Isolation, Purification, and the Amino Acid Sequence of a Secretory Trypsin Inhibitor from the Chicken Pancreas1

Isolation, Purification, and the Amino Acid Sequence of a Secretory Trypsin Inhibitor from the Chicken Pancreas1 MERTON H. PUBOLS Department of Animal Sciences, Washington State University, Pullman, Washington 99164-6320

ABSTRACT A trypsin inhibitor is secreted in the pancreatic juice of the chick. Extracts from tissue have an inhibitor that corresponds to die secreted inhibitor on die basis of chromatography on DEAE-cellulose. The secretory inhibitor was purified by anion- and cation-exchange chromatography and by preparative isoelectric focusing. The purified inhibitor has 69 amino acids and is highly homologous with die secretory inhibitor from die turkey pancreas. (Key words: chicken, pancreas, secretory, trypsin, inhibitor) 1990 Poultry Science 69:640-646 INTRODUCTION

A trypsin inhibitor stored in the zymogen granules of the pancreas and released with the zymogens in the pancreatic juice is present in mammalian species, including cows (Greene et al., 1966), pigs (Tschesche and Wachter, 1970), sheep (Hochstrasser et al., 1969), and humans (Pubols et al., 1974; Bartelt et al., 1977). The turkey has a similar inhibitor (Bogard and Laskowski, 1979). These secretory trypsin inhibitors have been termed Kazal inhibitors, and they are distincdy different from the Kunitz inhibitor found in the pancreas and other tissues of ruminants (Kunitz and Northrup, 1936). These differences are discussed in a review by Laskowski and Kato (1980). The Kazal inhibitors from mammalian species have been studied extensively (see Laskowski et al., 1980, for a review). Although the sequence of the pancreatic secretory trypsin inhibitor from the turkey has been established (Laskowski et al., 1980), the corresponding inhibitor from the chicken has not been purified. The purpose of the present research was to purify the chicken pancreatic

Published as scientific paper Number 7714, College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA. Pharmica Fine Chemicals, Piscataway, NJ. 3 Whatman Lab Sales, Inc., Hillsboro, OR. 4 Lot Number TRL9FC, Worthington Biochemical Corp., Freehold, NJ. 'CalbiochemCorp., La Jolla, CA. ^ i g m a Chemical Co., St. Louis, MO.

secretory trypsin inhibitor and compare its sequence to that of other species.

MATERIALS AND METHODS

The chemicals were reagent grade or better. Sephadex,2 QAE(quaternary aminoethyl)Sephadex,2 SP(sulphopropyl)-Sephadex,x2 and DEAE (diethylaminoethyl)-cellulose (De-52),3 microgranular, were used. Trypsin was used for me inhibitor assay.4 Tosyl arginine methyl ester (TAME)5 and Diisopropyl fluorophosphate (DFP)6 were also used. Trypsin Assay The trypsin activity was measured by following the hydrolysis of tosyl-L-arginine methyl ester (TAME); 1) spectrophotometrically at 247 nm with concentrations of 1.0 mA/ TAME, .1 M Ca++, and .04 M Tris [tris(hydroxymethyl)aminomethane], at pH 8.1 (Schwert and Takenaka, 1955); or 2) titrimetrically in the presence of .01 M TAME (Greene et al., 1966). The trypsin activity was measured at 25 C and was expressed as the number of micromoles of TAME hydrolyzed per minute. Trypsin-Inhibitor Assay The trypsin inhibitor was measured by following die decreased rate of TAME hydroly-

640

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(Received for publication May 26, 1989)

PANCREATIC SECRETORY TRYPSIN INHIBITOR

Collection of the Pancreatic Juice The pancreatic juice was obtained by the cannulation of one of the pancreatic ducts (Dal Borgo et ai, 1968) from anesthetized, 3-wk-old chicks (Hubbard by White Leghorn cross). Since the chick has 3 pancreatic ducts, no compensation was made for the loss of pancreatic juice or of electrolytes. Between .5 and 5 mL of juice were collected per day from each chick. The juice was collected daily, frozen, and stored until assayed. The trypsin inhibitor in the juice was determined without further purification. Preparation of Acetone Powder

supernatant was frozen in trays and then lyophilized. About 35 g of dry powder were obtained from 400 g of tissue. Ammonium Sulfate Precipitation About 50 g of acetone powder (representing about 575 g of pancreas) were sprinkled slowly, with stirring, into 1,200 mL of 10"4 M DFP. The suspension was stirred for 30 min, then centrifuged at 16,700 x g. This supernatant was made .3 saturated by adding solid ( N H ^ S O ^ 175.7 g per L, in three portions with stirring. The suspension was stirred for 15 min, then centrifuged. The .3 supernatant was made .7 saturated by adding 269.9 g of (NH^SC^ per L. The pellet collected by centrifugation was resuspended in a minimal volume of 10 -4 M DFP, then frozen until the next step. All operations were at 0 to 4 C. Gel-Filtration Chromatography The .3 to .7 ammonium sulfate precipitates from three preparations (representing about 1,800 g of pancreas) were combined, dissolved in 900 mL of 10"4 M DFP, and applied to a column 7.6 by 120 cm of Sephadex G-50 (medium) that had been equilibrated with .1 M Tris and .5 M KC1 (pH 8.2). The column was developed at a rate of about 2 mL per min (60mL fractions) with the same buffer. The fractions were assayed for the trypsin inhibitor, and the fractions with inhibitor were combined and were lyophilized.

To obtain sufficient quantities of the secretory trypsin inhibitor for purification and subsequent amino-acid analysis, acetone powders were prepared. Pancreata were obtained from freshly slaughtered broilers.7 The pancreata were frozen immediately in a mixture of dry ice and acetone (-70 C) and was kept frozen until Desalting processed. Frozen pancreata (200 g) were The lyophilized powder from the gel-filtrahomogenized in 2,000 mL of cold acetone for 3 min at -15 C. The precipitate was collected on a tion step was dissolved in 400 mL of .05 M filter paper in a Buchner funnel and was dried NH4HCO3 and applied to a column (7.6 by 71.5 under vacuum at room temperature. The dried cm) of Sephadex G-25 (fine). The column was powder is stable indefinitely when stored at -20 developed with the same buffer at a rate of 7.9 C. The powder from two batches (equivalent to mL per min (60-mL fractions). The fractions 400 g of tissue) was homogenized for 20 s in a having inhibitor were combined and lyophilized. blender at slow speed with 400 mL of 10~* M DFP to a smooth suspension. This suspension Ion-Exchange Chromatography was diluted to a final volume of 1,200 mL with 4 10" M DFP, stirred for 20 min at 4 C and then The details of the procedures for anion- and centrifuged for 30 min at 16,700 x g (4 C).8 The cation-exchange chromatography are described in the legends with the figures. In each of these steps, the inhibitor collected from several previous columns was combined for the subsePederson Farms, Inc., Tacoma, WA. 8 Model RC-2, GSA head, Sorvall Centrifuge, DuPont quent purification. All columns were developed at 4 C. Co., Wilmington, DE.

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sis, either spectrophotometrically or titrimetrically, in the presence of a trypsin standard. The mixture of trypsin and trypsin inhibitor was allowed to react for 1 min at room temperature (23 C) before the assay. The amount of inhibitor required to reduce by 50% the rate of TAME hydrolysis with a standard solution of trypsin was used for assays (Greene et al., 1966). A unit of trypsin inhibitor activity is defined as the reduction in micromoles of TAME hydrolyzed by trypsin in the presence of the inhibitor. The specific activity of the trypsin inhibitor is defined as the units of inhibitor activity per absorbance at 280 nm (A2so).

641

642

PUBOLS

TABLE 1. Purification steps and specific activity of the trypsin inhibitor isolated from the chick pancreas Specific activity (inhibitor units per absorbance at 280 nm)

Procedure

1 2 3 4

Acetone powder, supernatant Ammonium-sulfate precipitate, 30 to 70% saturated Gel-filtration, Sephadex G-50 Cation-exchange chromatography, diethylaminoethyl-cellulose, pH 9.0 [tris(hydroxymethyl)aminomethane] Cation-exchange chromatography, QAE(quaternary aminoethyl)-Sephadex, pH 7.5, ammonium acetate Anion-exchange chromatography, SP(sulphopropyl)-Sephadex, pH 4.0, ammonium acetate Cation-exchange chromatography, QAE-Sephadex, pH 7.5, ammonium acetate Preparative isoelectric focusing

5 6 7 8

.06 .2 2.5 30.9 459 1,280 1,611 2,170

'Pancreas, 7 u.g of inhibitor per g (fresh weight).

Isoelectric Focusing Preparative isoelectric focusing was done on the inhibitor from an anion (QAE-Sephadex) chromatographic column after several purifications. The sample was applied to a 100-mL, water-cooled column; for 20 h, 800 V were applied with an ampholyte mixture having a range of pH 4 to 6. Fractions 47 to 50 were combined and desalted on a G-25 Sephadex column (2 by 47 cm), equilibrated with .05 M ammonium bicarbonate. This inhibitor preparation was lyophilized before submission to the amino-acid analysis. Sequence Determination The purified inhibitor from the isoelectricfocusing step was analyzed for its amino-acid sequence by the Bioanalytical Center at Washington State University, Pullman. A protein sequencer 9 with a liquid-pulse update was used, according to protocols supplied by the manufacturer. A cyanogen-bromide cleavage at the methionines was used to obtain smaller peptides, which enabled a determination of the complete sequence. RESULTS AND DISCUSSION

The secretory-trypsin inhibitor was found in the pancreatic juice collected by cannulation.

'Model 470A, Applied Biosystems, Foster City, CA.

The average value was 13.4 inhibitor units per mL of juice. The pancreatic juice ranged between .2 and 4% protein. The pancreatic secretory-trypsin inhibitor elutes from the G-50 Sephadex column following the major peak of zymogens (Figure 1). Subsequent purification on DEAE-cellulose (Figure 2), QAE-Sephadex at pH 7.5 (Figure 3), SP-Sephadex at pH 4.0 (Figure 4), and again with QAE-Sephadex at pH 7.5 (Figure 5) provided a nearly pure preparation, having a specific activity of 1,611 inhibitor units per A280- The final purification was achieved by preparative isoelectric focusing (Figure 6). The purification steps are summarized in Table 1. The specific activity of the purified inhibitor was 2,170 units per A280. The concentration of the secretory-trypsin inhibitor was about 7 ng per g of fresh pancreas, which is about 1.5% of the trypsinogen present (unpublished data). Several minor peaks of inhibitor activity were detected (Figures 2, 3, 4, and 6). Whether these are artifacts of preparation or are endogenous inhibitors is not known. The secretory-trypsin inhibitor from the chicken pancreas has 69 amino acids; the one from the turkey has 72 (Laskowski et al., 1980). There are minor differences in sequence between the avian species and even greater differences between these and the inhibitors from the mammalian species, all of which have 56 amino acids and a high degree of homology (Bartelt et al., 1977; Laskowski et al., 1980). The amino-acid sequence of the secretorytrypsin inhibitor from the chicken pancreas is

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Step

643

PANCREATIC SECRETORY TRYPSIN INHIBITOR

l

\

- 1.4

- 1.2

- .6

- .4

20

40

60

80

100

120

o 20 O

1/ 140

160

FRACTION NUMBER (50 mL/tube)

10

60

80

100

120

140

FRACTION NUMBER (57 mL/tube)

FIGURE 1. Gel-filtration chromatography of the trypsin inhibitor by Sephadex G-50 (7.6 by 120 cm in length) using .5 M KC1, .025 M Tris[tris(hydroxymethyl)aminomethane], pH 8.2. The column was developed at 4 C. The inhibitor in Fractions 36 to 46 was combined and lyophilized.

shown in Figure 7. For comparison, the sequence of the secretory inhibitor from the turkey pancreas is also presented (Laskowski et al, 1980). The amino acids at Positions 45 and 48 could not be determined clearly. Cysteine is a good possibility for Position 45, but the signal was not distinct. Inexplicably, no single amino acid increased in concentration for Position 48. The sequences of these two peptides were highly homologous, differing only at Asp7, He19, Val 30 , Gly39, and Met62 of the chicken. There are differences in amide groups at Positions 35, 50, and 54. No amino acid(s) beyond Arg 69 could be detected. The sequence surrounding the reactive site, at Lys27 of the turkey, was identical for both species. A comparison of the amino-acid sequence surrounding the reactive site is shown in Figure 8 for the turkey (Laskowski et al, 1980), the human (Bartelt et al, 1977), the porcine I (Tschesche and Wachter, 1970), the porcine II (Schneider et al., 1973), the bovine

FIGURE 2. Anion-exchange chromatography of the secretory trypsin inhibitor by DEAE (diethylaminoethyl)cellulose (7.6 cm by 7 cm in length) using .025 M iris [tris(hydroxymethyl)aminomethane], pH 9.0. The inhibitor for this step was obtained by combining the pools from two gel-filtration columns. A gradient was started at Fraction 6 by adding 4,000 mL of eluting buffer (.08 M KC1, .025 M Tris, pH 9.0) to 4,000 mL of equilibration buffer (.025 M Tris, pH 9.0). Fractions 80 to 100, having the most abundant inhibitor, were combined and were lyophilized.

(Greene and Bartelt, 1969), the ovine (Hochstrasser et al, 1969) and the canine (Kikuchi et al, 1985) animals. Presumably, the secretory-trypsin inhibitor of the pancreas has a protective role in preventing a premature activation of trypsin in the zymogen granules. The secretory-trypsin inhibitor of the pancreas specifically inhibits trypsin but not trypsinogen, other serine proteinases secreted in the pancreatic juice, and enterokinase. The relatively low concentration of inhibitor, 1.5% of the potential trypsin, would not prevent normal activation of the zymogens but could scavenge the trypsin generated by a premature activation of trypsinogen. Avian species also secrete trypsin inhibitors into the egg white, the ovoinhibitor, and the ovomucoid inhibitor. Both (classed as Kazal inhibitors) are glycoproteins and are larger than the secretory inhibitorof the pancreas, but differ from each other (Laskowski et al, 1980; Laskowski and Kato, 1980; Kato et al, 1987; Laskowski et al, 1987). The physiological

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- .6

644

PUBOLS

160

200

240

280

320

FIGURE 3. Anion-exchange chromatography of the chicken pancreatic secretory trypsin inhibitor by QAESephadex (2 by 22 cm in length). The inhibitor pools from several diethylaminoethyl-cellulose columns were combined, desalted by a G-25 Sephadex column, and dissolved in the equilibration buffer. The column was equilibrated with .02 M ammonium acetate, pH 7.5. A gradient was started at Fraction 31 by adding 500 mL of .5 M ammonium acetate (pH 7.5) to 500 mL of the equilibration buffer. The inhibitor in Fractions 96 to 108 was pooled and were lyophilized.

20

40

60

80

100

120

140

40

80

120

160

200

F R A C T I O N NUMBER (2.0

240

280

320

mL/tube)

FIGURE 5. Anion-exchange chromatography of the major pool of the inhibitor from the SP(sulphopropyl)Sephadex column, performed by another column QAE(quaternary aminoethyl)-Sephadex (1 by 9 cm) that had been equilibrated with .02 Af ammonium acetate, pH 7.5. A gradient was formed by adding 250 mL of .5 M ammonium acetate (pH 7.5) to 250 mL of the equilibration buffer, starting at Fraction 20. Fractions 48 to 62 were combined and lyophilized.

16C

FRACTION NUMBER (2.4 mL/tube)

FIGURE 4. Cation-exchange chromatography of the secretory-trypsin inhibitor by SP(sulphopropyl)-Sephadex (1 by 10 cm length), equilibrated with .1 M ammonium acetate, pH 4.0. The sample from the previous anion column [QAE(quaternary aminoethyl)-Sephadex] (Figure 3) was diluted with the equilibration buffer. A gradient was started at Fraction 70 by adding 250 mL of .1 Af ammonium acetate (pH 6.0) to 250 mL of the equilibration buffer. The major peak of the inhibitor in Fractions 162 to 179 was combined and adjusted to pH 7.5 with dilute ammonium hydroxide.

FRACTION NUMBER, (1.0 mL/tube)

FIGURE 6. Preparative isoelectric focusing of the inhibitor from the previous (Figure 5) anion chromatographic column [QAE(quaternary aminoethyl)-Sephadex].

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120

FRACTION NUMBER, ( 4 mL/tube)

645

PANCREATIC SECRETORY TRYPSIN INHIBITOR

Turkey

Asn

- Ala

- Glu

- Pro

- Asp

- Gly

- Ala

- Ala

- Asn

Gln° 10 Gin

Chicken

Gly

- Thr

- Glu

- Ala

- Ala

- Cys

- Gly

- Asn

- Tyr

Asp

Turkey

Gly

- Thr

-

Glu

- Ala

- Ala

- Cys

- Gly

- Asn

- Tyr

20 Asp'

Chicken

He

- Arg

-

Lys -- Gly ••

Thr -- Lys

- Asn

- Phe

Asp

Turkey

Val

- Arg

- Lys •- Gly -- Cys •- Thr

Lys

• Asn

- Phe

Asp

Chicken

Pro

- Val

- Cys

•

Gly -- Thr

- Asp

- Asn

-' Val

- Leu

Tyr

Turkey

Pro

-

- Cys

- Gly •- Thr

- Asp

- Asp

- Val

- Leu

Tyr

Chicken

Gly •- Asn -- Glu -- Cys -•

Val

- Gin

Turkey

Ser ••

Cys

- Val

- Gin

Chicken

Met

- Glu

- Arg

•

His -- Thr -- Asp

• Val

- Arg

-

He

Turkey

Met

- Gin

- Arg

- His •- Thr -- Asn

- Val

- Arg

-

He

Chicken

Asn

- Arg

- Gly

- Met

- Glu

- Pro

- Ser

Pro

Turkey

Asn

- Arg

•

Cys -- Gin •• Glu

- Pro

• Ser

Pro

Chicken

Arg

Turkey

Arg

Chicken

Glu •- Pro ••

Asn

- Glu -•

••

Cys -•

Leu -•

Gly

•

- Ala

- Ala

- Asp

•

Leu -

Cys -- Leu -- Leu -•

- Cys -•

Gly •- Lys ••

Gin

18

28 30 38 40

Asn

50 58

60 Lys' 68 70

- Ser

FIGURE 7. The amino-acid sequence of the secretory-trypsin inhibitors from pancreas of the chicken and the turkey. The arrow after Lys*27 of the turkey indicates the reactive site.

Gly - Cys23

Thr

- Lys -- Asn

- Phe

- Asp -- Pro •- Val . .

Turkey . . .

Gly - Cys25

Thr

- Lys •- Asn

- Phe

- Asp

- Pro

-

Human. . . .

Gly - Cys 1

Thr

- Lys

He

- Tyr

- Asn

- Pro

- Val . .

Pro

- Lys -

He

- Tyr

- Asn •- Pro

- Val . .

Pro

- Lys -

He

- Tyr

- Asn -- Pro

- Val . .

Pro

- Arg -•

He

- Tyr

- Asn •- Pro

- Val . .

.

-

He . .

Bovine . . .

. Gly - Cys16 • Gly - Cys12 Gly - Cys16

Ovine. . . .

Gly - Cys16

Pro

- Arg •-

He

- Tyr

- Asn

- Pro

- Val . .

Canine . . .

. Gly - Cys17

Asn

- Lys •

He

- Tyr

- Asn

- Pro

-

Porcine I. . Porcine II .

He . .

H G U R E 88. The HGURE The amino-acid sequence near the reactive site of the secretory-trypsin inhibitors from the pancreas of amir several species. The reactive site for each species is indicated by the arrow following Lys 25 of the chicken.

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He

Asp

646

PUBOLS

function of these inhibitors in the egg white is not known. ACKNOWLEDGMENTS

REFERENCES Bartelt, D. C , R. Shapanka, and L. J. Greene, 1977. The primary structure of the human pancreatic secretory trypsin inhibitor. Arch. Biochem. Biophys. 179: 189-199. Bogard, W. C , Jr., and M. Laskowski, Jr., 1979. Turkey pancreatic secretory trypsin inhibitor. Fed. Proc. 38: 836. (Abstr.) Dal Borgo, G., P. C. Harrison, and J. McGinnis, 1968. A method for cannulation of pancreatic ducts in young chicks. Poultry Sci. 47:1818-1820. Greene, L. J., and D. C. Bartelt, 1969. The structure of the bovine pancreatic secretory trypsin inhibitor-Kazal's inhibitor. J. Biol. Chem. 244:2646-2657. Greene, L. J., M. Rigbi, and D. S. Fackre, 1966. Trypsin inhibitor from bovine pancreatic juice. J. Biol. Chem. 241:5610-5618. Hochstrasser, K., W. Schramm, H. Fritz, S. Schwartz, and E. Werle, 1969. Zur Primarstruktur des spezifischen Trypsininhibitors aus Schafpancreas. Hoppe-Seylers Z. Physiol. Chem. 350:893-896.

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The author appreciates the assistance of the following individuals: John Pyne for cannulating the pancreatic ducts; Beverly Conway and Loyd Falen for trypsin-inhibitor assays; Charlie Butts and Douglas McFarland for collecting tissue and preparing acetone powders; and Gerhard Munske for interpreting the sequence data.

Kato, I., J. Schrode, W. J. Kohr, and M. Laskowski, Jr., 1987. Chicken ovomucoid: Determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains. Biochemistry 26:193-201. Kikuchi, N., K. Nagata, N. Yoshida, T. Tanaka, M. Yamamoto, and Y. Saitoh, 1985. Purification and complete amino acid sequence of canine pancreatic secretory trypsin inhibitor. FEBS Lett. 191:269-272. Kunitz, M , and J. H. Northrup, 1936. Isolation from beef pancreas of a crystalline trypsinogen, trypsin, a trypsin inhibitor, and an inhibitor-trypsin compound. J. Gen. Physiol. 19:991-1007. Laskowski, M., Jr., and I. Kato, 1980. Protein inhibitors of proteinases. Annu. Rev. Biochem. 49:593-626. Laskowski M , Jr., I. Kato, W. Ardelt, J. Cook, A. Denton, M. W. Empie, W. J. Kohr, S. J. Park, K. Parks, B. L. Schatzley, O. L. Schoenberger, M. Tashiro, G. Vichot, H. E. Whatley, A. Wieczorek, and M. Wieczorek, 1987. Ovomucoid third domains from 100 avian species: Isolation, sequences, and hypervariability of enzyme-inhibitor contact residues. Biochemistry 26: 202-221. Laskowski, M , Jr., I. Kato, W. J. Kohr, C. J. March, and W. C. Bogard, 1980. Evolution of the family of serine proteinase inhibitors homologous to pancreatic secretory trypsin inhibitor (Kazal). Protides Biol. Fluids Proc. Colloq. 28:123-128. Pubols, M. H., D. C. Bartelt, and L. J. Greene, 1974. Trypsin inhibitor from human pancreas and pancreatic juice. J. Biol. Chem. 249:2235-2242. Schneider, S. L., L. Stasiuk, and M. Laskowski, Sr., 1973. Sequence of tryptic cleavages in porcine pancreatic secretory inhibitor II. J. Biol. Chem. 248:7207-7214. Schwert, G. W., and Y. Takenaka, 1955. A spectrophotometric determination of trypsin and chymotrypsin. Biochim. Biophys. Acta 16:570-575. Tschesche, H., and E. Wachter, 1970. The structure of the porcine pancreatic secretory trypsin inhibitor I. Eur. J. Biochem. 16:187-198.