The action against the human enzymes seems to be somewhat more affected (15-33%), but these differences could be partially due to a greater experimental error with the human duodenal juice. When raw seeds were first soaked in distilled water overnight and then boiled, no inhibitor activity at all could be detected in the extracts. This is in agreement with what we know from practice, i.e., the inhibitors are totally inactivated by conventional lentil meal preparation which recommends soaking for 12 h and boiling for 1- 2 h (Horn 1960; Krueger and Wolter 1973). Soaking in tap water for 12 h alone led to a 7-16% reduction of the trypsin inhibitor activity of lentil seeds, when the soaking fluid was discharged, while a 4% saline solution had a somewhat larger effect (Abou-Samaha et al. 1985). In full agreement with our results, exposure of lentils to a Bangladeshi home-cooking procedure (boiling 20 g of unsoaked seeds in 200 mL of water for about 45 min) resulted in a decrease in trypsin inhibitor activity of 57-88% (Sayeed and Njaa 1985). The increased heat lability of the whole soaked seeds in comparison to the extracts or the inhibitor


TABLE 6 . STABILITY OF LENTIL INHIBITORS IN A CRUDE PREPARATION, EXTRACTS AND

WHOLE SEEDS

Treatment Inhibitory act ivi t ya against H T ~ BT HCT BCT

Extract' treated with human

gastric juice for 4 h 145

LCI-C heated at 95OC for 1.5 h 1 3

Extract' heated at 95OC for 1 h 62

at 95'C for 2 h 63

145 104 88

92 86 19

95 56 1 2

87 47 62

SeedsC soaked overnight and

boiled for 2 h 1 0 0 1

~ ~~

a Percent activity related to untreated sample; means of three measurements HT: human trypsin, BT: bovine trypsin, HCT: human chymotrypsin, BCT: bovine chymotrypsin Experiments performed with another cultivar, Syrian local small lentils

preparations is obviously due to an involvement of other seed components in this inactivation process. This may be thiols present in the seeds which have been shown to participate in thermal inactivation of soybean trypsin inhibitors (Ellenrieder ef al. 1981) and to inactivate legume trypsin inhibitors, when added, under moderate conditions (Friedman and Gumbmann 1986). Altogether, the effects of thermal treatment on inhibitor activities against human and bovine proteinases are similar.

The experiments on lentil inhibitor stability show that although the inhibitors pass the stomach unaltered, they are destroyed by conventional food processing involving wet heating of the lentils, as are the inhibitors in other legume seeds (Liener and Kakade 1980; Sathe and Salunkhe 1984; Rackis et al. 1986). Because of the effect being highly dependent on the reaction conditions, new processes that will be introduced in future should be investigated anew. In general, results from experiments with purified inhibitor preparations or inhibitor extracts should be treated with caution when used as indicators for processing because of the


differences in stability between these experimental and the practical conditions (see also references above). Despite differences in action on human and bovine proteinases, the bovine enzymes can be used for an evaluation of a particular processing step on the inhibitor activity also when human enzymes are of interest, because the influences on inhibitor activity run in parallel for both human and bovine enzymes.

In the following paper (Weder and Mueller 1989), we report on the interactions of LCI-1 and LCI-4 with human and bovine trypsin and chymotrypsin. Complex formation and the influence of chemical and enzymatic modifications were studied to elucidate why these inhibitors inhibited those enzymes so differently.

ACKNOWLEDGMENTS

The authors thank Mrs. Anneliese Moedl and Mrs. Angelika Langwieser for performing the amino acid analyses and Mrs. Maria Valeria Bokor for assistance with the inhibitor stability experiments. The authors are indebted to Priv.-Doz. Dr. Peter Lehnert, Gastroenterologisches Labor der Medizinischen Klinik In- nenstadt der Universitaet Muenchen, for supplying the human duodenal juice and the human gastric juice, and to Dr. Mohan C. Saxena, ICARDA, Aleppo, Syria, for providing us with the Syrian local small lentils. Financial support of the work by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

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Documents

ISOLATION AND CHARACTERIZATION OF TWO TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM LENTIL SEEDS (LENS CULINARIS MEDIK.)