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Isolation and characterization of the proteolytic enzyme component from commercially available crude trypsins
ANALYTICAL
BIOCHEMISTRY
84, 205-217 (1978)
Isolation and Characterization of the Proteolytic Enzyme Component from Commercially Available Crude Trypsins DAVID Department
of Biochemistry
W. SPEICHER AND RICHARD and Biophysics, The Pennsylvania Park, Pennsylvania 16802
L. MCCARL State
University,
University
Received April 28, 1977; accepted July 29, 1977 A purification procedure for the isolation of a mixture of the major proteolytic pancreatic enzymes (trypsin, chymotrypsin, and elastase) from commercial crude trypsins is described. These enzymes are apparently the enzymes responsible for tissue dispersal in numerous cell culture systems. Materials toxic to cell cultures, present in certain crude trypsin samples, are removed during a purification involving centrifugation, dialysis, treatment with a cellulose ion-exchange resin, removal of salts, and lyophilization. While the fundamental use of this proteolytic mixture would be to prepare primary cell culture, the broad peptide bond specificity of this mixture would suggest application in cases where a general protease, free of other enzymatic activities, is required.
Previous work in our laboratory showed that trypsin, chymotrypsin, and elastase were the enzymes in crude trypsin samples responsible for disaggregation of neonatal rat heart tissue. It was observed that most commercial samples of trypsin contained these three enzymes at concentrations sufficient to disaggregate heart tissue. However, different samples of crude trypsin, even different lots of the same type of trypsin, contained varying amounts of unidentified toxic components which caused marked variability in effectiveness of tissue dispersal and cell viability. This variability could be minimized by using a mixture of trypsin, chymotrypsin, and elastase in concentrations comparable to those found in crude trypsin samples (1). Other researchers also have reported that the ability of crude trypsins to disperse a variety of tissues was due at least partially to enzymes other than trypsin (2-4). The enzymes involved in tissue dispersal have been characterized in some cell culture systems (23-8). Therefore, an enzyme preparation containing trypsin, chymotrypsin, and elastase is needed for rat heart cell culture as well as for certain other culture systems. The cost of purified individual enzymes makes mixing of the purified enzymes to obtain the desired activities impractical for routine, large-scale cell culture work. We report herein the facile isola205
0003.2697/78/0841-0205$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved
206
SPEICHER
AND
McCARL
tion of the desired enzymes, which provides a reproducible and lowcost means of disaggregating tissues for cell culture. The broad peptide bond specificity of this mixture of proteolytic enzymes would suggest applications in cases other than cell culture where a general protease, free of other enzymatic activities, is required. MATERIALS
AND METHODS
Materials
DEAE-cellulose (DE 52) and CM-cellulose (CM 52) were obtained from Whatman Inc., Clifton, N.J. Hollow fiber dialyzers were obtained from Bio-Rad Laboratories, Richmond, Calif. Conductivity was determined using a YSI Model 33 S-C-T meter, Yellow Springs Instruments, Yellow Springs, Ohio. Substrates for trypsin, chymotrypsin, and elastase activity assays and purified enzyme samples for electrophoresis were obtained from Sigma Chemical Co., St. Louis, MO. The lipase substrate (N-methyl indoxyl myristate) was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. Brilliant blue R-250 dye was obtained from Sigma Chemical Co., St. Louis, MO. Acrylamide, N,N’-methylenebisacrylamide (bis), and N,N,N’,N’-tetramethylenediamine (TEMED) were purchased from Bio-Rad Laboratories, Richmond, Calif. A number of crude enzymes were used for enzyme purification. They include several types of samples from Sigma Chemical Co., St. Louis, MO., which offers pancreatic enzyme samples in several stages of purity, and two samples from Nutritional Biochemicals Corporation, Cleveland, Ohio, commonly used for tissue culture work. The samples from Sigma Chemical Co. were: crude trypsin Type II, Lot 71C-2440 (crude trypsin A) and Lot 104C-0008 (crude trypsi‘n B); cY-amylase Type VI-A, Lot 71C0210 (crude amylase); and pancreatin Grade VI, Lot 71C-0670 (pancreatin A), Lot 112C-2780 (pancreatin B), and Lot 123C-1720 (pancreatin C). The two samples from Nutritional Biochemicals Corporation were trypsin 1:300, Control No. 5176 (trypsin 1:300), and trypsin 1:250, Control No. 6220 (trypsin 1:250). The abbreviated names in parentheses will be used to refer to the respective enzyme samples. Initial
Purification
of Crude Enzyme
All purification procedures, pH readings, and conductivity readings were performed at 2°C unless otherwise stated. Crude extract was prepared by adding buffer (100 mM glycine- 1 mM CaCl, titrated to pH 8.90 with 1 N NaOH) to the crude enzyme sample to give a final concentration of 50 mg (by weight)/ml. This mixture was stirred for 30 min, adjusted to pH 8.90 with 1 N NaOH, and centrifuged at 12,000g for 15 min. The pellet was discarded and the supematant solution was
PROTEASE
ENZYMES
OF CRUDE
TRYPSIN
207
dialyzed exhaustively against buffer using a Bio-Fiber dialyzer (nominal MW cutoff of 5000 daltons). A peristaltic pump was used to circulate buffer and crude extract through the respective chambers of the hollow fiber dialyzer. The buffer was replaced with fresh buffer when the conductivity of the dialysate approached that of the protein solution. During dialysis the sample solution was concentrated to half the original volume by maintaining positive pressure on the effluent tubing of the dialyzer. Dialysis was considered complete when a conductivity of 600 pmhos or less for the protein solution was obtained (starting buffer conductivity was approximately 400 pmhos). The sample solution was centrifuged at 12,000g for 15 min, and the supematant solution was retained for ion-exchange treatment. Analytical
DEAE-Ceffufose
Cofumn
Chromatography
Crude enzyme was purified as described above and then applied to a DEAE-cellulose column (1 x 100 cm) equilibrated with the buffer used above (100 mM glycine-1 mM CaCl,, pH 8.90). Buffer was used for the initial elution, followed by a linear salt gradient from 0 to 0.5 M NaCl in buffer, and then elution was completed with buffer containing 1 M NaCl. The column was regenerated with equilibration buffer. Samples were collected in lo-ml fractions and analyzed for enzyme activity. Prepurative
DEAE-Cellulose
Column
Chromatography
The above analytical procedure was modified to obtain gram quantities of purified enzymes for routine cell dissociation. Crude enzymes (50 to 200g of starting material) were purified as described in Initial Purification of Crude Enzyme and then applied to a DEAE-cellulose column (5.0 x 60 cm) equilibrated with buffer (100 mM glycine-1 mM CaCl,, pH 8.90). The first protein peak was eluted with buffer, collected, dialyzed against a 1: 100 dilution of buffer, concentrated to 10 mg of protein/ ml (OD,,, of a 10 mg/ml solution = 12.6), and lyophilized. The column was regenerated by washing with buffer containing 1 M NaCl followed by reequilibration with buffer containing no NaCl. Preparative
DEAE-Cellulose
Batch Puri$cation
Purification of enzyme samples by DEAE-cellulose batch purification was similar to the preparative column procedure except the sample, which was prepared as described in Initial Purification of Crude Enzyme, was added to degassed, preequilibrated DEAE-cellulose in a beaker and stirred at 2°C for 30 min. This mixture was filtered on a Btichner funnel, the filtrate was dialyzed against a 1: 100 dilution of buffer, concentrated to 10 mg of protein/ml (ODZB, = 12.6), and lyophilized. The ion-exchange resin
208 was regenerated by reequilibration
SPEICHER
AND
McCARL
by washing with buffer containing with buffer containing no NaCI.
1 M NaCl followed
Preparative CM-Cellulose Batch Purification Purification of enzyme samples by CM-cellulose batch purification was similar to the DEAE-cellulose batch purification except the buffer (100 mM glycine-1 mM CaCl,) was adjusted to pH 7.80 instead of pH 8.90, and CM-cellulose was substituted for DEAE-cellulose. Impurities were eluted with four washes of buffer (each wash was four times the settled resin volume), and subsequently the desired enzymes were released from the ion-exchange resin with two washes of buffer containing 2 M NaCl (each wash was equal to the settled resin volume). This latter solution was then dialyzed against a 1:lOO dilution of the pH 8.90 buffer without NaCl, concentrated to a protein concentration of 10 m&ml (OD,,, = 12.6), and lyophilized. Assay of Enzyme Activity Trypsin activity was determined by the method of Erlanger et al. (9), using N-benzoyl-arginyl-p-nitroanilide (bapna) as the substrate. One unit of activity was defined as the amount of enzyme required to hydrolyze 1 pmol of substrate in 1 min under the conditions of the assay (pH 8.2, 37°C). Chymotrypsin activity was determined by the method of Hummel (IO), using N-benzoyk-tyrosine ethyl ester (btee) as the substrate. One unit of activity was defined as the amount of enzyme required to hydrolyze 1 pmol of substrate in 1 min under the conditions of the assay (pH 7.8, 25°C). Elastase activity was determined by the method of Bieth and Meyer (11) using acetyl-L-alanyl-L-alanyl-L-alanine methyl ester as the substrate. One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 pmol of substrate in 1 min under the conditions of the assay (pH 7.0, 25°C). Amylase activity was assayed by the method of Bernfeld (12) using soluble potato starch as the substrate. A unit of activity was defined as the amount of enzyme required to liberate 1 pmol of reducing groups, calculated as maltose, per minute under the conditions of the assay (pH 6.9, 25°C). Lipase activity was measured by the method of Guilbault et al. (13) using Nmethyl indoxyl myristate as the substrate with modifications described previously (1). One unit of activity was defined as the amount of enzyme required to hydrolyze 1 pmol of substrate under the conditions of the assay (pH 7.8, 25°C). Gel Electrophoresis Electrophoresis was performed at 15°C on 10% acrylamide gels at pH 7.6 using glass tubes (5 by 130 mm). After sealing one end of the gel tubes
PROTEASE
ENZYMES
OF CRUDE
TRYPSIN
209
and clamping them in a vertical rack, gel solution was added to a height of 5 cm. A small amount of water was layered on top of the gel solution. Polymerization was complete in less than 30 min at room temperature. The water was then removed and sample was added. A j-cm gel was then formed above the sample and, after polymerization was complete, the gels were run at a constant 150 v for 3.5 hr. The gel composition was a modification of that given by Maize1 (14). Ten milliliters of gel was prepared from 3.33 ml of a 30% solution of acrylamide with 0.8% Bis as the cross-linker, 1.50 ml 0.66 M Tris-HCl buffer, pH 7.6, 5 ~1 of TEMED, 5.14 ml of HzO, and 30 ~1 of 10% ammonium persulfate. The electrode buffer was 0.1 M Tris-HCl, pH 7.6. Sample buffer contained 1.0 ml of 0.66 M Tris-HCl buffer, pH 7.6, 3 ml of glycerol, and 6 ml of H,O. Gels were stained with 200 mg of Coomassie blue (brilliant blue R-250) dye dissolved in 50 ml of methanol, 10 ml of glacial acetic acid, and 40 ml of H,O and destained in a solution of 70 ml of glacial acetic acid, 200 ml of methanol, and 730 ml of H,O. RESULTS
Chromatography
of Crude Trypsin on DEAE-Cellulose
In preliminary experiments several samples of crude trypsin were chromatographed on a DEAE-cellulose column using the procedure of Zelikson et al. (15). Using these conditions with crude porcine trypsin, the first protein peak contained trypsin, elastase, chymotrypsin, and amylase activities. In subsequent experiments the chromatography conditions were modified by raising the pH to 8.90 and changing the buffer to 100 mM glycine-1 mM CaCl,. The proteolytic enzymes (trypsin, elastase, and chymotrypsin) were found in the first protein peak (Fig. 1) and were clearly separated from amylase and lipase. The second protein peak contained only small amounts of chymotrypsin and amylase activities. Incubation of the crude trypsin sample at room temperature either for 1 hr or overnight at 2°C before column chromatography resulted in a decrease in the first protein peak and an increase in the second protein peak. More than 50% of the 280-nm-absorbing material in the second protein peak passed through a Bio-fiber 50 dialyzer (nominal MW cutoff of 5000 daltons). These results suggest the second peak was mostly inactive hydrolysis products of the proteins eluted in the first peak. Protein eluted by the salt gradient contained a mixture of amylase, lipase, and chymotrypsin (Figs. lb-d) and other proteins (detected by disc gel electrophoresis). A preparative DEAE-cellulose column purification was used to produce gram quantities of various column fractions. The protein elution profile for the purification of 5Og of pancreatin II is given in Fig. 2. The first pro-
210
SPEICHER AND McCARL 3.0
s N I. B
la --____---
I-----_c---
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,i
ID 3 E 1 OZ
Id 40
ELUTION
VOLUME
(ml)
FIG. 1. Analytical chromatography of crude porcine trypsin on DEAE-cellulose. Fivehundred milligrams of trypsin 1:300 was prepared for analytical chromatography (see Materials and Methods) and applied to a 1.O x 100~cm column which was equilibrated with buffer (100 mM glycine-1 mM CaCI,, pH 8230); protein was eluted using a salt gradient. Fractions from the column were collected and stored at 0°C until assayed (usually within 48 hr of the initial sample preparation). Enzyme assay procedures and definitions of enzyme units are given in Materials and Methods. (a) Protein elution profile (solid line) and salt gradient (broken line). (b) Elastase activity profile (solid line) and lipase activity profile (broken line). (c) Trypsin activity profile (solid line) and amylase activity profile (broken line). (d) Chymotrypsin activity profile (solid line).
tein peak contained trypsin, elastase, and chymotrypsin while the small peaks eluted between 1000 and 2000 ml contained elastase, chymotrypsin, and amylase. To prepare proteolytic enzymes free of amylase, which is not required for tissue disaggregation (l), a fraction eluting from 200 to 1200 ml was pooled and routinely used. This fraction contained trypsin, elastase, and chymotrypsin with a trace of amylase activity. The abbreviation TEC will subsequently be used to denote the mixture of trypsin, elastase, and chymotrypsin obtained by either DEAE-cellulose column purification or DEAE-cellulose batch purification since products of the
PROTEASE
ENZYMES
ELUTtON
211
OF CRUDE TRYPSIN
VCWME
(ml)
FIG. 2. A protein elution profile for preparative DEAE-cellulose column chromatography. Fifty grams of pancreatin B was prepared and applied to a 5 x 60-cm column of DEAE-cellulose equilibrated in 100 mM glycine-1 mM CaCI, buffer, pH 8.90. At the position indicated by the arrow, the buffer was replaced with buffer containing 1 M NaCI. The elution profile is typical of 1S purifications using six different crude enzyme samples as starting material.
two methods were similar. Since amylase was the first contaminating enzyme to be eluted from the ion-exchange column, its activity and trypsin, chymotrypsin, and elastase activities were routinely assayed in all samples. Enzyme recovery during purification on a preparative DEAE-cellulose column is given in Table 1. Small losses of the proteolytic enzymes occurred during initial centrifugation, dialysis, and subsequent centrifugation. Column chromatography resulted in larger losses. Trypsin loss was due largely to autodigestion during the column run since very little trypsin activity was recovered in other fractions. Chymotrypsin and elastase losses were larger since they elute later and some chymotrypsin and elastase activities were located in the discarded minor TABLE ENZYME
RECOVERY
DURING
PURIFICATION
1 B USING PREPARATIVE CHROMATOGRAPHY
OF PANCREATIN
DEAE-CELLULOSECOLUMN
Recovery of enzyme activity” (%) Purification step 1. Crude enzyme dissolved in buffer 2. First centrifugation (supernatant fluid) 3. Dialysis and second centrifugation (supernatant fluid) 4. Column chromatography 5. Dialysis and lyophilization
Trypsin
Chymotrypsin
Elastase
Amylase
100
100
100
100
86
90
95
95
72 52 49
80 27 28
82 34 32
85 0.8 0.1
a Recovery of the enzyme activities during purification of 50 g of pancreatin B is given. Recovery is reported as the percentage of the original enzyme activity retained following each purification step after corrections for sampling losses.
212
SPEICHER
AND McCARL
peaks. Also, in the case of chymotrypsin activity the acidic isozyme form of the enzyme (15) was bound to the column and eluted only with high salt concentration as indicated with the analytical column chromatography (Fig. 1). A large portion of the amylase activity retained after column chromatography was lost on lyophilization. The preparative DEAE-cellulose column procedure was used to purify 15 enzyme samples, of which eight are listed in Table 2. Trypsin recovery in the product (TEC) was 45-60% in all cases, while recovery of elastase and chymotrypsin was 25-40% except when different elution fractions were taken. On a weight basis, approximately 10% of crude trypsin samples were recovered as TEC. When pancreatin was purified, 2-4% of the original weight was recovered as TEC. The effect on enzyme composition of taking a smaller than normal elution fraction is illustrated by trypsin 1:250 and trypsin 1:300 purificaTABLE COMPARISON
OF ENZYME PREPARATIVE
2
ACTIVITIES BEFORE AND AFTER PURIFICATION DEAE-CELLULOSE COLUMN PROCEDURE
USING
Enzyme activitya Chymotrypsin
Trypsin
Elastase
Amylase
Samule”
Crude
Purified
Crude
Purified
Crude
Purified
Crude
PUIified
Crude amylase Pancreatin A Pancreatin B Pancreatin C Crude trypsin A Crude trypsin B Trypsin 1:250’ Trypsin 1:300’
0.30 0.60 0.43 0.51 2.32 2.21 1.70 1.90
10.0 12.6 10.5 11.4 10.5 10.8 13.8 13.3
0.41 0.83 1.5 1.3 2.9 2.3 1.9 2.3
9.3 10.1 9.5 9.0 9.0 9.3 5.4 2.0
0.40 0.92 0.75 1.0 0.8 2.0 2.4 2.5
11.0 12.0 8.0 10.5 7.4 8.1 7.2 6.5
25 54 34 63 1.0 2.0 4.6 12.3
0.3 0.3 1.0 0.8 0.2 0.1 0.3 0.3
a Enzyme activity was determined as described in Materials and Methods. Values reported are averages of triplicate determinations and are expressed as units of activity per milligram of dry weight of the sample. Protein values varied from 0.90 to 1.10 mg by the Lowry method per milligram of dry weight. Units of activity were defined in Materials and Methods. b Samples of crude enzymes ranging in size from 500 to 200 g were purified according to the procedure for preparative DEAE-cellulose column purification given in Materials and Methods. A more detailed description of the crude enzymes used as starting materials is also given in Materials and Methods. c The values for chymotrypsin activity in these samples were low after purification as a consequence of taking an earlier than normal fraction from the column. For these samples a fraction was taken which corresponded to the region from 200 to 900 ml of elution volume in Fig. 2. For all other samples, a fraction corresponding to the region from 200 to 1200 ml of elution volume was taken.
PROTEASE
ENZYMES
OF CRUDE
213
TRYPSIN
tion in Table 2. By taking a smaller fraction, significant amounts of elastase and chymotrypsin are lost with a small decrease in amylase activity. The first six purified enzyme samples listed in Table 2 consisted of similar column elution fractions. For these six enzyme samples, trypsin activity ranged from 10.0 to 12.6 units/mg after purification while the range was from 0.3 to 2.32 units/mg before purification. Similar results were observed for chymotrypsin (9.0 to 10.1 units/mg after purification and 0.41 to 2.9 unitsimg before purification) and elastase (7.4 to 12.0 unitslmg after purification and 0.80 to 2.0 units/mg before purification). Amylase activity after purification was low in all cases but was dependent upon the fraction taken, the amount present in the starting material, and the amount inactivated upon lyophilization. Comparison
of Enzyme Puri’cation
Methods
Samples of crude trypsin B were purified by DEAE-cellulose batch, and CM-cellulose batch procedures to compare the yield and composition of samples obtained by each purification procedure (Table 3). In both CM- and DEAE-cellulose batch purifications equilibrium between the ion-exchange resin and the enzymes was reached in less than 15 min. Routinely the resin was stirred with the enzymes for 30 min before filtration. Ratios of sample weight to ion-exchange resin weight as high as 0.2 were used in the batch purification. When crude trypsin samples were purified by the batch procedure, recovery of trypsin, elastase, and chymotrypsin was high. Amylase activity was slightly higher than in TEC samples prepared by column chromatography. When crude trypsin samples were purified by CM-cellulose batch purification, it was necessary to lower the buffer pH to 7.80 to allow significant binding of trypsin, chymotrypsin, and elastase to the resin. Lower pH TABLE COMPARISON
OF METHODS Trypsin
Purification method NOll‘3
DEAE preparative column DEAE batch CM batch
Recovery of weight (B) 100 11.2 10.0 4.0
ACfivity (Uhg)
3
OF ENZYME Chymotrypsin
Re-
AC-
(%)
fivity Wmg)
2.2
100
2.3
10.8 10.0 17.0
55 45 31
9.3 7.2 1.4
COVWy
ReCOVXY (%) 100 4s 31 2.5
PURIFICATION” Elastase
Amylase
ACtivity Ww)
Recovery cm
ACtivity Ww)
2.0
1M)
2.0
8.1 6.7 14
45 34 28
0.1 0.8 0.0
ReCOVfXY (%) 100 0.6 4.0 0
a One-hundred grams of crude trypsin B WBSpurified by each method using the procedures given in Mate&Is and Methods. Enzyme assay procedures and definitions of enzyme activity are alsogiven in Materials and Methods. Enzyme activity is reported as units of activity per milligram of dry weight. Each enzyme activity value is the mean of triplicate determinations.
214
SPEICHER
AND McCARL
values resulted in substantial contamination of the proteolytic enzymes with amylase. At pH 7.80, which was used routinely, the recovery of the three proteolytic enzymes was substantially lower than with the other methods, but chymotrypsin activity was drastically lower (Table 3). Amylase activity was not detectable. Gel Electrophoresis
of Enzyme Preparations
Electrophoresis of the crude trypsin samples resulted in bands for the five enzymes assayed and several proteins which migrated further toward the anode than either amylase or lipase (Figs. 3F and G). Protein bands on the gels were identified by comparison with purified protein standards electrophoresed under identical conditions and by slicing the gels, eluting
A
B
C
DEFGHIJK
FIG. 3. Native disc gel electrophoresis patterns. Samples were applied in the center of the running gels (at approximately the S.O-cm mark in the figure) by applying the sample before applying and polymerizing the top half of the gel. The anode was at the bottom of the gels as illustrated here. Reference samples A to E were purchased from Sigma Chemical Co.: (A) crystallized hog trypsin Type IX, 40 pg; (B) elastase Type III, 20 pg; (C) cr-chymotrypsin Type 1, 20 pg; (D) lipase Type VI, 80 pg; and (E) or-amylase Type I-A, 20 Fg. Gel (F) was Sigma crude trypsin B, 200 pg, and gel (G) was pancreatin C, 200 pg. Gels H, I, and J were purified from the Sigma crude trypsin B sample by: (H) DEAE-cellulose column chromatography, 100 pg; (I) DEAE-cellulose batch purification, 100 pg; and (J) CM-cellulose batch purification, 100 pg. Gel (K) was a sample of pancreatin C after purification by DEAE-cellulose column chromatography, 100 fig.
PROTEASE
ENZYMES
OF CRUDE
TRYPSIN
215
the enzymes, and determining enzyme activity of trypsin, chymotrypsin, elastase, amylase, and lipase as described in Materials and Methods. The gel protein patterns of enzyme preparations purified by the various methods (Figs. 3 H-K) agree with the results of the enzyme assays given in Tables 2 and 3. The protein bands below the origin (anode side) were determined to be amylase and not lipase. These bands had amylase activity but did not have lipase activity. Similar banding patterns could be obtained with purified amylase if it was first dialyzed and lyophilized in a manner similar to that of the samples purified from crude enzyme preparations. DISCUSSION
The present work shows that the three proteolytic pancreatic enzymes (trypsin, elastase, and chymotrypsin) can be readily isolated from crude samples of pancreatic enzymes using a purification technique employing batch or column DEAE-cellulose or batch CM-cellulose chromatography. This preparation, called TEC, removes the variability in disaggregation of heart tissue and several other tissues, encountered with many crude trypsin samples (Speicher and McCarl, in preparation). Purification on a DEAE-cellulose column has the advantage of giving the highest yield of enzymes. The DEAE-cellulose batch preparation is more rapid and also gives a high yield of enzymes, but a slightly larger amount of amylase contaminates the product. Purification on a CM-cellulose column yields a product which is free of detectable amylase but has less chymotrypsin than the product of the DEAE-cellulose preparations. The composition of the product isolated after DEAE-cellulose column purification can be controlled by varying the point at which collection of the sample is terminated. If collection is terminated on the descending slope of the first peak very little amylase is obtained but a large amount of the chymotrypsin activity is lost (see trypsin 1:250 and trypsin 1:300 in Table 2). Such preparations would be similar to CM-cellulose-purified samples. If the collection of column effluent is terminated at the base of the first peak, an enzyme preparation which has good recovery of trypsin, chymotrypsin, and elastase activities is obtained, but this preparation will contain a small amount of amylase activity. If collection of the column effluent includes all protein eluted with the equilibration buffer, the product will be similar to that obtained by the DEAE-cellulose batch procedure. Since the DEAE-cellulose batch purification procedure yields 4% recovery of initial amylase activity, pancreatin samples were not considered as good starting material for this method due to their high initial amylase content. When crude trypsin was used as a starting material, the amount of amylase contaminating the product was small. The increased speed of purification with the batch procedure was an advantage
216
SPEICHER AND McCARL
due to lower enzyme loss through proteolysis and increased ease in purification. The CM-cellulose batch purification procedure at pH 7.80 gave an enzyme preparation which was free of amylase activity but low in chymotrypsin activity. Lowering the pH to 6.80 gave a larger recovery of both chymotrypsin and amylase. The product obtained was similar to that from the DEAE-cellulose batch procedure and did not offer any advantages over the DEAE-cellulose procedure. When the purification is performed at pH 7.80, the product obtained offers a valuable alternative for those cases where an amylase-free proteolytic preparation is needed. Electrophoresis of other pancreatic enzymes and proteins including the carboxypeptidases, pancreatic trypsin inhibitor, deoxyribonuclease, and ribonuclease has shown that only ribonuclease will migrate in the region of the protein bands found in TEC preparations. This is consistent with results obtained for guinea pig exocrine pancreatic proteins (16). Several TEC samples tested for action against phospholipid, RNA, and lipase were negative. This suggests that any contaminants besides amylase which are present in the TEC samples are either inactive enzymatically or present in very minor amounts. TEC samples also contain small amounts of the buffer used in the final dialysis. In summary, crude trypsin samples can be purified using DEAE-cellulose or CM-cellulose to yield a product consisting of trypsin, chymotrypsin, and elastase. This preparation gives superior results in disaggregation of rat heart tissue, and other tissues have also been successfully disaggregated suggesting a wide application for this preparation in tissue dispersal. The DEAE-cellulose batch purification procedure is recommended for most cell culture applications because of high yield of enzymes and ease of preparation. The CM-cellulose batch purification procedure is recommended for other applications where low levels of amylase activity are undesirable. ACKNOWLEDGMENTS This research was partially funded by the United States Public Health Service Grant HL10018 and a grant from the Pennsylvania State University Agricultural Experiment Station. Authorized for publication as Paper 5268 in the journal series of the Pennsylvania Agricultural Experimental Station.
REFERENCES 1. Speicher, D. W., and McCarl, R. L. (1974) In Vitro 10, 30-41. 2. Pine, L., Taylor, G. C., Miller, D. M. Bradley, G., and Wetmore, H. R. (1969) Cyrobios
2, 197-207.
3. Rinaldini, L. M. J. (1958) Inr. Rev. Cyfol. 7, 587-647. 4. Willson, J. K. V., Pretlow, T. G., II, Zaremba, J. L., and Brattain, Immunology 30, 157- 160.
M. G. (1976)
PROTEASE
ENZYMES
OF CRUDE TRYPSIN
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j. Pine, L., Bradley, G., and Miller, D. (1969) Cytobios 4, 347-367. 5. Barofsky, A., Feinstein, M., and Halkerston, I. D. K. (1973) Exp. Cell Res. 79, 263-274. 7. Omar, A., and Krebs, A. (1975) Arch. Dermatol. Forsch. 253, 203-212. 3. Phillips, H. J. (1972) In Vifro 8, 101-105. J. Erlanger, B. F., Kokowsky, N., and Cohen, W. (1961) Arch. Biochem. Biophys. 95, 271-278. D. Hummel, B. C. W. (1959) Canad. J. B&hem. Physiol. 37, 1393-1399. 1. Bieth, J., and Meyer, J. F. (1973) Anal. Biochem. 51, 121-126. 2. Bernfeld, P. (1951) Advan. Enzymol. Relat. Areas Mol. Biol. 12, 379-428. 3. Guilbault, G. G., Hieserman, J., and Sadar, M. H. (1969) Anal. Left. 2, 185-196. 4. Maizel, J. V., Jr. (1971) in Methods in Virology (Maramorosch, K., and Koprowski, H., eds.), Vol. 5, pp. 179-246, Academic Press, New York. 5. Zelikson, R., Eilam-Rubin, G., and Kulka, R. G. (1971) J. Biol. Chem. 246, 6115-6120. 6. Scheele, G. A. (1975) J. Biol. Chem. 250, 5375-5385.
BIOCHEMISTRY
84, 205-217 (1978)
Isolation and Characterization of the Proteolytic Enzyme Component from Commercially Available Crude Trypsins DAVID Department
of Biochemistry
W. SPEICHER AND RICHARD and Biophysics, The Pennsylvania Park, Pennsylvania 16802
L. MCCARL State
University,
University
Received April 28, 1977; accepted July 29, 1977 A purification procedure for the isolation of a mixture of the major proteolytic pancreatic enzymes (trypsin, chymotrypsin, and elastase) from commercial crude trypsins is described. These enzymes are apparently the enzymes responsible for tissue dispersal in numerous cell culture systems. Materials toxic to cell cultures, present in certain crude trypsin samples, are removed during a purification involving centrifugation, dialysis, treatment with a cellulose ion-exchange resin, removal of salts, and lyophilization. While the fundamental use of this proteolytic mixture would be to prepare primary cell culture, the broad peptide bond specificity of this mixture would suggest application in cases where a general protease, free of other enzymatic activities, is required.
Previous work in our laboratory showed that trypsin, chymotrypsin, and elastase were the enzymes in crude trypsin samples responsible for disaggregation of neonatal rat heart tissue. It was observed that most commercial samples of trypsin contained these three enzymes at concentrations sufficient to disaggregate heart tissue. However, different samples of crude trypsin, even different lots of the same type of trypsin, contained varying amounts of unidentified toxic components which caused marked variability in effectiveness of tissue dispersal and cell viability. This variability could be minimized by using a mixture of trypsin, chymotrypsin, and elastase in concentrations comparable to those found in crude trypsin samples (1). Other researchers also have reported that the ability of crude trypsins to disperse a variety of tissues was due at least partially to enzymes other than trypsin (2-4). The enzymes involved in tissue dispersal have been characterized in some cell culture systems (23-8). Therefore, an enzyme preparation containing trypsin, chymotrypsin, and elastase is needed for rat heart cell culture as well as for certain other culture systems. The cost of purified individual enzymes makes mixing of the purified enzymes to obtain the desired activities impractical for routine, large-scale cell culture work. We report herein the facile isola205
0003.2697/78/0841-0205$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved
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tion of the desired enzymes, which provides a reproducible and lowcost means of disaggregating tissues for cell culture. The broad peptide bond specificity of this mixture of proteolytic enzymes would suggest applications in cases other than cell culture where a general protease, free of other enzymatic activities, is required. MATERIALS
AND METHODS
Materials
DEAE-cellulose (DE 52) and CM-cellulose (CM 52) were obtained from Whatman Inc., Clifton, N.J. Hollow fiber dialyzers were obtained from Bio-Rad Laboratories, Richmond, Calif. Conductivity was determined using a YSI Model 33 S-C-T meter, Yellow Springs Instruments, Yellow Springs, Ohio. Substrates for trypsin, chymotrypsin, and elastase activity assays and purified enzyme samples for electrophoresis were obtained from Sigma Chemical Co., St. Louis, MO. The lipase substrate (N-methyl indoxyl myristate) was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. Brilliant blue R-250 dye was obtained from Sigma Chemical Co., St. Louis, MO. Acrylamide, N,N’-methylenebisacrylamide (bis), and N,N,N’,N’-tetramethylenediamine (TEMED) were purchased from Bio-Rad Laboratories, Richmond, Calif. A number of crude enzymes were used for enzyme purification. They include several types of samples from Sigma Chemical Co., St. Louis, MO., which offers pancreatic enzyme samples in several stages of purity, and two samples from Nutritional Biochemicals Corporation, Cleveland, Ohio, commonly used for tissue culture work. The samples from Sigma Chemical Co. were: crude trypsin Type II, Lot 71C-2440 (crude trypsin A) and Lot 104C-0008 (crude trypsi‘n B); cY-amylase Type VI-A, Lot 71C0210 (crude amylase); and pancreatin Grade VI, Lot 71C-0670 (pancreatin A), Lot 112C-2780 (pancreatin B), and Lot 123C-1720 (pancreatin C). The two samples from Nutritional Biochemicals Corporation were trypsin 1:300, Control No. 5176 (trypsin 1:300), and trypsin 1:250, Control No. 6220 (trypsin 1:250). The abbreviated names in parentheses will be used to refer to the respective enzyme samples. Initial
Purification
of Crude Enzyme
All purification procedures, pH readings, and conductivity readings were performed at 2°C unless otherwise stated. Crude extract was prepared by adding buffer (100 mM glycine- 1 mM CaCl, titrated to pH 8.90 with 1 N NaOH) to the crude enzyme sample to give a final concentration of 50 mg (by weight)/ml. This mixture was stirred for 30 min, adjusted to pH 8.90 with 1 N NaOH, and centrifuged at 12,000g for 15 min. The pellet was discarded and the supematant solution was
PROTEASE
ENZYMES
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207
dialyzed exhaustively against buffer using a Bio-Fiber dialyzer (nominal MW cutoff of 5000 daltons). A peristaltic pump was used to circulate buffer and crude extract through the respective chambers of the hollow fiber dialyzer. The buffer was replaced with fresh buffer when the conductivity of the dialysate approached that of the protein solution. During dialysis the sample solution was concentrated to half the original volume by maintaining positive pressure on the effluent tubing of the dialyzer. Dialysis was considered complete when a conductivity of 600 pmhos or less for the protein solution was obtained (starting buffer conductivity was approximately 400 pmhos). The sample solution was centrifuged at 12,000g for 15 min, and the supematant solution was retained for ion-exchange treatment. Analytical
DEAE-Ceffufose
Cofumn
Chromatography
Crude enzyme was purified as described above and then applied to a DEAE-cellulose column (1 x 100 cm) equilibrated with the buffer used above (100 mM glycine-1 mM CaCl,, pH 8.90). Buffer was used for the initial elution, followed by a linear salt gradient from 0 to 0.5 M NaCl in buffer, and then elution was completed with buffer containing 1 M NaCl. The column was regenerated with equilibration buffer. Samples were collected in lo-ml fractions and analyzed for enzyme activity. Prepurative
DEAE-Cellulose
Column
Chromatography
The above analytical procedure was modified to obtain gram quantities of purified enzymes for routine cell dissociation. Crude enzymes (50 to 200g of starting material) were purified as described in Initial Purification of Crude Enzyme and then applied to a DEAE-cellulose column (5.0 x 60 cm) equilibrated with buffer (100 mM glycine-1 mM CaCl,, pH 8.90). The first protein peak was eluted with buffer, collected, dialyzed against a 1: 100 dilution of buffer, concentrated to 10 mg of protein/ ml (OD,,, of a 10 mg/ml solution = 12.6), and lyophilized. The column was regenerated by washing with buffer containing 1 M NaCl followed by reequilibration with buffer containing no NaCl. Preparative
DEAE-Cellulose
Batch Puri$cation
Purification of enzyme samples by DEAE-cellulose batch purification was similar to the preparative column procedure except the sample, which was prepared as described in Initial Purification of Crude Enzyme, was added to degassed, preequilibrated DEAE-cellulose in a beaker and stirred at 2°C for 30 min. This mixture was filtered on a Btichner funnel, the filtrate was dialyzed against a 1: 100 dilution of buffer, concentrated to 10 mg of protein/ml (ODZB, = 12.6), and lyophilized. The ion-exchange resin
208 was regenerated by reequilibration
SPEICHER
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by washing with buffer containing with buffer containing no NaCI.
1 M NaCl followed
Preparative CM-Cellulose Batch Purification Purification of enzyme samples by CM-cellulose batch purification was similar to the DEAE-cellulose batch purification except the buffer (100 mM glycine-1 mM CaCl,) was adjusted to pH 7.80 instead of pH 8.90, and CM-cellulose was substituted for DEAE-cellulose. Impurities were eluted with four washes of buffer (each wash was four times the settled resin volume), and subsequently the desired enzymes were released from the ion-exchange resin with two washes of buffer containing 2 M NaCl (each wash was equal to the settled resin volume). This latter solution was then dialyzed against a 1:lOO dilution of the pH 8.90 buffer without NaCl, concentrated to a protein concentration of 10 m&ml (OD,,, = 12.6), and lyophilized. Assay of Enzyme Activity Trypsin activity was determined by the method of Erlanger et al. (9), using N-benzoyl-arginyl-p-nitroanilide (bapna) as the substrate. One unit of activity was defined as the amount of enzyme required to hydrolyze 1 pmol of substrate in 1 min under the conditions of the assay (pH 8.2, 37°C). Chymotrypsin activity was determined by the method of Hummel (IO), using N-benzoyk-tyrosine ethyl ester (btee) as the substrate. One unit of activity was defined as the amount of enzyme required to hydrolyze 1 pmol of substrate in 1 min under the conditions of the assay (pH 7.8, 25°C). Elastase activity was determined by the method of Bieth and Meyer (11) using acetyl-L-alanyl-L-alanyl-L-alanine methyl ester as the substrate. One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 pmol of substrate in 1 min under the conditions of the assay (pH 7.0, 25°C). Amylase activity was assayed by the method of Bernfeld (12) using soluble potato starch as the substrate. A unit of activity was defined as the amount of enzyme required to liberate 1 pmol of reducing groups, calculated as maltose, per minute under the conditions of the assay (pH 6.9, 25°C). Lipase activity was measured by the method of Guilbault et al. (13) using Nmethyl indoxyl myristate as the substrate with modifications described previously (1). One unit of activity was defined as the amount of enzyme required to hydrolyze 1 pmol of substrate under the conditions of the assay (pH 7.8, 25°C). Gel Electrophoresis Electrophoresis was performed at 15°C on 10% acrylamide gels at pH 7.6 using glass tubes (5 by 130 mm). After sealing one end of the gel tubes
PROTEASE
ENZYMES
OF CRUDE
TRYPSIN
209
and clamping them in a vertical rack, gel solution was added to a height of 5 cm. A small amount of water was layered on top of the gel solution. Polymerization was complete in less than 30 min at room temperature. The water was then removed and sample was added. A j-cm gel was then formed above the sample and, after polymerization was complete, the gels were run at a constant 150 v for 3.5 hr. The gel composition was a modification of that given by Maize1 (14). Ten milliliters of gel was prepared from 3.33 ml of a 30% solution of acrylamide with 0.8% Bis as the cross-linker, 1.50 ml 0.66 M Tris-HCl buffer, pH 7.6, 5 ~1 of TEMED, 5.14 ml of HzO, and 30 ~1 of 10% ammonium persulfate. The electrode buffer was 0.1 M Tris-HCl, pH 7.6. Sample buffer contained 1.0 ml of 0.66 M Tris-HCl buffer, pH 7.6, 3 ml of glycerol, and 6 ml of H,O. Gels were stained with 200 mg of Coomassie blue (brilliant blue R-250) dye dissolved in 50 ml of methanol, 10 ml of glacial acetic acid, and 40 ml of H,O and destained in a solution of 70 ml of glacial acetic acid, 200 ml of methanol, and 730 ml of H,O. RESULTS
Chromatography
of Crude Trypsin on DEAE-Cellulose
In preliminary experiments several samples of crude trypsin were chromatographed on a DEAE-cellulose column using the procedure of Zelikson et al. (15). Using these conditions with crude porcine trypsin, the first protein peak contained trypsin, elastase, chymotrypsin, and amylase activities. In subsequent experiments the chromatography conditions were modified by raising the pH to 8.90 and changing the buffer to 100 mM glycine-1 mM CaCl,. The proteolytic enzymes (trypsin, elastase, and chymotrypsin) were found in the first protein peak (Fig. 1) and were clearly separated from amylase and lipase. The second protein peak contained only small amounts of chymotrypsin and amylase activities. Incubation of the crude trypsin sample at room temperature either for 1 hr or overnight at 2°C before column chromatography resulted in a decrease in the first protein peak and an increase in the second protein peak. More than 50% of the 280-nm-absorbing material in the second protein peak passed through a Bio-fiber 50 dialyzer (nominal MW cutoff of 5000 daltons). These results suggest the second peak was mostly inactive hydrolysis products of the proteins eluted in the first peak. Protein eluted by the salt gradient contained a mixture of amylase, lipase, and chymotrypsin (Figs. lb-d) and other proteins (detected by disc gel electrophoresis). A preparative DEAE-cellulose column purification was used to produce gram quantities of various column fractions. The protein elution profile for the purification of 5Og of pancreatin II is given in Fig. 2. The first pro-
210
SPEICHER AND McCARL 3.0
s N I. B
la --____---
I-----_c---
__----
,i
ID 3 E 1 OZ
Id 40
ELUTION
VOLUME
(ml)
FIG. 1. Analytical chromatography of crude porcine trypsin on DEAE-cellulose. Fivehundred milligrams of trypsin 1:300 was prepared for analytical chromatography (see Materials and Methods) and applied to a 1.O x 100~cm column which was equilibrated with buffer (100 mM glycine-1 mM CaCI,, pH 8230); protein was eluted using a salt gradient. Fractions from the column were collected and stored at 0°C until assayed (usually within 48 hr of the initial sample preparation). Enzyme assay procedures and definitions of enzyme units are given in Materials and Methods. (a) Protein elution profile (solid line) and salt gradient (broken line). (b) Elastase activity profile (solid line) and lipase activity profile (broken line). (c) Trypsin activity profile (solid line) and amylase activity profile (broken line). (d) Chymotrypsin activity profile (solid line).
tein peak contained trypsin, elastase, and chymotrypsin while the small peaks eluted between 1000 and 2000 ml contained elastase, chymotrypsin, and amylase. To prepare proteolytic enzymes free of amylase, which is not required for tissue disaggregation (l), a fraction eluting from 200 to 1200 ml was pooled and routinely used. This fraction contained trypsin, elastase, and chymotrypsin with a trace of amylase activity. The abbreviation TEC will subsequently be used to denote the mixture of trypsin, elastase, and chymotrypsin obtained by either DEAE-cellulose column purification or DEAE-cellulose batch purification since products of the
PROTEASE
ENZYMES
ELUTtON
211
OF CRUDE TRYPSIN
VCWME
(ml)
FIG. 2. A protein elution profile for preparative DEAE-cellulose column chromatography. Fifty grams of pancreatin B was prepared and applied to a 5 x 60-cm column of DEAE-cellulose equilibrated in 100 mM glycine-1 mM CaCI, buffer, pH 8.90. At the position indicated by the arrow, the buffer was replaced with buffer containing 1 M NaCI. The elution profile is typical of 1S purifications using six different crude enzyme samples as starting material.
two methods were similar. Since amylase was the first contaminating enzyme to be eluted from the ion-exchange column, its activity and trypsin, chymotrypsin, and elastase activities were routinely assayed in all samples. Enzyme recovery during purification on a preparative DEAE-cellulose column is given in Table 1. Small losses of the proteolytic enzymes occurred during initial centrifugation, dialysis, and subsequent centrifugation. Column chromatography resulted in larger losses. Trypsin loss was due largely to autodigestion during the column run since very little trypsin activity was recovered in other fractions. Chymotrypsin and elastase losses were larger since they elute later and some chymotrypsin and elastase activities were located in the discarded minor TABLE ENZYME
RECOVERY
DURING
PURIFICATION
1 B USING PREPARATIVE CHROMATOGRAPHY
OF PANCREATIN
DEAE-CELLULOSECOLUMN
Recovery of enzyme activity” (%) Purification step 1. Crude enzyme dissolved in buffer 2. First centrifugation (supernatant fluid) 3. Dialysis and second centrifugation (supernatant fluid) 4. Column chromatography 5. Dialysis and lyophilization
Trypsin
Chymotrypsin
Elastase
Amylase
100
100
100
100
86
90
95
95
72 52 49
80 27 28
82 34 32
85 0.8 0.1
a Recovery of the enzyme activities during purification of 50 g of pancreatin B is given. Recovery is reported as the percentage of the original enzyme activity retained following each purification step after corrections for sampling losses.
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peaks. Also, in the case of chymotrypsin activity the acidic isozyme form of the enzyme (15) was bound to the column and eluted only with high salt concentration as indicated with the analytical column chromatography (Fig. 1). A large portion of the amylase activity retained after column chromatography was lost on lyophilization. The preparative DEAE-cellulose column procedure was used to purify 15 enzyme samples, of which eight are listed in Table 2. Trypsin recovery in the product (TEC) was 45-60% in all cases, while recovery of elastase and chymotrypsin was 25-40% except when different elution fractions were taken. On a weight basis, approximately 10% of crude trypsin samples were recovered as TEC. When pancreatin was purified, 2-4% of the original weight was recovered as TEC. The effect on enzyme composition of taking a smaller than normal elution fraction is illustrated by trypsin 1:250 and trypsin 1:300 purificaTABLE COMPARISON
OF ENZYME PREPARATIVE
2
ACTIVITIES BEFORE AND AFTER PURIFICATION DEAE-CELLULOSE COLUMN PROCEDURE
USING
Enzyme activitya Chymotrypsin
Trypsin
Elastase
Amylase
Samule”
Crude
Purified
Crude
Purified
Crude
Purified
Crude
PUIified
Crude amylase Pancreatin A Pancreatin B Pancreatin C Crude trypsin A Crude trypsin B Trypsin 1:250’ Trypsin 1:300’
0.30 0.60 0.43 0.51 2.32 2.21 1.70 1.90
10.0 12.6 10.5 11.4 10.5 10.8 13.8 13.3
0.41 0.83 1.5 1.3 2.9 2.3 1.9 2.3
9.3 10.1 9.5 9.0 9.0 9.3 5.4 2.0
0.40 0.92 0.75 1.0 0.8 2.0 2.4 2.5
11.0 12.0 8.0 10.5 7.4 8.1 7.2 6.5
25 54 34 63 1.0 2.0 4.6 12.3
0.3 0.3 1.0 0.8 0.2 0.1 0.3 0.3
a Enzyme activity was determined as described in Materials and Methods. Values reported are averages of triplicate determinations and are expressed as units of activity per milligram of dry weight of the sample. Protein values varied from 0.90 to 1.10 mg by the Lowry method per milligram of dry weight. Units of activity were defined in Materials and Methods. b Samples of crude enzymes ranging in size from 500 to 200 g were purified according to the procedure for preparative DEAE-cellulose column purification given in Materials and Methods. A more detailed description of the crude enzymes used as starting materials is also given in Materials and Methods. c The values for chymotrypsin activity in these samples were low after purification as a consequence of taking an earlier than normal fraction from the column. For these samples a fraction was taken which corresponded to the region from 200 to 900 ml of elution volume in Fig. 2. For all other samples, a fraction corresponding to the region from 200 to 1200 ml of elution volume was taken.
PROTEASE
ENZYMES
OF CRUDE
213
TRYPSIN
tion in Table 2. By taking a smaller fraction, significant amounts of elastase and chymotrypsin are lost with a small decrease in amylase activity. The first six purified enzyme samples listed in Table 2 consisted of similar column elution fractions. For these six enzyme samples, trypsin activity ranged from 10.0 to 12.6 units/mg after purification while the range was from 0.3 to 2.32 units/mg before purification. Similar results were observed for chymotrypsin (9.0 to 10.1 units/mg after purification and 0.41 to 2.9 unitsimg before purification) and elastase (7.4 to 12.0 unitslmg after purification and 0.80 to 2.0 units/mg before purification). Amylase activity after purification was low in all cases but was dependent upon the fraction taken, the amount present in the starting material, and the amount inactivated upon lyophilization. Comparison
of Enzyme Puri’cation
Methods
Samples of crude trypsin B were purified by DEAE-cellulose batch, and CM-cellulose batch procedures to compare the yield and composition of samples obtained by each purification procedure (Table 3). In both CM- and DEAE-cellulose batch purifications equilibrium between the ion-exchange resin and the enzymes was reached in less than 15 min. Routinely the resin was stirred with the enzymes for 30 min before filtration. Ratios of sample weight to ion-exchange resin weight as high as 0.2 were used in the batch purification. When crude trypsin samples were purified by the batch procedure, recovery of trypsin, elastase, and chymotrypsin was high. Amylase activity was slightly higher than in TEC samples prepared by column chromatography. When crude trypsin samples were purified by CM-cellulose batch purification, it was necessary to lower the buffer pH to 7.80 to allow significant binding of trypsin, chymotrypsin, and elastase to the resin. Lower pH TABLE COMPARISON
OF METHODS Trypsin
Purification method NOll‘3
DEAE preparative column DEAE batch CM batch
Recovery of weight (B) 100 11.2 10.0 4.0
ACfivity (Uhg)
3
OF ENZYME Chymotrypsin
Re-
AC-
(%)
fivity Wmg)
2.2
100
2.3
10.8 10.0 17.0
55 45 31
9.3 7.2 1.4
COVWy
ReCOVXY (%) 100 4s 31 2.5
PURIFICATION” Elastase
Amylase
ACtivity Ww)
Recovery cm
ACtivity Ww)
2.0
1M)
2.0
8.1 6.7 14
45 34 28
0.1 0.8 0.0
ReCOVfXY (%) 100 0.6 4.0 0
a One-hundred grams of crude trypsin B WBSpurified by each method using the procedures given in Mate&Is and Methods. Enzyme assay procedures and definitions of enzyme activity are alsogiven in Materials and Methods. Enzyme activity is reported as units of activity per milligram of dry weight. Each enzyme activity value is the mean of triplicate determinations.
214
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values resulted in substantial contamination of the proteolytic enzymes with amylase. At pH 7.80, which was used routinely, the recovery of the three proteolytic enzymes was substantially lower than with the other methods, but chymotrypsin activity was drastically lower (Table 3). Amylase activity was not detectable. Gel Electrophoresis
of Enzyme Preparations
Electrophoresis of the crude trypsin samples resulted in bands for the five enzymes assayed and several proteins which migrated further toward the anode than either amylase or lipase (Figs. 3F and G). Protein bands on the gels were identified by comparison with purified protein standards electrophoresed under identical conditions and by slicing the gels, eluting
A
B
C
DEFGHIJK
FIG. 3. Native disc gel electrophoresis patterns. Samples were applied in the center of the running gels (at approximately the S.O-cm mark in the figure) by applying the sample before applying and polymerizing the top half of the gel. The anode was at the bottom of the gels as illustrated here. Reference samples A to E were purchased from Sigma Chemical Co.: (A) crystallized hog trypsin Type IX, 40 pg; (B) elastase Type III, 20 pg; (C) cr-chymotrypsin Type 1, 20 pg; (D) lipase Type VI, 80 pg; and (E) or-amylase Type I-A, 20 Fg. Gel (F) was Sigma crude trypsin B, 200 pg, and gel (G) was pancreatin C, 200 pg. Gels H, I, and J were purified from the Sigma crude trypsin B sample by: (H) DEAE-cellulose column chromatography, 100 pg; (I) DEAE-cellulose batch purification, 100 pg; and (J) CM-cellulose batch purification, 100 pg. Gel (K) was a sample of pancreatin C after purification by DEAE-cellulose column chromatography, 100 fig.
PROTEASE
ENZYMES
OF CRUDE
TRYPSIN
215
the enzymes, and determining enzyme activity of trypsin, chymotrypsin, elastase, amylase, and lipase as described in Materials and Methods. The gel protein patterns of enzyme preparations purified by the various methods (Figs. 3 H-K) agree with the results of the enzyme assays given in Tables 2 and 3. The protein bands below the origin (anode side) were determined to be amylase and not lipase. These bands had amylase activity but did not have lipase activity. Similar banding patterns could be obtained with purified amylase if it was first dialyzed and lyophilized in a manner similar to that of the samples purified from crude enzyme preparations. DISCUSSION
The present work shows that the three proteolytic pancreatic enzymes (trypsin, elastase, and chymotrypsin) can be readily isolated from crude samples of pancreatic enzymes using a purification technique employing batch or column DEAE-cellulose or batch CM-cellulose chromatography. This preparation, called TEC, removes the variability in disaggregation of heart tissue and several other tissues, encountered with many crude trypsin samples (Speicher and McCarl, in preparation). Purification on a DEAE-cellulose column has the advantage of giving the highest yield of enzymes. The DEAE-cellulose batch preparation is more rapid and also gives a high yield of enzymes, but a slightly larger amount of amylase contaminates the product. Purification on a CM-cellulose column yields a product which is free of detectable amylase but has less chymotrypsin than the product of the DEAE-cellulose preparations. The composition of the product isolated after DEAE-cellulose column purification can be controlled by varying the point at which collection of the sample is terminated. If collection is terminated on the descending slope of the first peak very little amylase is obtained but a large amount of the chymotrypsin activity is lost (see trypsin 1:250 and trypsin 1:300 in Table 2). Such preparations would be similar to CM-cellulose-purified samples. If the collection of column effluent is terminated at the base of the first peak, an enzyme preparation which has good recovery of trypsin, chymotrypsin, and elastase activities is obtained, but this preparation will contain a small amount of amylase activity. If collection of the column effluent includes all protein eluted with the equilibration buffer, the product will be similar to that obtained by the DEAE-cellulose batch procedure. Since the DEAE-cellulose batch purification procedure yields 4% recovery of initial amylase activity, pancreatin samples were not considered as good starting material for this method due to their high initial amylase content. When crude trypsin was used as a starting material, the amount of amylase contaminating the product was small. The increased speed of purification with the batch procedure was an advantage
216
SPEICHER AND McCARL
due to lower enzyme loss through proteolysis and increased ease in purification. The CM-cellulose batch purification procedure at pH 7.80 gave an enzyme preparation which was free of amylase activity but low in chymotrypsin activity. Lowering the pH to 6.80 gave a larger recovery of both chymotrypsin and amylase. The product obtained was similar to that from the DEAE-cellulose batch procedure and did not offer any advantages over the DEAE-cellulose procedure. When the purification is performed at pH 7.80, the product obtained offers a valuable alternative for those cases where an amylase-free proteolytic preparation is needed. Electrophoresis of other pancreatic enzymes and proteins including the carboxypeptidases, pancreatic trypsin inhibitor, deoxyribonuclease, and ribonuclease has shown that only ribonuclease will migrate in the region of the protein bands found in TEC preparations. This is consistent with results obtained for guinea pig exocrine pancreatic proteins (16). Several TEC samples tested for action against phospholipid, RNA, and lipase were negative. This suggests that any contaminants besides amylase which are present in the TEC samples are either inactive enzymatically or present in very minor amounts. TEC samples also contain small amounts of the buffer used in the final dialysis. In summary, crude trypsin samples can be purified using DEAE-cellulose or CM-cellulose to yield a product consisting of trypsin, chymotrypsin, and elastase. This preparation gives superior results in disaggregation of rat heart tissue, and other tissues have also been successfully disaggregated suggesting a wide application for this preparation in tissue dispersal. The DEAE-cellulose batch purification procedure is recommended for most cell culture applications because of high yield of enzymes and ease of preparation. The CM-cellulose batch purification procedure is recommended for other applications where low levels of amylase activity are undesirable. ACKNOWLEDGMENTS This research was partially funded by the United States Public Health Service Grant HL10018 and a grant from the Pennsylvania State University Agricultural Experiment Station. Authorized for publication as Paper 5268 in the journal series of the Pennsylvania Agricultural Experimental Station.
REFERENCES 1. Speicher, D. W., and McCarl, R. L. (1974) In Vitro 10, 30-41. 2. Pine, L., Taylor, G. C., Miller, D. M. Bradley, G., and Wetmore, H. R. (1969) Cyrobios
2, 197-207.
3. Rinaldini, L. M. J. (1958) Inr. Rev. Cyfol. 7, 587-647. 4. Willson, J. K. V., Pretlow, T. G., II, Zaremba, J. L., and Brattain, Immunology 30, 157- 160.
M. G. (1976)
PROTEASE
ENZYMES
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217
j. Pine, L., Bradley, G., and Miller, D. (1969) Cytobios 4, 347-367. 5. Barofsky, A., Feinstein, M., and Halkerston, I. D. K. (1973) Exp. Cell Res. 79, 263-274. 7. Omar, A., and Krebs, A. (1975) Arch. Dermatol. Forsch. 253, 203-212. 3. Phillips, H. J. (1972) In Vifro 8, 101-105. J. Erlanger, B. F., Kokowsky, N., and Cohen, W. (1961) Arch. Biochem. Biophys. 95, 271-278. D. Hummel, B. C. W. (1959) Canad. J. B&hem. Physiol. 37, 1393-1399. 1. Bieth, J., and Meyer, J. F. (1973) Anal. Biochem. 51, 121-126. 2. Bernfeld, P. (1951) Advan. Enzymol. Relat. Areas Mol. Biol. 12, 379-428. 3. Guilbault, G. G., Hieserman, J., and Sadar, M. H. (1969) Anal. Left. 2, 185-196. 4. Maizel, J. V., Jr. (1971) in Methods in Virology (Maramorosch, K., and Koprowski, H., eds.), Vol. 5, pp. 179-246, Academic Press, New York. 5. Zelikson, R., Eilam-Rubin, G., and Kulka, R. G. (1971) J. Biol. Chem. 246, 6115-6120. 6. Scheele, G. A. (1975) J. Biol. Chem. 250, 5375-5385.