THE JOURNAL OF BIOLOGICAL CHEMIRTRY Vol. 249, No. 6, Issue of Maroh 10, pp. 155G1665, 1974

Printed in U.S.A.

Pepsin Inhibitors from Ascaris Zumbricoides

ISOLATION, PURIFICATION, AND SOME PROPERTIES*

(Received for publication, September 27, 1973)

GHALEB M. ABU-ERREISH~ AND ROBERT J. PEANASKY

From the Department of Biochemistry, School of Medicine, The University of South Dakota, Vermillion, South Dakota 67069

SUMMARY

Extracts of the body walls of the adult Ascaris lumbri- coides var. suis inhibit pepsin between pH 1 and 6. Four in- hibitors of pepsin were isolated and purified as follows. The crude extract of Ascaris was incubated at 37” and pH 2.0 for 75 min. The pepsin-inhibiting activity was obtained in a fraction precipitated at 0.65 saturation with ammonium sul- fate at pH 5.35, and sequentially chromatographed on Bio- Gel P-30 at pH 2.1 and Cellex SE at pH 4.7 and resolved into four inhibitors on DEAE-Sephadex at pH 8.8. The degree of purity of each inhibitor was demonstrated by disc gel electrophoresis. The over-all yield of pepsin inhibitors was 44 % of the initial extract. Inhibitor I was 7 % ; Inhibitor II, 10% ; Inhibitor III, 20% ; and Inhibitor IV, 7 %. The over-all purification of Inhibitors I + III was 6,800-fold and that of Inhibitor IV, 3,300-fold. The molecular weights of these inhibitors were calculated from their amino acid com- position and were in agreement with the values obtained from both 5% and 10% polyacrylamide gels in sodium dode- cyl sulfate. The values obtained from the amino acid com- position were: 17,515 ( =160 amino acid residues), 15,584 ( =142 amino acid residues), 16,124 ( =I47 amino acid residues), and 31,719 ( =290 amino acid residues) for In- hibitors I, II, III, and IV, respectively. The NH2-terminal amino acid residue of each of these proteins was histidine. The ability of each of these inhibitors to inactivate pepsin was lost by its treatment with either trypsin or chymotrypsin. Each inhibitor inhibited porcine, bovine, and human pepsins, and porcine gastricsin, but not human gastricsin.

* Thii study was supported by Grant GM-16203 from the Na- tional Institute of General Medical Sciences and by a cooperative agreement with the United States Department of Agriculture. Preliminary reports were presented at the International Research Conference on Proteinase Inhibitors at Munich, 1970; the 13th and 14th West Central States Biochemistry Conferences at Kansas State University, Manhattan, 1970, and the University of Kansas, Lawrence, 1971; and the American Society of Biological Chemists’ 62nd Annual Meeting at San Francisco in June, 1971 (1).

$ Data are taken ;n part from a dissertation submitted to the Graduate Facultv of The Universitv of South Dakota in martial fulfillment of the’ requirements for ‘the degree of Doctor of Phi- losophy. Present address, Boston Biomedical Research Institute, Boston, Mass. 02114.

The stoichiometric interaction of a serine proteinase with a protein other than a y-globulin to form a unique complex which behaves as a new species of protein and is devoid of proteinase activity is a well known phenomenon. The ubiquity of these serine proteinase inhibitors is indicated in a few selected refer- ences (2-5). These proteinase-protein complexes, which may be isolated, have apparent dissociation constants less than lo-* M;

some of these complexes can regulate the level of the active form of an enzyme. An example of this function is the presence of the secretory pancreatic trypsin inhibitor in the zymogen granules of the pancreas which minimizes the premature activation of trypsinogen and then the activation of the other zymogens in the granules by trypsin. In contrast to these widely studied sys- tems, there are no examples of the inhibition of pepsin (which is not a serine proteinase) by well characterized non-y-globulin proteins, although one of the activation products obtained in the conversion of pepsinogen to pepsin inactivates the milk-clotting activity of pepsin at pH 5.0. This polypeptide, obtained in the activation of pepsinogen, is digested and ineffective at pH 2.0 where pepsin functions in the process of digestion (6).

Werle et al. (7) observed the inhibition of pepsin by a trypsin- inhibiting factor obtained by ammonium sulfate fractionation of a potato extract; Hilliard and West (8) and Carsten and Pierce (9) observed the inhibition of pepsin by extracts of bovine pituitary. In none of these three reports was the pepsin-in- hibiting fraction purified and characterized. Recently some organic compounds have been obtained from among the metabo- lites of certain strains of actinomycetes which inhibit pepsin (10, 11). These pepsin-inhibiting metabolites have molecular weights of 685 and 643. The metabolite from Streptomyces ar- genteolus var. toyokoensis is called pepstatin and contains six organic units connected through five secondary amide bonds. Its structure is isovaleryl-L-valyl-L-valyl-L-4-amino-3-hydroxy- 6 - methylheptanoyl - L-alanyl-C amino-3 - hydroxy-6 - methyl hep- tanoic acid. The metabolite of Streptomyces EF-44-201 is called S-PI and it also contains twice as much valine as alanine in “addition to other fatty acids.”

This communication reports that protein inhibitors of pepsin, able to inactivate pepsin at pH 2.0, have now been obtained from Ascaris lumbricoides. These substances were first recognized in 1910 by Mendel and Blood (12) who showed that extracts of Ascuris inhibited pepsin as well as trypsin, but not the plant proteinase, papain. Collier (13) obtained a trypsin-inhibiting preparation from Ascaris which also inhibited pepsin. When

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the trypsin-inhibiting fraction was purified further by treatment with hot 2.5% trichloroacetic acid and fractionated with am- monium sulfate, the pepsin-inhibiting activity was lost. Rola and Pudles (14) also prepared an extract of Ascaris able to inhibit pepsin but following treatment with trichloroacetic acid they, too, lost the pepsin-inhibiting activity.

We are now reporting the isolation of four pepsin inhibitors. Each of these inhibitors can form a stable stoichiometric complex with pepsin capable of being isolated (15). The purity of these pepsin inhibitors will be demonstrated and their molecular weight, amino acid composition, stability in the presence of other pro- teolytic enzymes, and their interaction with acid proteinases from stomachs of different species reported.

EXPERIMENTAL PROCEDURE

Materials

A. lumbricoides var suis were collected at a slaughterhouse and were kept live in the salt medium of Baldwin and Moyle (16) at 3740”. The body walls of the adult worms were prepared as de- scribed by Peanasky and Laskowski (17). Porcine pepsin (EC 3.4.4.1) twice crystallized with an activity of 2500 units per mg, bovine carboxypeptidase A (EC 3.4.2.1)) chymotrypsin (EC 3.4.4.5), chymotrypsinogen A, and trypsin (EC 3.4.4.4) were purchased from Worthington. Pepsinogen (technical) from hog stomach mucosa was obtained from Nutritional Biochemicals, activated, and used to prepare porcine gastricsin. Bovine pep- sin was a generous gift from Dr. B. Kassell, Department of Bio- chemistrv. Medical College of Wisconsin. Milwaukee. Porcine pepsin was passed through a Sephadex G-25 column equilibrated with 100 rni acetate, pH5.3, before use. Bovine serum albumin, horse heart cvtochrome c. eee white lvsozvme. PTHl-Phe. thio- glycolic acid,“and Tris (base)-were obtainedfrom Sigma. Human pepsin and gastricsin and porcine gastricsin were prepared accord- ing to Tang and co-workers (l&20). Porcine gatriesin and human gastricsin did not hydrolyze N-acetyl-n-phenylalanyl-n-diiodo- tyrosine but hydrolyzed hemoglobin with a AA per 10 min per mg of protein of 200 f 25.

Hemoglobin powder was prepared from outdated human red cells according to Drabkin (21). All amino acids, the phenyl- thiohydantoins of most amino acids, and phenylisothiocyanate were obtained from Mann Research Laboratories. PTH-Thr was prepared according to Levy and Chung (22). Ellman’s re- agent, 5,5’-dithiobis(2-nitrobenzoic acid), was supplied by Aldrich Chemical Co. Bio-Gel P-30 (200 to 400 mesh) and Cellex SE, in the sodium form, were obtained from Bio-Rad Laboratories. DEAE-Sephadex A-25 and Sephadex G-25 were supplied by Phar- macia. All other chemicals were reagent grade. Double-distilled water was used throughout.

Zsolation of Pepsin Inhibitors-The procedure of Peanasky and Laskowski (i7) for the preparation of a 59,000 X g super&ant was followed. The bodv walls of Ascaris were homoeeniaed with water (400 ml/100 g of tissues) in a Waring Blendor. The homoge- nate was centrifuged in a Sorvall angle centrifuge at 11,000 X g ?or 20 min, and then in a Beckman model L2-65B ultracentrifuge at 59,000 X B (using a rotor tvne 19) for 3 hours. The sunernatant

Methods

Enzymatic Assays-The activity of the inhibitor(s) was meas- ured during purification studies by incubating 1 to 3 Mg of inhibitor with 6 pg of pepsin in 10 mM acetate adjusted to pH 2.0 in a final volume of 1.0 ml at 37” for 3 min. Residual pepsin activity was determined by adding 1.0 ml of 2.5% hemoglobin solution to each tube at 37” and stopping the reaction after 5 min by adding 2.0 ml of cold 50/, trichloroacetic acid solution. After standing in an ice bath for 15 to 30 min, the tubes were centrifuged for 40 min at 20,000 X g in a Sorvall angle centrifuge. The absorbance of the clear supernatant was determined at 280 nm in a Beckman model DU spectrophotometer and was proportional to the digestion of hemoglobin by pepsin. One unit of inhibitor was defined as that amount of inhibitor which inactivates 1.0 rg of pepsin under the conditions of the assay. Specific activity was defined as units of inhibitor per 1.0 unit of absorbance of the protein as measured through a l-cm light path at 280 nm.

solution was filtered through Whatman No. 1 paper. - The pH of the 59,000 X g supernatant was brought to 2.0 with

5 N HCl, and the solution was then incubated at 37” for 75 min. The solution was cooled to room temperature in an ice bath and 5 N NaOH solution was added to raise the IJH to 5.5. A heavv nre- cipitate was removed by centrifuging at il,OOO X g for 30 m&and was discarded.

The amount of solid ammonium sulfate required to bring the heat-treated solution to 0.65 saturation at 0” was calculated ac- cording to Noda and Kuby (31). After standing at 0” for at least 3 hours, the precipitated protein was separated by centrifugation at 11,000 X B for 40 min. This packed precipitate was dissolved in a minimum -volume of water and was c&rifuged at 33,000 X g for 20 min to remove some insoluble material. The nH (5.3) was then dropped to 2.1, solid Tris (base) was added to aconcentration of 100 mm, and the pH was readjusted to 2.1.

Carbohvdrates were determined as hexoses and pentoses by the

Any precipitate that formed was removed. The final volume of the protein solution was adjusted to >&, of the volume of the 59,000 X g supernatant.

Chromatography 011 Bio-Gel P-SO-P-30 was swelled in 100 mM Tris-HCl solution, adjusted to pH 2.1. A column (Pharmacia) (10 X 100 cm) was packed according to the instructions of Pharma- cia and was washed with 3 void volumes (6 liters per void volume) before the application of the protein sample. The volume of the protein solution applied to the column was 4% of the volume of the

anthrone met,hod (23). Disc eel electroohoresis was nerformed bv the method of Orn-

stein and Davis $4). I

Molecular weights were determined in 5 and 10% polyacrylamide

i The abbreviation used is: PTH, phenylthiohydantoin.

gels in sodium dodecyl sulfate following the method of Dunker and Reuckert (25).

NH,-terminal analysis was done by Edman’s method as de- scribed by Schroeder (26).

Performic acid oxidation of protein samples was done by the procedure of Hirs (27).

Sulfhydryl groups were determined with Ellman’s reagent in the presence of either sodium dodecyl sulfate, urea, or both (28).

Spectral analysis of essentially salt-free solutions of pepsin in- hibitors was done at pH 2.0 (in one case at pH 7.0) with a Cary model 14 recording spectrophotometer.

Amino Acid Analysis-The amino acid composition of the in- hibitors was determined on a Beckman-Spinco model 116 C amino acid analyzer following the method of Moore and Stein (29). The buffers were made in double-distilled water passed through a resin column (Hi-Rez ammonia filtration system with DC-3 resin sup- plied by Pierce Chemical Co., Rockford, Ill.).

Eight samples, two aliquots of each of the four solutions of the inhibitors, containing an amount of protein which inhibited 1210 rg of the same pepsin solution, were dried in ignition tubes. To one sample of each inhibitor was added 1.0 ml of 5.7 N HCl solu- tion (triple distilled). One milliliter of 2yo thioglycolic acid in 5.7 N HCl solution was added to each of the other four samples. Four additional samnles. one aliauot of each of the four inhibitor solut,ions, containing an amount of protein which inhibited 605 Gg of pepsin, were dried in ignition tubes and performic acid-oxidized. To each of these dried samules was added 1.0 ml of 5.7 N HCl. The 12 samples were sealed under vacuum and were placed in boiling toluene. Those samples which were performic acid- oxidized and the ones which did not have thiodveolic acid were hydrolyzed for 24 hours. The remaining samples were hydrolyzed for 84 hours. Thioglycolic acid was added to these samples (64- hour hydrolysis) to prevent the destruction of tryptophan by acid (30). HCl (5.7 N) and thioglycolic acid were removed on a Buchler evaporator. The residue was dissolved in ammonia-free water and evaporated to dryness four t,imes. The dried hydrolysates were dissolved in 500 ~1 of water and 10 11 of 5.7 N HCl were added. Two hundred microliters of the hydrolysate of Inhibitors I, II, and III. which would have inhibited 0.0142 umoles of nensin. were then applied to each column for amino acid analysis. ‘One hun- dred mikoliters of the hydrolysate of Inhibitor iv, which would have inhibited 0.0071 rtmoles of nensin. were analvzed. The dried residue from the performic acid:oxidizkd samples”was dissolved in 250 ~1 of water, 5 ~1 of 5.7 N HCl added, and a 200~~1 aliquot ana- lyzed on the long column.

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gel in the column (250 ml) and the concentration of the protein charged was 8 to 12 A oer ml. The column was operated by gravity flow at a rate of 180 ml per hour.

(Ihrom.atoaraohu on Cellex SE-Cellex SE was nrenared in 20 mM

Tris-HCl-0.i I& acetic acid at pH 3.65. A column (2.4 X 45 cm) was equilibrated and washed with this solution before it was used. The protein solution was adjusted to 0.5 A per ml in 20 mM Tris- HCl, pH 3.65, before it was charged onto the column. (When the solution was too dilute, it was concentrated on a UM-05 membrane under 40 p.s.i.)

Chromatography on DEAE-Sephadex A-25-Dry DEAE-Sepha- dex A-25 was soaked in 100 mM Tris-HCl buffer, pH 8.8, for 48 hours at room temperature. A column (0.9 X 60 cm) was packed and equilibrated with the same buffer before it was used. All columns were operated in a cold room at 7” unless otherwise indicated.

RESULTS AND DISCUSSION

Isolation of Pepsin Inhibitors

The possibility that the pepsin inhibitor was uniquely located in the body walls of Ascaris was considered, but no difference was found in the distribution of pepsin-inhibiting activity when the worm was subdivided into three equal parts (heads, middles, and tails). Therefore, entire Ascaris from which the urogenital tract had been dissected were used in the preparations.

Step 1. Ammonium Sulfate Fractionation-Fig. 1 summarizes the steps employed to obtain the ammonium sulfate fraction of the pepsin inhibitor. Special precautions must be taken in sep- arating the residue and the last 20 ml of the supernatant solution from the centrifugate at 59,000 x g. Careless decantation results in the addition of unwanted water-soluble materials which adversely affect the succeeding chromatography steps.

Attempts to extract or precipitate the inhibitors with trichloro- acetic acid solution (as was described for trypsin inhibitors by Collier (13) and by Rola and Pudles (14)) failed. Instead, it was discovered that trichloroacetic acid inactivates both pepsin and the inhibitors.

The inhibitors are not precipitated in 70% MgS04 or in 70% ethanol, and are poorly precipitated in 90 y. acetone.

Step 2. Chromatography on Bio-Gel P-SO-The ammonium sulfate fraction from Step 1 was applied to a Bio-Gel P-30 col- umn, as summarized in Fig. 2.

The inhibitors were found on the descending side of the major protein peak. Carbohydrate was separated from the major protein peak and this facilitated the pnrification of the inhibitors in the next step. After the P-30 column was used several times, the separation of the proteins became poor. This was due to the irreversible absorption of the protein onto the column, i.e. about 70% of the first sample of protein charged into a fresh column

ASCARIS BODY WALLS

I Centrduge 01 i9.000 x g for 3 hours

n I R&e

(discardI

QH 2.0

75 mlnules at 37O pti 5.5

. . Precipitate S”p&lGM

(dasolve in If,01 (dmrd)

FIG. 1. Initial steps in the isolation of pepsin inhibitors from Ascaris body walls. SAS, saturation with ammonium sulfate.

was retained on the gel. Attempts to regenerate the column by boiling the gel at pH 8.0 and at high salt concentrations and at high and low pH were unsuccessful. Therefore, pepsin-inhibit- ing fractions were collected from this column until the specific activity of the combined fractions reached a limiting specific activity of 40 to 50. These fractions were concentrated on the UM-05 membrane and rechromatographed on a previously un- used P-30 column (4.5 x 50 cm) under the same conditions for elution as the first column. This recycling step was effective, and a final recovery of better than 70% of the pepsin-inhibiting activity with a specific activity of the combined fractions of > 120 was obtained.

Step 3. Chromatography on Cellex-SE-Fractions of pepsin inhibitors from Step 2 were applied to a column of Cellex SE as summarized in Fig. 3. Seventy per cent of the pepsin-inhibiting activity was eluted in a small peak before 40% of the protein applied was eluted.

Step /t. Chromatography on DEAE-Sephadex-Pepsin-in- hibiting fractions from the Cellex column were concentrated, ad- justed to pH 8.8, and charged onto a DEAE-Sephadex column. Fig. 4 shows the elution pattern. Each of the four peaks con- tained pepsin inhibitors.

Peaks I to III have the same apparent specific activity (3020 to 3400)) but Peak IV appears to have half of the specific activity (1680) of the other peaks. The yield and specific activities of the fractions obtained in the purification are shown in Table I.

Polyacrylamide gel electrophoresis was performed on each pepsin-inhibiting fraction. Peaks I and IV appeared as single components and were easily separated by electrophoresis. Peaks II and III migrated as one component even in 5 and 10% gels in sodium dodecyl sulfate. When these four pepsin inhibitor peaks were mixed and electrophoresed, only three bands were obtained (Fig. 5).

Molecular Weights

Fig. 6 shows the relative mobility of pepsin Inhibitors I, II, and III in 5 and 10% polyacrylamide gel in sodium dodecyl sul- fate. (Inhibitor IV was unavailable at this time and was not run.) The corresponding molecular weight was calculated as

0’ FRACTION NUMBER

FIG. 2. Chromatography on Bio-Gel P-30 of Ascaris pepsin- inhibiting fraction precipitated between 0 and 0.65 saturation with ammonium sulfate. Column (100 X 10 cm) was equilibrated and eluted with 100 mM Tris-HCl solution at pH 2.1. Fractions, each 17 ml, were collected at a flow rate of 180 ml per hour. Left ordinate is per cent of charge of protein (-) or per cent of charge of units of pepsin inhibitor (Q- - -0). Right ordinate is per cent of charge of carbohydrate (- - -).

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60

-4

6C 0 125 130 203 210 247 250 2

FRACTION NUMBER

TABLE I Recovery of pepsin inhibitors from 6

Step of isolation Units of inhibitor

59,000 X g supernatant.. . Step 1 (ammonium sulfate frac-

tion)....................... Step 2 (Bio-Gel P-30). . Step 3 (Cellex SE). Step 4 (DEAE-Sephadex)

Peak I.. Peak II...................... Peak III..................... Peak IV. .

126,000

100,800 81,000 64,000

g,ooo 13,000 25,000 10,000

1561

I of Ascaris

Specific activity RKWXy

W&S/A protein

0.5

%

(100)

6 80 120 65

1600 50

3020 7 3409 10 3400 20 1680 7

FIG. 3. Chromatography on Cellex SE of Ascaris pepsin- inhibiting fraction from Bio-Gel P-30 column. Seventy A at 280 nm of total protein (= 10,000 units of inhibitors) were applied to a column (2.4 X 45 cm) which was equilibrated and eluted with 20 mM Tris-HCl-0.1 mM acetic acid solution at pH 3.65. At Frac- tion 30 elution was continued with 20 mM Tris-HCl-0.1 mM acetic acid, pH 4.6. At Fraction 125 a five-chamber gradient was used in which each of the first three chambers contained 300 ml of 20 mM Tris-HCl-0.1 mM acetic acid, pH 4.6, and the last two chambers contained 10 mM Tris-HCl-10 mM acetic acid, pH 5.0. Twelve- milliliter fractions were collected at a flow rate of 17 ml per hour. Ordinate is per cent of charge of protein (-) or per cent of charge of pepsin inhibitor (o- - -0).

/

0 230 260 290 330 360 390 420 460

FRACTION NUMBER

FIG. 4. Chromatography of Ascaris pepsin inhibitors on DEAE- Sephadex (A-25) after chromatography on Cellex SE. Inhibitor, 28.7 A (= 64,000 units of inhibitors), in 100 ml of 100 mM Tris-HCl buffer, pH 8.8, was applied to the column (0.9 X 60 cm) which was equilibrated with 100 mM Tris-HCl buffer, pH 8.8, at a rate of 14 ml per hour. After the protein solution passed into the gel, elu- tion was continued with the same buffer. As soon as the absorb- ance at 280 nm dropped to the base-line (Position A), the elution was continued with a five-chamber gradient system in which the chambers contained 300 ml of 100, 200, 300, 400, and 500 mM Tris- HCl buffer, pH 8.8, respectively. At Position B, elution was con- tinued with 500 mM Tris-HCl-10 mM KCl, pH 8.8. Ordinate is per cent of charge of protein (--) or per cent of charge of units of pepsin inhibitors (O- - -0).

A B C D FIG. 5. Polyacrylamide gel electrophoresis of Ascaris pepsin

inhibitors after chromatography on DEAE-Sephadex. Approxi- mately 50 pg of inhibitor were applied to 7.5oj, cross-linked gels at pH 9.05. Electrophoresis was performed at 5 ma per gel for 60 min in Tris-glycine buffer, pH 8.9 and 25”. The gels were stained in 1% aniline blue-black and destained in 7% acetic acid. Pro- tein bands migrated towards the anode. A is Peak I, B is Peaks II and III, C is Peak IV, and D is a mixture of Peaks I to IV.

15,800 to 16,800 on the 5% gel (Fig. 6A) and 14,200 on the 10% gel (Fig. 6B). The accuracy of the determination of the molecu- lar weight of a protein by this method was found to vary from ~1.5 to lt9.7% of the actual value (25). The proteins which

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RELATIVE MOBILITY RELATIVE MOBILITY

FIG. 6. Determination of the molecular weight of Ascaris pepsin inhibitors on 5y0 (A) and on 10% (B) polyacrylamide gel in sodium dodecyl sulfate at pH 7.2. All points represent the average of two experiments. The points represent the following proteins: 1, bovine serum albumin; 8, carboxypeptidase A; S, trypsin; 4, chymotrypsinogen A; 6, pepsin inhibitor (I, II, or III); 6, lyso- zyme; 7, cytochrome c. The points (5, 5) represent the limits of uncertainty of measurement of the distance the band of an in- hibitor migrated in the gel.

were chosen in this study were in this category. Therefore, we felt that an assignment of 15,500 (average of 14,200 and 16,800) for the molecular weight of the pepsin inhibitors (I, II, or III) could be within the mean of the experimental error.

Amino Acid Analysis

Table II shows the amino acid composition of each inhibitor. Half-cystine residues were assigned from the analysis for cysteic

acid following oxidation with performic acid. No free sulfhydryl groups were found when the inhibitors were tested with Ellman’s reagent even in the presence of urea and sodium dodecyl sulfate for 1 day. From this it was concluded that all of the half-cystine residues existed in the intact molecule of each inhibitor as di- sullide bonds. No hexosamines were detected when the long column run of the amino acid analyzer was extended.

The minimal molecular weights calculated from amino acid compositions of these inhibitors were 17,515 ( = 160 amino acid residues), 15,584 ( = 142 residues), 16,124 ( = 147 residues), and 31,719 (= 290 residues) for Inhibitors I, II, III, and IV, respec- tively. These values for Inhibitors I, II, and III were in agree- ment with the values obtained by the polyacrylamide gel method. The finding that Inhibitor IV had about twice the molecular weight of any of the other inhibitors was supported by the finding that Inhibitor IV had half of the specific activity of the other inhibitors. The inhibitor exhibited one band on polyacrylamide gel electrophoresis. The possibility that Inhibitor IV exists as a dimer with one active center is not likely because a number of the amino acid residues in this inhibitor are not double their number in any of the other inhibitors or any combination of two of the inhibitors. For example, Inhibitor IV has 31 alanine residues, while Inhibitors II and III each have 10 and Inhibitor I has 12 residues. Discrepancies can be found in comparing half- cystine, glycine, isoleucine, methionine, phenylalanine, and valine. The possibility that Inhibitor IV is a combination dimer of one of these three inhibitors and a fourth one, or a dimer of a fourth inhibitor, is not ruled out.

Since Inhibitors II and III could be separated (at least par- tially) by DEAELSephadex and not by disc gel electrophoresis,

it was speculated that these two inhibitors might differ in their amino acid composition by a minimum number of amino acid residues. The amino acid analysis of both inhibitors revealed that Inhibitor III might have 1 more residue of aspartic acid (or asparagine), proline, and valine, and 2 more residues of glutamic acid (or glutamine) than Inhibitor II. It is likely that the acidic residues were asparagine and glutamine because a separation on disc gel electrophoresis was not achieved. This led to the con- clusion that the separations of Inhibitors II and III on DEAE- Sephadex were due to stronger hydrophobic interaction between the DEAE-Sephadex matrix and Inhibitor III, which is richer in the hydrophobic amino acid residues than Inhibitor II.

Spectral Analysis

The spectra of solutions of the four inhibitors (at pH 2.0) are shown in Fig. 7. The fine structure of phenylalanine, the broad maximum of tryptophan (276 to 283 nm), and the shoulder at 290 nm in every spectrum indicate the presence of these two aromatic amino acids in each of the four inhibitors.

The spectrum of a mixture of cystine, phenylalanine, tyrosine, and tryptophan in the molar proportions 3: 10 : 1: 1 at pH 2.0 is shown in Fig. 70 for comparison with the spectra of the other four inhibitors. The maximum at 263 nm is missing in the spec- trum of each inhibitor. There are no differences in the spectra of these four Ascaris pepsin inhibitors. I f Inhibitor IV had 4 tyrosine residues and 1 tryptophan, we might observe a sharper maximum in the spectrum at 275 nm. This argument supports the assignment of 2 tryptophan residues to the molecule. Wet- laufer (32) pointed out that the validity of the assignment of the aromatic amino acids in a protein, particularly tryptophan and tyrosine, may be checked by the ratio of the observed molar absorption, e&s, and the calculated molar absorption, E,~I~, of the protein, which theoretically should be 1.00. The ratio of e&s:&& was computed from the observed molar absorption of the inhibitor at 280 nm and from its calculated molar absorp- tion (etryptoghan = 5550, etyro.ine = 1340, and ecystine = 150, all at pH 2). Table III shows the e&s : e&c ratios of each Ascaris pepsin inhibitor and of a number of proteins listed for comparison. The ratio of 1.00 supports the assignment of 2 tryptophan resi- dues per molecule of Inhibitor IV. The ratio 1.16 for Inhibitors II and III is within the normal value when compared to carbonic anhydrase (1.13) or to ribonuclease (1.13)) and less than that obtained for As-3-ketosteroid isomerase (1.37). The value 1.31 for Inhibitor I is still acceptable and is supported by the finding that this inhibitor exhibits a specific activity of 3020 compared to 3400 for either II or III.

NHz-terminal Analysis

The reaction of phenylisothiocyanate with each Ascaris pepsin inhibitor produced PTH-histidine. This indicated that the NHz-terminal amino acid in each inhibitor was histidine.

Stability of Pepsin Inhibitors

One of the characteristics which protease inhibitors exhibit is their resistance to the proteolytic action of other enzymes. Kas- sell and Laskowski (33,34) found that the basic trypsin inhibitor (bovine) was not inactivated by pepsin or by chymotrypsin. Chymotrypsin inhibitors from Ascuris also were not inactivated by either trypsin or pepsin (35). We examined this phenomenon with pepsin inhibitors from Ascaris. Fig. 8 shows the inactiva- tion of pepsin Inhibitor III by chymotrypsin and by trypsin (this also was true for Inhibitors I, II, and IV). When the en- zyme and the inhibitor were mixed in 1: 1 molar ratio at pH 7.5

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TABLE II Amino acid composition of As&a?% pepsin inhibitors

Integer values (unless otherwise indicated) are the averages obtained from the 24-hour hydrolysate and the 64-hour hydrolysate in the presence of 2% thioglycolic acid. The number of residues was assigned on the basis that the sample analyzed contained 0.0142 Foles of inhibitor (1 &mole of inhibitor reacts with 1 pmole of pepsin; see Table IV).

Amino

Acid

- II Inhibitor I i-

24 Hr

r

I:

Lysine 9.7

Histidine 3.1

Arginine 3.1

Aspartic Acid 14.5

Ttweonine 8.8

Serine 6.6

Glutamic Acid 20.6

Proline 15.8

Glycine 10.3

Alanine 12.0

Half-Cystine 5.4

Valine 11.4

Methionine 4.7

Isoleucine 4.0

Leucine 7.3

Tyrosine 1.0

Phenylalanine 11.8

Tryptophan --

64 Hr

Z

9.9

3.2

3.2

14.9

8.5

5.5

21.9

18.4

10.9

11.9

--

12.6

_-

4.0

7.8

1.2

12.3

0.8

[nte- 9er

Z

10

3

3

15

9=

7a

22b

18b

11

12

6'

1P

5d

4

8

1

12

lb

Total Residues 160

K Inhibitor I

24 Hr

Z

8.9

2.7

3.0

13.9

7.6

6.0

17.3

14.3

9.3

9.9

4.7

11.0

3.8

3.7

6.7

1.0

10.9

--

64 Hr

-i

8.6

2.7

2.6

13.3

6.6

4.5

16.4

16.8

8.6

9.6

__

10.2

--

3.2

6.5

1.0

10.0

0.7

r\te- 9er

E

9

3

3

14

8=

7=

17

17b

9

10

6'

11

4d

4

7

1

11

lb

- c

142

Inhibitor III

24 64 Hr Hr

8.0 9.0

2.5 3.0

2.5 2.8

14.7 14.4

8.1 7.5

5.4 4.9

17.1 19.2

13.8 la.5

10.0 9.7

9.9 9.8

4.9 --

11.6 11.4

3.9 __

3.2 3.5

6.8 6.8

0.9 1.1

10.5 11.4

-- 1.0

- :nte- 9er

E

9

3

3

15

8=

6a

19b

18b

10

10

6'

12

4d

4b

7

1

11

lb

-

147

T Inhibitor IV

24 Hr

!2.0

5.1

4.8

Z9.8

15.2

10.9

11.2

!5.2

17.0

H.2

6.6

17.0

5.7

10.0

15.2

2.2

18.1

--

64 Hr

E

21.4

5.1

4.7

29.2

14.5

9.2

42.6

26.8

17.3

31.1

_-

18.6

mm

10.2

15.8

3.5

la.1

1.1

i - nte- 9er

=

22

5

5

30

16a

12a

42

27b

17

31

a=

19b

6d

10

16

4b

18

2b

- c

290

Amnonia (19) (17) (17) (36)

Molecular Weight 17,515 15,584 16,124 31,719

a Obtained by extrapolation to zero time. ) 64-hour hydrolysate only. 0 As cysteic acid from a 24-hour hydrolysate following performic acid oxidation. d As methionine sulfone from a 24-hour hydrolysate following performic acid oxidation.

and 37” for I hour the inhibitor was almost 100% inactivated in the presence of chymotrypsin and nearly 70% inactivated by trypsin.

Interaction of Ascaris Pepsin Inhibitors with Bovine and Human Gastric Enzymes

Peanasky and Abu-Erreish (35) observed that trypsin inhibi- tors isolated from pork Ascaris were unable to inhibit human trypsin and they suggested that this observation could be re- lated to the survival of the adult Ascaris in its proper host. The species specificity of the pepsin inhibitors was therefore tested against bovine and human gastric enzymes.

Bovine pepsin was inhibited by each of the four Ascaris in-

hibitors, just like porcine pepsin. The inhibition of human and porcine pepsins by each of these Ascaris pepsin inhibitors is compared in Table IV. The inhibitors interact with porcine and with human pepsins in a stoichiometric (1 :l molar) ratio. A comparison of the inhibition of human and porcine gastricsin by these Ascaris pepsin inhibitors shows that porcine gastricsin is inhibited stoichiometrically while human gastricsin is not (Table IV). This is the second example of a species specificity which has been observed at the molecular level and which is in agreement with the host specificity of this parasite. Experi- ments are currently underway to relate these observations to an explanation of parasitism on a molecular basis.

Some other studies of the specificity of these pepsin inhibitors

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06

240 260 2ho 360 3io 240 260 2kO 360 320

06

B

s z 0.4

2

8

2

a

0.2 l!fzi.l 0.0 240 260 260 300 320

06

D

04

02

0.0

“;r- i- b

k

230 250 270 290 310 33c

WAVE LENGTH (nm)

0 60 120 180

TIME (min.) FIG. 8. Inactivation of Ascaris pepsin inhibitors with intestinal

proteinases. Pepsin inhibitor (9 X 10-4amoles) and enzyme (9 X lO+~~moles) were mixed in 200 mM Tris-HCI, pH 7.6, and incubated _ _

WAVE LENGTH (nm)

FIG. 7. Ultraviolet absorption spectra of Ascaris pepsin in- at 37”. One hundred-microliter aliquots were removed at the

hibitors. Spectra were scanned on a Cary model 14 Spectro- indicated times and the pH was adjusted to 2.0. Pepsin inhibitor

photometer at a speed of 0.25 nm per s. All solutions were meas- activity was determined against 6 pg of pepsin using the hemoglo-

ured through a l-cm light path and were in 10 mM HCl (pH 2.0) bin assay method. Pepsin inhibitor + chymotrypsin, A- - -h;

except C-a which was in 0.1 mM Tris-HCl, pH 7.0. A, inhibitor pepsin inhibitor + trypsin, O- - -0. I; B, inhibitor II or III; C-b, inhibitor IV; C-a, inhibitor IV (pH 7.0); D, solution of 6.3 X lob4 M cystine, 21 X 10-d M phenylalanine, 2.1 X lo-4 M tyrosine, and 2.1 X 1W4 M tryptophan, all at pH 2.0. TABLE IV

Comparison of inhibition of porcine and human

pepsins by Asearis pep& inhibitors@ - - TABLE III

Comparison of observed and calculated absorption at I. Ratio ena me inhl lted 4

to inhibitor added

Ascaris pepsin inhibitor

Inhibitor 1 EIYZ.pIltT added il nhibited* 880 nrn of Ascaris pepsin inhibitors and Enzyme

I

-

a number ’ OJ f other proteins”

Residues/mole 1 1

rw -

- :ys - .-

3 3 3 4

0 4

0

TY~ 1 I --- I I

1 1 9,650 7,34C 1 1 8,600 7,34c 1 1 8,600 7,34C 2 4 17,200 17,06C

6 8 49,60044,OoC 0 6 9,800 8,64C

0 10 18,300 13,4-M I I

I -

-

I

-

_-

I moles x 10"

0.80 1.44 1.03 1.60 0.80 1.44 1.96 1.60 0.80

12.70 1.60 1.73 1.35 1.78

‘“% x 0.81 1.40 1.00 1.61 0.80 1.34 1.73 1.37 0.27 0.40 0.07 1.51 1.46 1.48

Protein

Ascaris pepsin in- hibitor:

I . . . II. . . . , . III. . . . . . . IV

Carbonic anhy- drase. .

Ribonuclease.. . A6-3-Ketosteroid

isomerase.

Porcine pepsin (

-

I II

III IV I II

III IV I II + III

IV I II + III

IV

Human pepsin

Human gastricsin

Porcine gastricsin

1.00 0.97 0.97 1.00 1.00 0.93 0.88 0.86 0.32 0.03 0.04 0.87 1.08 0.83

~Pepsins, gastricsins, and inhibitors were allowed to react at pH 2.0 and 37” for 5 min. Free pepsin or gastricsin was estimated by the rate at which it hydrolyzed hemoglobin for 5 min.

b Molecular weights were taken as follows: porcine pepsin,

33,500; human pepsin, 34,000; porcine gastricsin, 32,500; human gastricsin, 31,500.

17,359 15,533 16,207 31,505

28,000 13,600

40,800

1.31 1.16 1.16 1.00

1.13 1.13

1.37

D From Wetlaufer (32).

have already appeared. Keilova and Tomalek (36) prepared a mixture of the four pepsin inhibitors according to an earlier report by us (35). This preparation inhibited cathepsin D but not cathepsin E or rennin, although all three of these proteinases have been classified as acidic proteinases. These observations show that the inhibitors from Ascaris exhibit a far greater speci- ficity than the fermentation product of actinomycetes, pepsta- tin, which inhibits all of the above named acid proteinases (37). The Ascaris pepsin inhibitors may prove to be useful tools in the characterization of acidic proteinases of tissues.

AcrEnowledgments-We thank Dr. Karl Wegner for the supply of outdated human blood and human gastric juice. We also thank Dr. Donald R. Babin at Creighton University for valuable help in establishing the technique of the Edman degradation. The cooperation of the Sioux Quality Packers Co., Sioux City,

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Iowa, and the John Morrell Co., Sioux Falls, S. D., is gratefully

acknowledged.

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Ghaleb M. Abu-Erreish and Robert J. PeanaskyAND SOME PROPERTIES

: ISOLATION, PURIFICATION,Ascaris lumbricoidesPepsin Inhibitors from

1974, 249:1558-1565.J. Biol. Chem. 

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