[72]
H U M A NPANCREATIC SECRETORY TRYPSIN INHIBITOR
813
taining 67 amino acid residues 12 (Table I I ) and comprising a sugar moiety of more than 30% of its molecular weight. 2° The sugar is composed of fucose, mannose, galactose, glucose, galactosamine, glucosamine, and sialic acid. 2° Hog colostrum trypsin inhibitor has a similar composition. 1° As far as the multiple forms of both the cow and hog inhibitors are concerned, some variations could be shown in the lysine ~,26,27 and threonine ~ contents, and different forms containing 1, 2, or 3 lysine residues could be isolated. Differences in content of other amino acids ~s,27 are also likely. The p r i m a r y structures of two inhibitor forms have alr e a d y been determined, 12,28 differing only by two amino acids: replacement of threonine in position 3 by lysine. ~8 There is a 40% homology in the p r i m a r y structure of the colostrum inhibitor with t h a t of the basic pancreatic trypsin inhibitor, 12 and a comparison of the two structures is given in Fig. 2. The disulfide bonds in the molecule of cow colostrum trypsin inhibitor are shown in Fig. 3; their positions are identical with the disulfides in the basic pancreatic trypsin inhibitor, s 26U. Kucich, Ph.D. Thesis, State University of New York at Buffalo, 1972. 27M. Laskowski, Sr., personal communication. ~8V. Jon~kov~. and D. ~echov£, Collect. Czech. Chem. Commun., in press.
[72] H u m a n
Pancreatic
Secretory Trypsin
Inhibitor 1
B y LEWIS J. GREENE, MERTON H. PUBOLS,2 and DIANA C. BARTELT
Pancreatic juice, the exocrine secretion of pancreas, contains a polypeptide trypsin inhibitor 3-s in addition to hydrolytic enzymes and inactive enzyme precursors (zymogens). The pancreatic secretory trypsin inhibitor ( P S T I ) prevents the premature trypsin-catalyzed activation of zymogens within the pancreas and the pancreatic duct. In the small in1Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission. ~Visiting Biochemist at Brookhaven National Laboratory 1968 to 1969, supported by the National Institutes of Health Special Fellowship GM-40285-01. 3M. H. Kalser and M. Grossman, Gastroenterology 29, 35 (1955) 4B. J. Haverback, B. Dyce, H. Bundy, and H. A. Edmondson, Am. J. Med. 29, 424 (1960). 5L. J. Greene, M. Rigbi, and D. S. Fackre, J. Biol. Chem. 241, 5610 (1966). 6H. Fritz, F. Woitinas, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 345, 168 (1966). 7L. J. Greene, J. J. I)iCarlo, A. J. Sussman, and D. C. Bartelt, J. Biol. Chem. 243, 1804 (1968). 8 M. H. Pubols, D. C. Bartelt, and L. J. Greene, J. Biol. Chem. 249, 2235 (1974).
814
NATURALLY OCCURRING PROTEASE INHIBITORS
[72]
testine, enterokinase initiates the activation of trypsinogen, which is responsible for the activation of all other zymogens. Enterokinase is not inhibited by P S T I 2 K a z a l et al. '° isolated the first pancreatic secretory trypsin inhibitor ll from bovine pancreas in 1948. I t could be distinguished from the bovine t r y p s i n - k a l l i k r e i n inhibitor (Kunitz inhibitor) 1~ on the basis of its inability to inhibit bovine chymotrypsin or porcine pancreatic kallikrein and because the inhibition is t e m p o r a r y in the presence of excess trypsin. Bovine and porcine P S T I , 13 as well as the bovine t r y p s i n - k a l l i krein inhibitor, 1~ have been described in this treatise. More recent information m a y be found in review articles 1~,16 and reports of conferences. 1ms The trypsin inhibitor activity present in human pancreas and p a n creatic juice was first demonstrated by H a v e r b a c k et al. ~ Partially purified inhibitor was later identified as K a z a l - t y p e on the basis of its inhibitory properties2 ,'J,2° This assignment is independently indicated by the amino acid sequence of the h u m a n inhibitor, which is homologous to the pancreatic secretory trypsin inhibitors of bovine, porcine, and ovine origin. A comparison of these structures is included in this article. The procedure s detailed here for the isolation of multiple chromatographic forms of h u m a n P S T I utilizes inactive pancreatic juice or tissue as the starting material. The inhibitor is not exposed to proteolytic enzymes and is isolated by gel filtration and ion exchange c h r o m a t o g r a p h y in the free form (not complexed with trypsin). The use of trypsin-affinity c h r o m a t o g r a p h y for the isolation of inhibitor from autolyzed h u m a n pancreas has recently been reported? t S. Maroux, J. Baratti, and P. Desnuelle, J. Biol. Chem. 246, 5031 (1971). ~°L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948). ~lPancreatic secretory trypsin inhibitors are also referred to in the literature as "Kazal4ype" inhibitors or specific inhibitors from pancreatic juice. 1~M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). 13p. j. Burck, this series Vol. 19 [67]. ~4B. Kassell, this series Vol. 19 [66b]. ~5M. Laskowski, Jr., and R. W Sealock, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. III, p. 375. Academic Press, New York, 1971. 1~H. Tschesche, Angew. Chem. 13, 10 (1974). 17"Proteinase Inhibitors, Proceedings of the First International Research Conference (Bayer Symposium V) Munich, 1970. de Gruyter, Berlin, 1971. 18Proteinase Inhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973. Springer-Verlag, Berlin and New York, 1974. ~ H. Fritz, I. ttiiller, M. Wiedemann, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 348, 405 (1967). ~°P. J. Keller and B. J. Allan, J. Biol. Chem. 242, 281 (1967). 2~G. Feinstein, R. ttofstein, J Koifmann, and M. Sokolovsky, Eur. J. Biochem. 43, 569 (1974).
[72]
HUMAN
PANCREATIC
SECRETORY
TRYPSIN
INHIBITOR
815
Assay Method
The inhibition of the trypsin hydrolysis of p-toluenesulfonyl-Larginine methyl ester (TAME) is used as the quantitative assay for the trypsin inhibitor. The pH-stat method is recommended because it can be used to assay trypsin inhibitor in tissue extracts where nucleotide absorption interferes with the spectrophotometric determination. The assay is carried out with a final substrate concentration of 0.008 M T A M E in 0.005 M Tris-HC1 buffer, 0.1 M KC1, 0.02 M CaC12 at pH 7.8, 250. 5 Crystalline bovine trypsin (Worthington Biochemical Co., Freehold, New Jersey) is used. A description of the procedure used in our laboratory has appeared in a previous volume of this treatise. 1~ Definition of Unit and Specific Activity. One inhibition unit is the a m o u n t of inhibition t h a t has caused the reduction of T A M E hydrolysis by 1 tLmole/min. Specific activity is defined as inhibitor units per a~ 280 l°m• Preparation of H u m a n PSTI s
Individual samples of pancreatic juice and tissue are initially screened for trypsin and trypsin inhibitor activity to ensure that no tryptic activation has occurred. This precaution is taken so that the inhibitor can be separated in the "free" form from trypsin, trypsinogen, or inhibitor-trypsin complex by gel filtration on Sephadex G-75. The inhibitor is then purified and separated into five chromatographic forms by gradient elution chromatography on DEAE-cellulose and SP-Sephadex. Pancreatic tissue is subjected to a preliminary fractionation by ammonium sulfate precipitation and then processed in the same manner as the pancreatic juice. Step la. Collection of Pancreatic Juice. H u m a n pancreatic juice is collected by catheterization of the pancreatic duct after related surgical procedures. 2-0,23 The pancreatic juice is frozen as soon as possible after collection. Trypsin activity is determined by the pH-stat method on an aliquot of juice containing 250-500 t~g protein. Samples that do not have demonstrable trypsin activity and contain inhibitor activity are used. Pancreatic juice was stored at --22 ° after lyophilization. Step lb. Extraction o] PSTI ]rom Pancreas. Postmortem human pancreas from approximately 100 individuals who did not have diagnosed pancreatic disease were stored at --22 ° for up to 12 months. Minced tissue in 50-100 g portions is suspended in 5 volumes (w/v) of chilled 0.1 mM diisopropyl phosphofluoridate ( D F P ) and homogenized in a ~"A. Morgan, L. A. Robinson, and T. T. White, Am. J. Surg. 115, 131 (1968). 23C. Figarella and T. Robeiro, Scan~d.J. Gastroenterol. 6, 133 (1971).
816
NATURALLY OCCURRING PROTEASE INHIBITORS
[72]
Waring blender for 2 min at maximum speed. The suspension is adjusted to pH 4.5 with 6 N HC10~ and centrifuged at 13,000 g for 45 min at 5 ° in a Sorvall GSA rotor. If the pH of the tissue suspension is brought to pH 3 or less, large losses of trypsin inhibitor activity occur. The sediment is reextracted, and the combined supernatants are brought to 70% saturation by the slow addition of 472 g of solid ammonium sulfate per liter of supernatant at 5 °. After centrifugation at 13,000 g, 5 °, for 45 min the supernatant is discarded. The precipitate is resuspended in 3 volumes of 0.1 mM DFP and stored at --22 °. Each preparation is assayed for trypsin and inhibitor activity before being combined with others for gel filtration on Sephadex G-75. The DFP present does not inhibit the trypsin under the conditions of the assay. Step 2. Gel Filtration on Sephadex G-75. Two 7.6 X 180 cm columns are prepared from 1.5 kg of Sephadex G-75 which has been swelled overnight in 50 liters of 50% acetic acid. The fines are removed by suction provided by a water pump. The acetic acid is replaced with distilled water and then by the eluting buffer (0.5 M KCI, 0.01 M Tris-HC1, pH 8.1, and 0.1 mM DFP) by settling, followed by decantation with suction. Equilibration of the gel is completed by storage at 4 ° for 4-5 days. Two centimeters of glass beads are overlaid on top of the sintered-glass disks fitted at the bottom of each column. The gel suspension is deaerated at 4 ° with a water pump before the columns are poured in sections. Thirty liters of buffer are passed through each column before the columns are connected in series. The flow rate is limited to 120 ml/hr by a peristaltic pump attached to the outlet of the second column. Effluent is collected in 60-ml fractions. The columns are operated in a cold room at 2o-4 °. Lyophilized human pancreatic juice is suspended in 200-400 ml of cold distilled water at 4 ° to give a final protein concentration of 1-2%. One milliliter is removed for the determination of inhibitor activity later to be used for the calculation of inhibitor recovery. The solution is then made 1 mM with respect to D F P by the addition of 0.1 M D F P (in isopropyl alcohol) and held at 4 ° for 2 hr with stirring before gel filtration on Sephadex G-75 (Fig. 1). The inhibitor activity, corresponding to a molecular weight of ~6000, is retarded relative to most of the secretory protein. Amylase activity is anomalously eluted after the inhibitor in the effluent corresponding to fractions 240-270. Effluent with inhibitorspecific activity greater than 12 (fractions 193-203, indicated by the solid bar) is combined and lyophilized. This fraction is denoted "low molecular weight fraction." Step 3. Gel Filtration on Sephadex G-25. Sephadex G-25 is swelled and degassed in 10 volumes of distilled wafer'by placing the beaker in a boiling water bath for 2 hr. After settling, the supernatant and fines are
[72]
HUMAN
PANCREATIC
SECRETORY
TRYPSIN
,
i
817
INHIBITOR Z
~1
,,
,
I
,
,
,I
f
,
I,,
,
,,
,
i
,,
,
i )F-
A
I 1.5
OA
z~
I
Oo~
-,5.0 E~
E c
rno
o 1.0 GO o,J
i,1 0
zO.5
< cn nO cD
5.0 E o
E ::k
~0.1 60
I00 140 180 220 260 FRACTION NUMBER, 60 ml/FRACTION
300
FIG. 1. Gel filtration of human pancreatic juice on Sephadex G-75. Sample, 34 A~so 1cm × 190 ml. @ - - - - 0 , Absorbance at 280 nm; O - - - - O , trypsin inhibition. TAME, p-toluenesulfonyl-L-arginine methyl ester. From M. H. Pubols, D. C. Bartelt, and L. J. Greene, J. Biol. Chem. 249, 2235 (1974). decanted by suction. The eluting buffer (0.05 M ammonium bicarbonate, pH 8.1) is then added to the swelled gel at 4 or 5 times the settled gel volume. The Sephadex is gently stirred and allowed to settle; and fines are removed by suction. Buffer addition, gel settling, and decantation are repeated three times. The equilibrated gel is kept at 20-4 ° overnight before packing the columns as described for Sephadex G-75. The lyophilized low-molecular-weight fraction from the Sephadex G-75 column (Fig. 1) is dissolved in 400 ml of 0.1 m M D F P and desalted on a Sephadex G-25 (medium) column, 7.6 }( 72 cm, equilibrated and developed with 0.05 M ammonium bicarbonate buffer, pH 8.1, containing 0.1 m M D F P at 200 ml/hr, 4 °. Fractions of 30 ml are collected. The effluent is monitored by measurement of absorbance at 280 nm, trypsin-inhibitor activity, and effluent conductivity. Fractions with inhibitor specific activity greater than 20 are pooled and lyophilized. Because the inhibitor activity is partially included within the matrix of Sephadex G-25, this preparation is then resubmitted to gel filtration on two columns (1.8 X 160 cm) of Sephadex G-25 (Superfine) connected in series. The columns are equilibrated and developed with 0.05 M ammonium bicarbonate, pH 8.1, at 15 ml/hr. Effluent with a specific activity greater than 90 is combined and lyophilized twice to remove the volatile buffer.
818
N A T U R A L L Y OCCURRING P R O T E A S E I N H I B I T O R S
[72]
Step 4. Chromatography on DEAE-Cellulose. To prepare resin for two 1.8 }( 45 cm columns, 120 g DE-52 resin (Whatman) is suspended in 2 liters of 0.280 Tris-HCl buffer, pH 9.0 (ten times the concentration of the starting elution buffer). After the suspension has settled for 10 rain, the supernatant is removed by suction. The resin is resuspended with one additional liter of buffer, and the pH of the slurry is adjusted, if necessary. The resin suspension is degassed with a water pump for 30 rain and stored at 2o-4 °. The column is poured in the cold room at 20-4 ° using a final slurry volume which is 1.5 times of the wet settled volume. The column is then equilibrated with the starting elution buffer (0.028 M Tris-HC1, pH 9.0) by passing 2 liters or more of buffer through the column, until the effluent pH and conductivity are exactly the same as that of the starting elution buffer. The lyophilized fraction containing inhibitor activity derived from the Sephadex G-25 column is suspended in 25 ml of 0.014 M Tris-HC1 buffer, pH 9.0. The pH is corrected to pH 9.0, and the conductivity is reduced, if necessary, by dilution to a value below that of the starting elution buffer. After sample application, the column is operated with starting elution buffer at 40 ml/hr with a piston pump until 1320 ml have passed through the column (tube 66, indicated by the arrow in Fig. 2). A linear gradient is then applied to the column. It is prepared from 2 liters each of equilibrating buffer and 0.028 M Tris-HC1 buffer, 0.2 M potassium chloride, pH 9.0, using mixing chambers of the same cross sectional area. 2~ The inhibitor activity from both pancreatic juice (Fig. 2, top) and tissue (Fig. 2, bottom) are each resolved into two chromatographic components, A and B, which are eluted with the same effluent conductivity and present in similar proportions. The effluent corresponding to each peak of inhibitor activity is combined, lyophilized, desalted by gel filtration on Sephadex G-25 (Superfine), 1.8 by 72 cm, and developed with 0.05 M ammonium bicarbonate buffer, pH 8.1, at 4 °. After gel filtration, tile active material, located by inhibitor activity and absorbance at 280 nm, is pooled and lyophilized. Step 5. Gradient Elution Chromatography on SP-Sephadex. To prepare resin for two 0.9 X 70 cm columns, 15 g of SP-Sephadex G-25 are suspended in 1 liter of 0.5 M acetic acid overnight, and fine particles are removed by settling several times in distilled water using suction to remove the supernatant. The resin is equilibrated by suspending it in 1 liter of 0.1 M ammonium acetate, pH 4.5 (0.1 M in acetic acid) overnight. The supernatant is removed and the equilibration process is repeated three times. The column is poured at 4 ° with glass beads overlaying the sintered-glass disks at the bottom of the column. ~ R. M. B o c k a n d N.-S. Ling, Anal. Chem. 26, 1546 (1954).
[721
HUMAN
I
0.4
PANCREATIC
I
SECRETORY
I
'
I
TRYPSIN
i
PANCREATIC JUICE B
819
INHIBITOR
'
Z
6 ~o - 2 0 0
>_aD.-
0.5
2
150 ~
0.2
2 o
IOOz~
0. I
0 ~
O~
'~
LIJ (.9
z
H U M A NPANCREATIC SECRETORY TRYPSIN INHIBITOR
813
taining 67 amino acid residues 12 (Table I I ) and comprising a sugar moiety of more than 30% of its molecular weight. 2° The sugar is composed of fucose, mannose, galactose, glucose, galactosamine, glucosamine, and sialic acid. 2° Hog colostrum trypsin inhibitor has a similar composition. 1° As far as the multiple forms of both the cow and hog inhibitors are concerned, some variations could be shown in the lysine ~,26,27 and threonine ~ contents, and different forms containing 1, 2, or 3 lysine residues could be isolated. Differences in content of other amino acids ~s,27 are also likely. The p r i m a r y structures of two inhibitor forms have alr e a d y been determined, 12,28 differing only by two amino acids: replacement of threonine in position 3 by lysine. ~8 There is a 40% homology in the p r i m a r y structure of the colostrum inhibitor with t h a t of the basic pancreatic trypsin inhibitor, 12 and a comparison of the two structures is given in Fig. 2. The disulfide bonds in the molecule of cow colostrum trypsin inhibitor are shown in Fig. 3; their positions are identical with the disulfides in the basic pancreatic trypsin inhibitor, s 26U. Kucich, Ph.D. Thesis, State University of New York at Buffalo, 1972. 27M. Laskowski, Sr., personal communication. ~8V. Jon~kov~. and D. ~echov£, Collect. Czech. Chem. Commun., in press.
[72] H u m a n
Pancreatic
Secretory Trypsin
Inhibitor 1
B y LEWIS J. GREENE, MERTON H. PUBOLS,2 and DIANA C. BARTELT
Pancreatic juice, the exocrine secretion of pancreas, contains a polypeptide trypsin inhibitor 3-s in addition to hydrolytic enzymes and inactive enzyme precursors (zymogens). The pancreatic secretory trypsin inhibitor ( P S T I ) prevents the premature trypsin-catalyzed activation of zymogens within the pancreas and the pancreatic duct. In the small in1Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission. ~Visiting Biochemist at Brookhaven National Laboratory 1968 to 1969, supported by the National Institutes of Health Special Fellowship GM-40285-01. 3M. H. Kalser and M. Grossman, Gastroenterology 29, 35 (1955) 4B. J. Haverback, B. Dyce, H. Bundy, and H. A. Edmondson, Am. J. Med. 29, 424 (1960). 5L. J. Greene, M. Rigbi, and D. S. Fackre, J. Biol. Chem. 241, 5610 (1966). 6H. Fritz, F. Woitinas, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 345, 168 (1966). 7L. J. Greene, J. J. I)iCarlo, A. J. Sussman, and D. C. Bartelt, J. Biol. Chem. 243, 1804 (1968). 8 M. H. Pubols, D. C. Bartelt, and L. J. Greene, J. Biol. Chem. 249, 2235 (1974).
814
NATURALLY OCCURRING PROTEASE INHIBITORS
[72]
testine, enterokinase initiates the activation of trypsinogen, which is responsible for the activation of all other zymogens. Enterokinase is not inhibited by P S T I 2 K a z a l et al. '° isolated the first pancreatic secretory trypsin inhibitor ll from bovine pancreas in 1948. I t could be distinguished from the bovine t r y p s i n - k a l l i k r e i n inhibitor (Kunitz inhibitor) 1~ on the basis of its inability to inhibit bovine chymotrypsin or porcine pancreatic kallikrein and because the inhibition is t e m p o r a r y in the presence of excess trypsin. Bovine and porcine P S T I , 13 as well as the bovine t r y p s i n - k a l l i krein inhibitor, 1~ have been described in this treatise. More recent information m a y be found in review articles 1~,16 and reports of conferences. 1ms The trypsin inhibitor activity present in human pancreas and p a n creatic juice was first demonstrated by H a v e r b a c k et al. ~ Partially purified inhibitor was later identified as K a z a l - t y p e on the basis of its inhibitory properties2 ,'J,2° This assignment is independently indicated by the amino acid sequence of the h u m a n inhibitor, which is homologous to the pancreatic secretory trypsin inhibitors of bovine, porcine, and ovine origin. A comparison of these structures is included in this article. The procedure s detailed here for the isolation of multiple chromatographic forms of h u m a n P S T I utilizes inactive pancreatic juice or tissue as the starting material. The inhibitor is not exposed to proteolytic enzymes and is isolated by gel filtration and ion exchange c h r o m a t o g r a p h y in the free form (not complexed with trypsin). The use of trypsin-affinity c h r o m a t o g r a p h y for the isolation of inhibitor from autolyzed h u m a n pancreas has recently been reported? t S. Maroux, J. Baratti, and P. Desnuelle, J. Biol. Chem. 246, 5031 (1971). ~°L. A. Kazal, D. S. Spicer, and R. A. Brahinsky, J. Am. Chem. Soc. 70, 3034 (1948). ~lPancreatic secretory trypsin inhibitors are also referred to in the literature as "Kazal4ype" inhibitors or specific inhibitors from pancreatic juice. 1~M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 991 (1936). 13p. j. Burck, this series Vol. 19 [67]. ~4B. Kassell, this series Vol. 19 [66b]. ~5M. Laskowski, Jr., and R. W Sealock, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. III, p. 375. Academic Press, New York, 1971. 1~H. Tschesche, Angew. Chem. 13, 10 (1974). 17"Proteinase Inhibitors, Proceedings of the First International Research Conference (Bayer Symposium V) Munich, 1970. de Gruyter, Berlin, 1971. 18Proteinase Inhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973. Springer-Verlag, Berlin and New York, 1974. ~ H. Fritz, I. ttiiller, M. Wiedemann, and E. Werle, Hoppe-Seyler's Z. Physiol. Chem. 348, 405 (1967). ~°P. J. Keller and B. J. Allan, J. Biol. Chem. 242, 281 (1967). 2~G. Feinstein, R. ttofstein, J Koifmann, and M. Sokolovsky, Eur. J. Biochem. 43, 569 (1974).
[72]
HUMAN
PANCREATIC
SECRETORY
TRYPSIN
INHIBITOR
815
Assay Method
The inhibition of the trypsin hydrolysis of p-toluenesulfonyl-Larginine methyl ester (TAME) is used as the quantitative assay for the trypsin inhibitor. The pH-stat method is recommended because it can be used to assay trypsin inhibitor in tissue extracts where nucleotide absorption interferes with the spectrophotometric determination. The assay is carried out with a final substrate concentration of 0.008 M T A M E in 0.005 M Tris-HC1 buffer, 0.1 M KC1, 0.02 M CaC12 at pH 7.8, 250. 5 Crystalline bovine trypsin (Worthington Biochemical Co., Freehold, New Jersey) is used. A description of the procedure used in our laboratory has appeared in a previous volume of this treatise. 1~ Definition of Unit and Specific Activity. One inhibition unit is the a m o u n t of inhibition t h a t has caused the reduction of T A M E hydrolysis by 1 tLmole/min. Specific activity is defined as inhibitor units per a~ 280 l°m• Preparation of H u m a n PSTI s
Individual samples of pancreatic juice and tissue are initially screened for trypsin and trypsin inhibitor activity to ensure that no tryptic activation has occurred. This precaution is taken so that the inhibitor can be separated in the "free" form from trypsin, trypsinogen, or inhibitor-trypsin complex by gel filtration on Sephadex G-75. The inhibitor is then purified and separated into five chromatographic forms by gradient elution chromatography on DEAE-cellulose and SP-Sephadex. Pancreatic tissue is subjected to a preliminary fractionation by ammonium sulfate precipitation and then processed in the same manner as the pancreatic juice. Step la. Collection of Pancreatic Juice. H u m a n pancreatic juice is collected by catheterization of the pancreatic duct after related surgical procedures. 2-0,23 The pancreatic juice is frozen as soon as possible after collection. Trypsin activity is determined by the pH-stat method on an aliquot of juice containing 250-500 t~g protein. Samples that do not have demonstrable trypsin activity and contain inhibitor activity are used. Pancreatic juice was stored at --22 ° after lyophilization. Step lb. Extraction o] PSTI ]rom Pancreas. Postmortem human pancreas from approximately 100 individuals who did not have diagnosed pancreatic disease were stored at --22 ° for up to 12 months. Minced tissue in 50-100 g portions is suspended in 5 volumes (w/v) of chilled 0.1 mM diisopropyl phosphofluoridate ( D F P ) and homogenized in a ~"A. Morgan, L. A. Robinson, and T. T. White, Am. J. Surg. 115, 131 (1968). 23C. Figarella and T. Robeiro, Scan~d.J. Gastroenterol. 6, 133 (1971).
816
NATURALLY OCCURRING PROTEASE INHIBITORS
[72]
Waring blender for 2 min at maximum speed. The suspension is adjusted to pH 4.5 with 6 N HC10~ and centrifuged at 13,000 g for 45 min at 5 ° in a Sorvall GSA rotor. If the pH of the tissue suspension is brought to pH 3 or less, large losses of trypsin inhibitor activity occur. The sediment is reextracted, and the combined supernatants are brought to 70% saturation by the slow addition of 472 g of solid ammonium sulfate per liter of supernatant at 5 °. After centrifugation at 13,000 g, 5 °, for 45 min the supernatant is discarded. The precipitate is resuspended in 3 volumes of 0.1 mM DFP and stored at --22 °. Each preparation is assayed for trypsin and inhibitor activity before being combined with others for gel filtration on Sephadex G-75. The DFP present does not inhibit the trypsin under the conditions of the assay. Step 2. Gel Filtration on Sephadex G-75. Two 7.6 X 180 cm columns are prepared from 1.5 kg of Sephadex G-75 which has been swelled overnight in 50 liters of 50% acetic acid. The fines are removed by suction provided by a water pump. The acetic acid is replaced with distilled water and then by the eluting buffer (0.5 M KCI, 0.01 M Tris-HC1, pH 8.1, and 0.1 mM DFP) by settling, followed by decantation with suction. Equilibration of the gel is completed by storage at 4 ° for 4-5 days. Two centimeters of glass beads are overlaid on top of the sintered-glass disks fitted at the bottom of each column. The gel suspension is deaerated at 4 ° with a water pump before the columns are poured in sections. Thirty liters of buffer are passed through each column before the columns are connected in series. The flow rate is limited to 120 ml/hr by a peristaltic pump attached to the outlet of the second column. Effluent is collected in 60-ml fractions. The columns are operated in a cold room at 2o-4 °. Lyophilized human pancreatic juice is suspended in 200-400 ml of cold distilled water at 4 ° to give a final protein concentration of 1-2%. One milliliter is removed for the determination of inhibitor activity later to be used for the calculation of inhibitor recovery. The solution is then made 1 mM with respect to D F P by the addition of 0.1 M D F P (in isopropyl alcohol) and held at 4 ° for 2 hr with stirring before gel filtration on Sephadex G-75 (Fig. 1). The inhibitor activity, corresponding to a molecular weight of ~6000, is retarded relative to most of the secretory protein. Amylase activity is anomalously eluted after the inhibitor in the effluent corresponding to fractions 240-270. Effluent with inhibitorspecific activity greater than 12 (fractions 193-203, indicated by the solid bar) is combined and lyophilized. This fraction is denoted "low molecular weight fraction." Step 3. Gel Filtration on Sephadex G-25. Sephadex G-25 is swelled and degassed in 10 volumes of distilled wafer'by placing the beaker in a boiling water bath for 2 hr. After settling, the supernatant and fines are
[72]
HUMAN
PANCREATIC
SECRETORY
TRYPSIN
,
i
817
INHIBITOR Z
~1
,,
,
I
,
,
,I
f
,
I,,
,
,,
,
i
,,
,
i )F-
A
I 1.5
OA
z~
I
Oo~
-,5.0 E~
E c
rno
o 1.0 GO o,J
i,1 0
zO.5
< cn nO cD
5.0 E o
E ::k
~0.1 60
I00 140 180 220 260 FRACTION NUMBER, 60 ml/FRACTION
300
FIG. 1. Gel filtration of human pancreatic juice on Sephadex G-75. Sample, 34 A~so 1cm × 190 ml. @ - - - - 0 , Absorbance at 280 nm; O - - - - O , trypsin inhibition. TAME, p-toluenesulfonyl-L-arginine methyl ester. From M. H. Pubols, D. C. Bartelt, and L. J. Greene, J. Biol. Chem. 249, 2235 (1974). decanted by suction. The eluting buffer (0.05 M ammonium bicarbonate, pH 8.1) is then added to the swelled gel at 4 or 5 times the settled gel volume. The Sephadex is gently stirred and allowed to settle; and fines are removed by suction. Buffer addition, gel settling, and decantation are repeated three times. The equilibrated gel is kept at 20-4 ° overnight before packing the columns as described for Sephadex G-75. The lyophilized low-molecular-weight fraction from the Sephadex G-75 column (Fig. 1) is dissolved in 400 ml of 0.1 m M D F P and desalted on a Sephadex G-25 (medium) column, 7.6 }( 72 cm, equilibrated and developed with 0.05 M ammonium bicarbonate buffer, pH 8.1, containing 0.1 m M D F P at 200 ml/hr, 4 °. Fractions of 30 ml are collected. The effluent is monitored by measurement of absorbance at 280 nm, trypsin-inhibitor activity, and effluent conductivity. Fractions with inhibitor specific activity greater than 20 are pooled and lyophilized. Because the inhibitor activity is partially included within the matrix of Sephadex G-25, this preparation is then resubmitted to gel filtration on two columns (1.8 X 160 cm) of Sephadex G-25 (Superfine) connected in series. The columns are equilibrated and developed with 0.05 M ammonium bicarbonate, pH 8.1, at 15 ml/hr. Effluent with a specific activity greater than 90 is combined and lyophilized twice to remove the volatile buffer.
818
N A T U R A L L Y OCCURRING P R O T E A S E I N H I B I T O R S
[72]
Step 4. Chromatography on DEAE-Cellulose. To prepare resin for two 1.8 }( 45 cm columns, 120 g DE-52 resin (Whatman) is suspended in 2 liters of 0.280 Tris-HCl buffer, pH 9.0 (ten times the concentration of the starting elution buffer). After the suspension has settled for 10 rain, the supernatant is removed by suction. The resin is resuspended with one additional liter of buffer, and the pH of the slurry is adjusted, if necessary. The resin suspension is degassed with a water pump for 30 rain and stored at 2o-4 °. The column is poured in the cold room at 20-4 ° using a final slurry volume which is 1.5 times of the wet settled volume. The column is then equilibrated with the starting elution buffer (0.028 M Tris-HC1, pH 9.0) by passing 2 liters or more of buffer through the column, until the effluent pH and conductivity are exactly the same as that of the starting elution buffer. The lyophilized fraction containing inhibitor activity derived from the Sephadex G-25 column is suspended in 25 ml of 0.014 M Tris-HC1 buffer, pH 9.0. The pH is corrected to pH 9.0, and the conductivity is reduced, if necessary, by dilution to a value below that of the starting elution buffer. After sample application, the column is operated with starting elution buffer at 40 ml/hr with a piston pump until 1320 ml have passed through the column (tube 66, indicated by the arrow in Fig. 2). A linear gradient is then applied to the column. It is prepared from 2 liters each of equilibrating buffer and 0.028 M Tris-HC1 buffer, 0.2 M potassium chloride, pH 9.0, using mixing chambers of the same cross sectional area. 2~ The inhibitor activity from both pancreatic juice (Fig. 2, top) and tissue (Fig. 2, bottom) are each resolved into two chromatographic components, A and B, which are eluted with the same effluent conductivity and present in similar proportions. The effluent corresponding to each peak of inhibitor activity is combined, lyophilized, desalted by gel filtration on Sephadex G-25 (Superfine), 1.8 by 72 cm, and developed with 0.05 M ammonium bicarbonate buffer, pH 8.1, at 4 °. After gel filtration, tile active material, located by inhibitor activity and absorbance at 280 nm, is pooled and lyophilized. Step 5. Gradient Elution Chromatography on SP-Sephadex. To prepare resin for two 0.9 X 70 cm columns, 15 g of SP-Sephadex G-25 are suspended in 1 liter of 0.5 M acetic acid overnight, and fine particles are removed by settling several times in distilled water using suction to remove the supernatant. The resin is equilibrated by suspending it in 1 liter of 0.1 M ammonium acetate, pH 4.5 (0.1 M in acetic acid) overnight. The supernatant is removed and the equilibration process is repeated three times. The column is poured at 4 ° with glass beads overlaying the sintered-glass disks at the bottom of the column. ~ R. M. B o c k a n d N.-S. Ling, Anal. Chem. 26, 1546 (1954).
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