Biochem. J. (1981) 193, 29-36Printed in Great Britain

Natural plant enzyme inhibitorsCharacterization of an unusual a-amylase/trypsin inhibitor from ragi (Eleusine coracana Geartn.)

B. SHIVARAJ and T. N. PATTABIRAMANDepartment ofBiochemistry, Kasturba Medical College, Manipal-576 119, India

(Received 3 March 1980/Accepted 1 September 1980)

An inhibitor I-1, capable of acting on both a-amylase and trypsin, was purified tohomogeneity from ragi (finger-millet) grains. The factor was found to be stable to heattreatment at 100°C for 1 h in the presence of NaCl and also was stable over the wide pHrange 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in gradual loss ofboth the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex wasdemonstrated by chromatography on a Bio-Gel P-200 column. This indicated that theinhibitor is 'double-headed' in nature. The inhibitor was retained on a trypsin-Sepharose4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery ofamylase-inhibitory and trypsin-inhibitory activities. a-Amylase was retained on atrypsin-Sepharose column to which inhibitor I-1 was bound, but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-tri-nitrobenzenesulphonic acid resulted in complete loss of amylase-inhibitory activity butonly 40% loss in antitryptic activity. Modification of arginine residues by cyclo-hexane-1,2-dione led to 85% loss of antitryptic activity after 5h, but no effect onamylase-inhibitory activity. The results show that a single bifunctional protein factor isresponsible for both amylase-inhibitorydifferent reactive sites.

Protein proteinase inhibitors are widely distri-buted in the plant kingdom (Vogel et al., 1968).Most of these factors are known to inactivatetrypsin, chymotrypsin and related enzymes (Vogel etal., 1968). A number of such inhibitors have beenisolated and characterized from legume seeds,cereals and tubers (Birk, 1976; Sumathi & Pat-tabiraman, 1977, 1979). Proteinaceous a-amylaseinhibitors of plant origin have received increasingattention during the last few years. a-Amylaseinhibitors have been studied in detail from wheat(Buonocore et al., 1977), rye (Granum, 1978) andkidney bean (Marshall & Lauda, 1975). Previouslywe reported the isolation and characterization of twoproteinaceous factors from ragi (finger-millet) grainsthat inhibited human salivary, human pancreatic andpig pancreatic amylases (Shivaraj & Pattabiraman,1980). One of the inhibitors, designated inhibitorI-1, was found to be resistant to tryptic digestion,whereas the second one, inhibitor I-2, was in-activated by trypsin. In the present paper we report aremarkable property of inhibitor I-1, namely itscapacity to inhibit both a-amylase and trypsin. To

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and trypsin-inhibitory activities with two

the best of our knowledge, this is the first report onthe existence of such an unusual bifunctional protein.

ExperimentalMaterials

Pig pancreatic amylase (EC 3.2.1.1), pig pepsin(EC 3.4.4.1), Pronase (type VI proteinase),lysozyme (hen's egg-white, EC 3.2.1.17), myo-globin (horse heart), a-chymotrypsinogen A (bovine)and the sodium salt of 2,4,6-trinitrobenzenesul-phonic acid were purchased from Sigma ChemicalCo., St. Louis, MO, U.S.A. Bovine trypsin (EC3.4.21.4), salt-free and twice-crystallized, and bovinea-chymotrypsin (EC 3.4.21.1), salt-free and thrice-crystallized, were the products of WorthingtonBiochemical Corp., Freehold, NJ, U.S.A. Solublepotato starch was obtained from E. Merck,Darmstadt, W. Germany. Cyclohexane- 1,2-dionewas purchased from Aldrich Chemical Co., Mil-waukee, WI, U.S.A. CNBr-activated Sepharose 4Band Sephadex G-50 were procured from Pharmacia

0306-3275/81/010029-08$01.50/1 (© 1981 The Biochemical Society

B. Shivaraj and T. N. Pattabiraman

Fine Chemicals, Uppsala, Sweden. Bio-Gel P-60 andP-200 were obtained from Bio-Rad Chemicals,Richmond, CA, U.S.A. Bovine serum albuminfraction V was the product of Fluka, Buchs,Switzerland. All other reagents were of analyticalgrade.

Assay methodsAmylase activity was measured as described by

Bernfeld (1955), with the following modification. Aportion of amylase (0.1 ml; 0.2,ug of protein of pigpancreatic amylase) was added to a solutioncontaining 30,umol of sodium phosphate buffer,pH 6.9, and 10.5,umol of NaCl in a volume of 1.5 ml.After 20min incubation at 370C, 0.5ml of 1.0%starch solution was added. The enzyme reaction wasarrested after 5min incubation by the addition of1 ml of dinitrosalicylate reagent (Bernfeld, 1955).The solution was kept in a boiling-water bath for10min, cooled and diluted to 11.0ml with water.Amylolytic activity was calculated as maltoseequivalents liberated. One unit of enzyme is definedas the amount of enzyme that liberated Iumol ofmaltose equivalent under the assay conditions.To measure the amylase-inhibitory activity, suit-

able amounts of the purified ragi inhibitors werepreincubated with the enzyme solution in a totalvolume of 1.5 ml containing 30,umol of phosphatebuffer, pH6.9, and 10.5,amol of NaCl for 20min at370C. Controls without inhibitors were run simul-taneously. The enzyme reaction was initiated by theaddition of starch solution, and the systems wereprocessed as described above. The decrease inenzyme activity was a measure of the inhibitoryactivity. One unit of inhibitor is defined as theamount that decreased amylase activity by 1 unit.

Trypsin and chymotrypsin activities were assayedby the caseinolytic method as described before(Sumathi & Pattabiraman, 1975). The assay systemcontained 20.0mg of casein, 12Oumol of phosphatebuffer, pH 7.6, and trypsin or chymotrypsin (6-lO,ug of active enzyme) in a total volume of 2.Oml.After 10min incubation at 370C, the reaction wasstopped by the addition of 3.0ml of 5% (w/v)trichloroacetic acid. After 1 h standing, the precipit-ate was sedimented by centrifugation at 2500g for10 min. Then 1 ml samples of the supernatant wereanalysed by the method of Lowry et al. (1951). Oneunit of proteolytic activity is defined as the amountof enzyme that released 1 mg of trichloroaceticacid-soluble peptide fragments under the assayconditions. For the purpose of calculation bovineserum albumin was used as standard in this method.Proteinase inhibitory activities of the purified ragiinhibitors were determined by including suitableamounts of inhibitor solutions in the assay system.Decrease in caseinolytic activity in the test systemcompared with the control was a measure of the

inhibitory activity. One unit of inhibitor is defined asthe amount that suppressed proteolytic activity by 1unit.

Protein content was determined by the method ofLowry et al. (1951), with bovine serum albumin asstandard.

Purification ofinhibitorsThe details of purification of amylase inhibitors

I-1 and I-2 from ragi grains were as describedpreviously (Shivaraj & Pattabiraman, 1980).Ground ragi grains were homogenized with 3 vol.(w/v) of 0.15M-NaCl and filtered. To the filtrate,solid (NH4)2SO4 was added to 55% saturation. Theprecipitate formed was collected by centrifugationand dissolved in 2 mM-sodium acetate buffer, pH 5.0,containing 75 mm-NaCl and dialysed overnightagainst the same buffer. The dialysis residue wascentrifuged, and the clear supernatant was sub-jected to chromatography on a CM-cellulose columnequilibrated with 2 mM-acetate buffer, pH 5.0, con-taining 75 mM-NaCl. The two inhibitors that wereexchanged on the column were eluted separatelywith 20mM-acetate buffer, pH 5.0, containing0.15M-NaCl and the same buffer containing 0.3M-NaCl. The two fractions were further purified bygel chromatography on Sephadex G-50 andrechromatography on CM-cellulose. Inhibitor I-1accounted for 86.5% of the recovered activity andinhibitor I-2 for 13.5% of the activity.

Molecular-weight determinationThe molecular weights of purified inhibitors I-1

and I-2 were determined by electrophoresis onpolyacrylamide gel (10% acrylamide) in the pre-sence of sodium dodecyl sulphate as described byWeber et al. (1972). Lysozyme (egg-white, mol.wt.14 300), myoglobin (horse heart, mol.wt. 17 200),a -chymotrypsinogen A (bovine, mol.wt. 25 700) andpepsin (pig, mol.wt. 35000) were used as standardproteins.

Complex-formationComplex-formation between inhibitor I-1 and

trypsin or amylase was demonstrated by gelchromatography on a Bio-Gel P-200 column(0.9cm x 53.5 cm, bed volume 34.0ml) equilibratedwith 20mM-phosphate buffer, pH 7.0, containing0.15M-NaCl. In each experiment the componentswere applied to the column in a total volume of0.3 ml containing 6,mol of phosphate buffer,pH 7.0, and 45,umol of NaCl. The column waseluted with equilibration buffer at a flow rate of3 ml/h, and 1 ml fractions were collected. To obtainthe trypsin-inhibitor I-1 complex, 400,g of proteinof bovine trypsin was incubated with 400,g ofprotein of inhibitor I-1 at room temperature (280C)

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a-Amylase/trypsin inhibitor from ragi

for 30min, and the mixture was applied to thecolumn. The fractions were assayed for proteincontent and a-amylase-inhibitory activity. Similarly,the mixture of pig pancreatic amylase (550,pg ofprotein) and inhibitor I-1 (400jug of protein) wasapplied to the column after incubation for 30min inorder to obtain the amylase-inhibitor I-1 complex.Formation of trimer complex between typsin,inhibitor I-1 and amylase was shown by passing amixture of 400,ug of protein of trypsin, 400,ug ofprotein of inhibitor I-1 and 550jag of protein of pigpancreatic amylase through the column after in-cubation for 30min at pH7.0. Protein content wasdetermined in each fraction. In separate experi-ments inhibitor I-1 (400,ug of protein), bovinetrypsin (400,g of protein) and pig pancreaticamylase (550 ug of protein) were passed through thecolumn to determine their elution patterns.

Affinity chromatographyAffinity chromatography of inhibitor I-1 was

performed at 4°C on a trypsin-Sepharose 4Bcolumn. Trypsin (25mg) was treated with CNBr-activated Sepharose 4B (3.5ml wet volume) in thepresence of 0.1 M-sodiumfl borate buffer, pH 8.3,containing 0.5M-NaCl. The trypsin-Sepharose 4Bwas packed into a column (0.9cm x 4.7 cm, bedvolume 3.0ml) and equilibrated with 20mM-phos-phate buffer, pH 7.0, containing 5OmM-NaCl. Then0.5mg of protein ofinhibitor I-I in a total volume of0.4 ml containing 8,umol of phosphate buffer,pH7.0, and 20,umol of NaCl was applied to thecolumn. The column was washed with 15.6ml of theequilibration buffer. The inhibitor bound to thecolumn was then eluted with 50mM-HCl containing50mM-NaCl. Fractions of volume 2ml were collec-ted during washing and elution at a flow rate of4ml/h. Protein and inhibitory activities were deter-mined in the fractions.

Formation of trimer complex between trypsin,inhibitor I-1 and a -amylase was also demonstratedon a trypsin-Sepharose column. First, 0.5mg ofprotein of inhibitor I-1 was bound to the trypsin-Sepharose column (bed volume 3.0ml) as indicatedabove. The column was washed extensively with20mM-phosphate buffer, pH 7.0, containing 0.15 M-NaCl. Then 250pg of protein of pig pancreaticamylase in a volume of 0.25 ml containing 5,umol ofphosphate buffer, pH 7.0, and 37.5pmol of NaClwas applied to the column. The column was againwashed with 20mM-phosphate buffer, pH7.0, con-taining 0.15M-NaCl. The washings (20ml) wereassayed for protein content and a-amylase activity.In a separate control experiment 250,ug of protein ofpig pancreatic amylase alone was applied to atrypsin-Sepharose column, and protein as well asa-amylase activity were determined in the washings.

Vol. 193

Effect oftemperatureFor studying the effect of heat treatment on the

amylase-inhibitory and trypsin-inhibitory activitiesof inhibitor I-1, 3.2,ug of protein of the inhibitor inthe presence of 1.5 ,mol of NaCl in a volume of 20,1was kept in a boiling-water bath at 1000C for lh.The contents were cooled and assayed for residualinhibitory activities.

Effect ofpHEffect of pH on the inhibitory activities of

inhibitor I-1 was measured as follows. A sample(8,ug of protein) of inhibitor I-1 was preincubatedwith 0.1M buffers of different pH values (sodiumcitrate, pH3.0; acetate, pH5.0; phosphate, pH6.9and 8.0; glycine/NaOH, pH9.0 and 10.0), 0.2M-HCI, pH0.9, and 0.2M-NaOH, pH 12.6, for 16h at40C in a total volume of 0.1 ml. After addition of0.5 ml of 0.2M-phosphate buffer, pH 7.6, suitablesamples were withdrawn and assayed for residualinhibitory activities.

Preincubation ofinhibitor I-l with trypsin atpH3. 7Inhibitor I-1 (8.0ug of protein) was preincubated

with 26jug of trypsin at 250C in a volume of 0.l mlcontaining 2.Opmol of acetate buffer, pH 3.7, and10,umol of NaCl for different time intervals. The pHof the system was adjusted to 7.6 by adding 0.4ml of0.2 m-phosphate buffer, pH 7.6. The contents weremade up to 1.0ml with water and assayed forresidual trypsin activity. Unlike the procedurementioned above, the reaction was started by adding1.Oml of substrate (casein) and incubated for 10min.Controls were run simultaneously without additionof the inhibitors. Similarly the preincubation mix-tures were assayed for amylase-inhibitory activityafter neutralization of the contents by adding 0.4 mlof 0.2M-phosphate buffer, pH6.9.

Results

The isolation of two proteinaceous a-amylaseinhibitors, I-I and 1-2, in homogeneous forms fromragi has already been described (Shivaraj & Pat-tabiraman, 1980). Both the inhibitors were basicproteins, and the apparent molecular weights basedon gel chromatography on Sephadex G-50 andBio-Gel P-60 were about 9000 and 8000 respectivelyfor inhibitors I-1 and 1-2. However, the inhibitorswere non-diffusible on dialysis. This led to are-investigation of the molecular weights of theinhibitors by sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis. The electrophoretic pat-terns of inhibitors I-1 and 1-2 are shown in Fig. 1.The calculated molecular weights based on gelelectrophoresis of inhibitors I-1 and I-2 were 14 300and 16 500 respectively, which are at wide variance

31

B. Shivaraj and T. N. Pattabiraman

++

1-1 1-2

Fig. 1. Polyacrylamide-gel electrophoresis of inhibitorsI-i and I-2 in thepresence ofsodium dodecyl sulphateThe electrophoresis was performed on polyacryl-amide gel (10% acrylamide) in the presence of 0.1%sodium dodecyl sulphate in both gel and electrodebuffer as described by Weber et al. (1972). Thesamples (inhibitors I-1 and I-2) were treated with1% sodium dodecyl sulphate and 1% ,-mercapto-ethanol for 2h at 370C at pH 7.2, and subjected toelectrophoresis for 4h with a current of 6 mA/tube.The protein bands were stained with CoomassieBrilliant Blue R-250 (0.1% in methanol/aceticacid/water, 5:1:5, by vol.).

with the values obtained by gel-permeationchromatography. The discrepancy is probably dueto the fact that the two inhibitors, being basicproteins, are retarded on Sephadex G-50 andBio-Gel P-60 columns, exhibiting anomalous elutionbehaviour. Similar observations have been reportedfor other proteins (Starkey & Barrett, 1976).When inhibitor I-2 (16,ug of protein) was

incubated with bovine trypsin (10ug) for 2h atpH 7.6 and at 37°C, the amylase-inhibitory activitywas almost completely lost. On the other hand,inhibitor I-1 was fully resistant to tryptic attackunder identical conditions. This prompted us tostudy the effects of inhibitors I-1 and I-2 on trypticactivity. Although inhibitor I-2 had no effect on thecaseinolytic activity of bovine trypsin even whenused in a molar ratio of 4: 1, inhibitor I-1 was found

(a) (b)

Fig. 2. Polyacrylamide-gel electrophoresis of inhibitor I-i(a) At pH 5.0 with 40 mM-acetate buffer (con-tinuous buffer system). The electrophoresis was runfor 3 h with a current of 3 mA/tube. (b) At pH 8.6with 25 mM-Tris/Veronal buffer (continuous buffersystem) for 45 min with a current of 3 mA/tube. Theprotein bands were stained with 0.1% AmidoSchwartz in 7% (v/v) acetic acid.

to be a powerful inhibitor of trypsin. This suggestedthat inhibitor I-1 is an unusual bifunctional inhi-bitor capable of acting on a-amylase as well as ontrypsin. The following studies corroborated thisconclusion.

In order to prove that inhibitor I-I is a singleprotein responsible for both trypsin-inhibitory andamylase-inhibitory activities, the homogeneity of thepreparation was established by several criteria.Inhibitor I-I moved as a single band towards thecathode at both pH 5.0 and pH 8.6 when electro-phoresis was performed on polyacrylamide gel (Fig.2) and cellulose acetate strips (Fig. 3). Inhibitor I-1was also found to be homogeneous on the basis ofgel-filtration studies on Sephadex G-50 in thepresence and in the absence of 6M-urea (Fig. 4).Similar elution behaviour was also obtained duringgel filtration on Bio-Gel P-60.When inhibitor I-1 was passed through a column

of trypsin-Sepharose 4B, both the inhibitory ac-tivities were bound to the immobilized enzyme. Thebinding was not due to weak interaction, sinceelution with 0.5 M-NaCl did not dissociate theamylase-inhibitory or trypsin-inhibitory activities.The inhibitor protein was eluted with 50mM-HClcontaining 50mM-NaCl. Complete recovery oftrypsin-inhibitory activity and 92% recovery ofamylase-inhibitory activity were obtained in a single

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.1

a -Amylase/trypsin inhibitor from ragi

+

4, 4-

(a) (b)Fig. 3. Cellulose acetate electrophoresis ofinhibitor I-iThe cellulose acetate electrophoresis was performedboth at (a) pH 5.0 (0.1 M-acetate buffer) and (b)pH 8.6 (40mM-sodium barbitone buffer) onCelagram (Shandon) strips. The time of run was 1 hat 200V. The strips were stained with 0.5% AcidRed in 5% (v/v) acetic acid.

fraction (elution volume 20 ml) during affinitychromatography. The eluted inhibitor I-1 resembledthe native inhibitor with respect to chromatographicbehaviour on Sephadex G-50 and electrophoreticpattern on polyacrylamide gel. Moreover the frac-tion could be again immobilized on a trypsin-Sepharose column at neutral pH. There was nointeraction or binding of inhibitor I-1 to Sepharosealone, since the inhibitor was not retained on achymotrypsin-Sepharose column.

a-Amylase was not retained on a trypsin-Sepharose column and was eluted in the washings(20mM-phosphate buffer, pH 7.0, containing 0.15M-NaCi). However, when amylase was passedthrough the column on which inhibitor I-I wasimmobilized, no amylase activity or protein waseluted in the washings. These findings show thatamylase is bound to immobilized inhibitor I-1.This demonstrates that amylase-inhibitory activityand trypsin-inhibitory activity are associated withone and the same protein.When inhibitor I-i solution was subjected to heat

treatment in the presence of 75 mM-NaCl at 1000Cfor 1 h, there was no loss of antitryptic activity aswell as amylase-inhibitory activity. Dialysis of theinhibitor against water resulted in complete pre-cipitation, indicating that salt (NaCl) is essential forthe solubility of the inhibitor.

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0 12

60 1,

8 1 6 24 32Fraction no.

Fig. 4. Gelfiltration ofinhibitorFI-I on a Sephadex G-50column (0.9cm x 60cm, bed volume 38 ml)

0, Inhibitor I-1 (0.9mg of protein) in 0.4mlcontaining 8,umol of phosphate buffer, pH7.6, and60umol of NaCl was applied to the columnpreviously equilibrated with 20mM-phosphate buf-fer, pH 7.6, containing 0.3M-NaCl. 9, Inhibitor I-I(0.9mg of protein) was incubated with 6M-urea in avolume of 0.4ml containing 8#umol of phosphatebuffer, pH7.6, and 60,umol ofNaCl for 2h at 370C.The' sample was then applied to the columnpreviously equilibrated with 20mM-phosphate buf-fer, pH 7.6, containing 0.15 M-NaCl and 6M-urea. Inboth cases the inhibitor was eluted with equlibrationbuffers at a flow rate of 7ml/h, and 1 ml fractionswere collected. The fractions were assayed forprotein content.

When the inhibitor was exposed to different pHvalues varying from 1 to 10 for 16h at 40C therewas no loss of either inhibitory activity. However, atpH 12.6 about 30% of the amylase-inhibitory ac-tivity was lost, though the antitryptic activity wasnot diminished.

The effects of pepsin and Pronase treatment onthe inhibitory activities of inhibitor I-1 are shown inFigs. 5 and 6 respectively. There was a gradual lossof the activities with increasing time of treatmentwith the proteinases. However, pepsin destroyedpreferentially the amylase-inhibitory activity ofinhibitor I-1, whereas the antitryptic activity waspreferentially removed by Pronase.

It has been shown previously that treatment ofinhibitor I-1 with starch for lOmin resulted incomplete abolition of amylase-inhibitory activity

33

B. Shivaraj and T. N. Pattabiraman

'S.0

c:a

0 25 50 75 100

Time of pepsin treatment (min)125

Fig. 5. Changes in amylase-inhibitory and trypsin-in-hibitory activities of inhibitor I-1 on treatment with

pepsinInhibitor I-1 (15 pug of protein) was treated with 5jugof pepsin in the presence of 20,umol of HCl/KClbuffer, pH2.0, in a total volume of 0.2ml. Thecontents were incubated for different time intervalsat 370C. Pepsin action was arrested by the additionof 0.2ml of 0.2 M-phosphate buffer, pH 7.6. Residualamylase-inhibitory and trypsin-inhibitory activitieswere measured in the samples. 0, Amylase-inhi-bitory activity; 0, trypsin-inhibitory activity.

25 50 75 100

Time of Pronase treatment (min)

Fig. 6. Changes in amylase-inhibitory and trypsin-in-hibitory activities of inhibitor I-1 on treatment with

PronaseInhibitor I-1 (15,pg of protein) was incubated withS5pg of protein of Pronase in a volume of 0.2mlcontaining 20umol of phosphate buffer, pH7.6, at370C. At different time intervals the Pronase wasinactivated by heat treatment at 750C for lOmin.The contents were assayed for residual inhibitoryactivities. 0, Amylase-inhibitory activity; *,trypsin-inhibitory activity.

Table 1. Modiflcation ofinhibitor I-1 with 2,4,6-trinitrobenzenesulphonic acid and cyclohexane-1,2-dione and changes inamylase-inhibitory and trypsin-inhibitory activities

Amino groups were modified by treatment with 2,4,6-trinitrobenzenesulphonic acid at pH 7.6 (Sumathi &Pattabiraman, 1977). A sample (520jug of protein) of inhibitor I-1 was treated with 3.3mg of 2,4,6-trinitro-benzenesulphonic acid in the presence of 400,umol of phosphate buffer, pH7.6, in a volume of 4.Oml at 280C.Samples were withdrawn at intervals of 1, 2, 3, 4 and 5h and each was dialysed against 2 litres of 50mM-NaCIfor 5h at 40C. The dialysed samples were assayed for residual amylase-inhibitory and trypsin-inhibitory activities.Arginine residues of inhibitor I-1 were modified by treatment with cyclohexane-1,2-dione (Toi et al., 1967).Inhibitor I-1 (520,ug of protein) was treated with 1.7mg of cyclohexane-1,2-dione in the presence of 200jumol ofsodium borate buffer, pH 9.0, at 280C in a total volume of 4.0ml. At intervals samples were withdrawn, dialysedas described above and tested for residual inhibitory activities.

Assay componentsInhibitor I-IInhibitor I-I + cyclohexane-1,2-dione

Inhibitor I-I + 2,4,6-trinitrobenzene-sulphonic acid

Time(h)

Control1234512345

Residual inhibitory activity (%)

Antiamylase Antitrypsin100 100100 64100 49100 28100 19100 16

6 783 680 660 600 60

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a-Amylase/trypsin inhibitor from ragi

(Shivaraj & Pattabiraman, 1980). To study theeffect of such a treatment on the antitryptic activity,4.Oug of protein of inhibitor I-i was mixed with0.3 ml of starch solution (1%) at pH6.9 and at 7.6.After 10min, the contents were assayed for trypsin-inhibitory activity. There was no detectable loss ofproteinase-inhibitor activity after preincubation withstarch, indicating that the binding site for trypsin oninhlbitor I-1 is mutually exclusive of the binding sitefor amylase. This was also proved by preincubationof inhibitor I-i with amylase. A sample (4.0,g ofprotein) of inhibitor I-i was incubated with lOpug ofprotein of pig pancreatic amylase in 0.3 ml in thepresence of 6,umol of phosphate buffer, pH6.9, and15umol of NaCl at 370C for 20min. When thismixture was directly assayed for antitryptic activity,100% of the proteinase-inhibitory activity wasretained compared with the control without amylase.The results on complex-formation between inhi-

bitor I-1 and the enzymes are depicted in Fig. 7.When trypsin/inhibitor I-1 mixture was subjected togel chromatography on Bio-Gel P-200, a peak withmaximal protein values at an elution volume of 24 mlwas seen. In addition, there was a shoulder region(27-31 ml). Trypsin itself when subjected to gelchromatography was eluted with a peak at 27ml,and the inhibitor alone was eluted with a peak at30ml. The trypsin-inhibitor complex (tube no. 24).displayed amylase-inhibitory activity. The amylase/inhibitor mixture was eluted with a protein peak at22ml. A second peak (30ml) was also noticedduring this experiment. This might correspond tonon-reacted amylase inhibitor. Amylase alone whensubjected to gel chromatography was eluted with anelution volume of 25ml. Trypsin/amylase/inhibitormixture was also subjected to gel chromatography,and two protein peaks were observed with maximalprotein values at 18ml and 23ml. The first peakshould represent the trimer complex, and the secondpeak may represent the mixture of amylase-inhibitorand trypsin-inhibitor complexes. The molecularweights of the inhibitor-enzyme complexes could notbe ascertained by gel chromatography owing to theanomalous behaviour of the inhibitor during gelchromatography.The findings from chemical modification also

supported the conclusion that binding sites fortrypsin and amylase on inhibitor I-i are mutuallyexclusive. The effects of treatment of inhibitor I-1with cyclohexane-1,2-dione and 2,4,6-trinitro-benzenesulphonic acid on the inhibitory activities areshown in Table 1. Modification of the arginineresidues with cyclohexane-1,2-dione by treatmentfor up to 5 h did not result in any loss ofamylase-inhibitory activity, whereas 85% of theantitryptic activity was lost under this condition.Only 7% of the trypsin-inhibitory activity wasretained after 20h treatment with cyclohexane-

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1254

0

500

25 o \

125 A

1, 14

0 1 5 20 25 30 35 40Fraction no.

Fig. 7. Complex-formation between inhibitor I-l, trypsinand amylase on a Bio-Gel P-200 column

(0.9cm x 53.5 cm, bed volume 34.0ml)The column was equilibrated with 20mM-phosphatebuffer, pH 7.0, containing 0.15M-NaCl. (a) 0, Pigpancreatic amylase (550,g of protein); *, inhibitorI-1 (400#g of protein). (b) 0, Bovine trypsin(400pag of protein); A, trypsin-inhibitor I-1 com-plex (400,g of protein of each of trypsin andinhibitor I-1). (c) 0, Amylase-inhibitor I-1 com-plex (550,ug of protein of amylase and 400,ug ofprotein of inhibitor I-1); A, trypsin-inhibitor I-I-amylase trimer complex (400pag of protein ofeach of trypsin and inhibitor I-i and 550,ug ofprotein of amylase). To obtain the complexes thecomponents were mixed and incubated for 30min at280C at pH 7.0 and applied to the column. Then1 ml fractions were collected at a flow rate of 3 ml/h.

1,2-dione. Conversely, modification of amino groupsby 2,4,6-trinitrobenzenesulphonic acid resulted inalmost complete loss of amylase-inhibitory activity

35

36 B. Shivaraj and T. N. Pattabiraman

in 1 h, whereas only 40% of the antitryptic activitywas destroyed after 5 h treatment.When inhibitor I-1 was preincubated with trypsin

at pH 3.7 for 30min, there was about 50% loss ofantitryptic activity, which remained constant up to1 h. This loss of activity could be due to limitedproteolysis of inhibitor at lower pH (Smirnoff et al.,1976; Sumathi & Pattabiraman, 1979). However,this treatment had no effect on amylase-inhibitoryactivity.The inhibitory pattern against trypsin was found

to be linear with respect to inhibitor concentrationup to 75%. In the linear range of inhibition 1.0molof inhibitor I-1 was found to interact stoicheio-metrically with 1.0 mol of bovine trypsin. InhibitorI-1 did not exhibit any inhibitory activity againstbovine chymotrypsin even when excess (40jug ofprotein) of the inhibitor was used. With pigpancreatic amylase, the inhibition by inhibitor I-Iwas linear only up to 40%, as reported previously(Shivaraj & Pattabiraman, 1980).

Discussion

Proteinase inhibitors capable of acting on trypsinand chymotrypsin and possessing distinct bindingsites for the two enzymes have been reported (Birk etal., 1967; Smirnoff et al., 1976). However, thepresent studies establish for the first time theexistence of an unusual 'double-headed' enzymeinhibitor that inhibits two unrelated enzymes,namely a-amylase and trypsin. The inhibitor wasfound to be a basic protein with mutually exclusivebinding sites for the two enzymes. The results ofchemical modification suggest that arginine residuesare essential for the trypsin-inhibitory activity,whereas they are not involved in the inactivation ofamylase by the inhibitor. On the other hand, amino

groups on the inhibitor were found to be necessaryfor binding and inactivation of a-amylases.

This work was supported by a grant-in-aid, B & M125/78, from the Department of Atomic Energy, Govern-ment of India. The authors are grateful to Dr. A. KrishnaRao, Dean, Kasturba Medical College, Manipal, for hiskeen interest and encouragement.

References

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