Chicken chymotrypsin and Turkey trypsin

ARCHIVES

OF

BIOCHEMISTRY

Chicken

AND

BIOPHYSICS

110,

Chymotrypsin

169-174

(19%)

and

Turkey

Trypsin

Part I: Purification C. A. RYAN’ Western

Regional

iZgricultura1

Western Utilization Reoearch Laboratory, Research Service, United StateP Department

Research and Development Division, of Agriculture, Albany, California

Received November 25. 19G4 Ammonium sulfate fractionation and column chromatography were used to obtain highly purified chicken chymotrypsin from the protein extract,ed from chicken pancreas by acid. Turkey t,rypsin was isolated and purified by the same methods and, in addition, was crystallized from solutions of magnesium sulfate or ammonium sulfate. Turke) chymotrypsin and chicken trypsin were not purified. The zymogens of these enzymes were not found in good yield in the proteins extracted by acid from pancreas homogenates. They were demonstrated, however, in the proteins extract,ed by neutral sodium phosphate buffer. The yields and properties of the avian endopeptidases suggest that the enzymes perform the same role in intestinal digestion in birds that chymotrypsin and t,rypsin perform in mammals. Recent studies demon&rated 90% of the protein in bovine

that over pancreatic zymogen granules or intestinal pancreatic secretions is digestive enzymes and enzyme precursors (1, 2). Less is krlowr~ of the intestinal pancreatic secretions from other mammals or from members of ot.her classes. Pancreatic enzymes from birds have received limited attention, and when they have, whole tissue honlogenates or intestinal contents have been assayed. It seemed logical that digestion of protein in the small intestine proceeds as in t’he nlanlnlal (3) since pepsin has for years been known in the

proventriculus and ventriculus of birds (4). Furthermore, the presence in the chicken of a well-organized pancreas containing zymogen granules (5) suggested an intestinal digest,ive process similar to the mammal’s. A recent) study (6) demonstrated that chymotrypsin-like and t,rypsin-like enzymic activities were present) in ammonium sulfate1 Present address: Ijepartment of Agricultura,l Chemistry, Washington St,ate University, Pullman, Washington.

precipitated fractions from water extracts of chicken pancreas. Although impure, these enzymes hydrolyzed several ester substrates in a manner analogous to mammalian trypsin and chymotrypsin and were inhibited by DFP as well as by ot,her inhibitors of natural origin. The purpose of the present communication is to report the isolation and purification of chymotrypsin from chicken (Gallus dotnesticus) pancreas, and trypsin from turkey (Meleaguh gatlopavo) pancreas. When similar methods were used for isolations, chicken pancreas preferentially yielded chymotrypsin and turkey pancreas preferentially yielded trypsin, alt’hough both enzymes could be act,ivated from whole homogenates of pancreatic tissue from either species. MATERIALS

ANU METHOI,S

Chymotryptic and tryptic activities were determined titrimetrically (7) at 25°C on 30 rrnoles 2 Abbreviat,ions used : I)FP, diisopropyl fluorophosphate; TEE, r,-tyrosine ethyl ester; TAME, tosyl-r,-arginine methyl ester.

170

RYAN TABLE

I

ESTERASE ACTIVITIES FROM EXTILKTS OF CHICKEN AND TURKEY P.\NCRE.IS Specific

Turkey extract” TEE TAME Chicken extract* TEE TAME

activity

X 1OP after?

0 hour

5 hours

20 hours

0 0

3.8 1.3

4.1 3.0

0 0

4.2 0.8

3.4 0.9

0 In all cases 1 pg bovine trypsin per 500 rg protein was used to activat,e at pH 7.0 and 5”. * A fraction precipitated by ammonium sulfate saturation from 0.2 to 0.8 of an extract prepared at pH 7.0 with 0.067 M sodium phosphate from chicken or turkey pancreatic Gssue. 22.5 gm of chicken pancreas and 24.5 gm. turkey pancreas yielded 570 and 870 mg protein, respectively, in this fraction. TEE in 3.0 ml of 0.01 M CaC12 at pH 6.3 for chymotrypsin, and 30 /*moles TAME in 3.0 ml of 0.005 M tris-chloride at pH 7.9 for trypsin. Specific activity is the micromoles substrate hydrolyzed per minute per microgram enzyme protein. Protein was determined by the Biuret, method (8) with chymotrypsin as a standard; TEE was purchased from the Aldrich Chemical CO.~ and recrystallized twice from ethyl acetate as the free base; TAME was purchased from the California Biochemical Corporation. Chicken pancreata were a gift of Reif and Brody, Inc., Petaluma, California. The organs were collected from g-week-old fryers of the Van Tress, Arbor Acres strain. Turkey pancreata were a gift of Armour and Co., Turlock, California. The turkeys were 24-28-week-old males of the Broad Breasted Bronze variety. Pancreata from both species were recovered within 5 minutes after death and frozen over dry ice in plastic bags. The tissue was stored frozen until used. RESULTS

Preliminary Observations of Chymotryptic and Tryptic Activities in Chicken and Turkey Pancreas Frozen chicken pancreat,ic t,issue equivalent to about ten pancreases (24.5 gm), and 3 Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture t,o the exclusion of others that may be suitable.

frozen turkey pancreatic tissue equivalent to about five pancreata (22.5 gm), each in 37.5 ml of 0.067 M sodium phosphate buffer, pH 7.0, were allowed to thaw for 30 minutes at’ room temperature. When thawed, each sample was homogenized at 4’ in a Waring Blendor and allowed to remain at this temperature for 20 hours. The homogenat,e was filtered through cheesclot’h and then through Whatman no. 1 filt,er paper wit’h the aid of Celite. A fraction that precipitat)ed at 0.2 sat,uration of ammonium sulfate and a second that precipitated at 0.8 saturation were collected from each filt’rate at 4” and dissolved in a total volume of 19 ml wit,h water. Enzymes were activated at pH 7.0 by beef trypsin using 1 pg t>rypsin per 500 rg protein at 5”. Enzyme activities resembling chymotrypsin (on TEE) and trypsin (on TAME) were activated from both chicken and turkey preparations (Table I). Preparations from either species were completely inactive until treated with bovine trypsin, suggesting that t#he enzymes were present in the pancreatic tissues as inactive zymogens. Based on known specific activit’ies of bovine trypsin and bovine alpha chymotrypsin the act,ivable avian enzyme proteins represented about l-2% of the t’otal protein of the pancreatic tissue. This activity was somewhat less than that’ found from mammalian pancrerctic tissue (11) but] shows that chicken or turkey pancreatic tissue is a reasonable starting material for isolating these avian enzymes. Extraction

and Fractionation of Chicken and Turkey Endopeptidases

During all of the following operations the tissue or extract’s therefrom were below 5”. Approximately 600 gm of frozen pancreata from either chickens or turkeys were broken into pieces and allowed to thaw in 1200 ml of ice cold 0.25 N sulfuric acid for 30 minutes. The acid was decanted and saved, and the tissue was passed through a meat grinder. The ground material was redispersed in the acid and homogenized at high speed for 1 minute in a Waring Blendor. The homogenate was allowed to steep in the cold for 2 more hours, and was then filtered through cheesecloth. The material retained on the cloth was resuspended in 600 ml of ice cold

A\-IAN

CHYMOTRYPSIN TABLE

AMMoNII.M

~ULF.\TE

FK.WTIONS

ANI)

TRYPSIN.

II

OF ACIWSOLVBLE ~'RO'L'EIN TITRKEY P.\NCRE.LS

~~IYL\~~I'~I~

mg protein/gm whole pancreas

Specific

Fraction

CHICKEN

.\NL,

activity TAMI?

Turkey Chicken

0.4-0.8 saturation

FROM

TEE Chicken

Extracted with acid Precipitated with ammonium sulfate 0.2-0.4 saturation

171

I

10.5

11.2

2.8

5.1

3.1

2.1

(l),~ 0.025 (2)h 0.019 0

Turkey

Chicken

Turkey

0.005 0

0.005 0.009 0

0.128 ~ 0

11Two-hour act,ivation, 1 rg bovine trypsin per 100 pg protein, pH 7.0, 25”. h Activated with chicken intestinal juice. 20 ml intestinal juice from a freshly killed chicken was buffered with 5 ml 0.2 M sodium phosphate, pH 7.0. Juice equal 1.01 gg equivalent of trypsin was added per 1000 pg protein. One-hour activation time, pH 7.0, 25”.

0.25 N sulfuric acid, refiltered, and discarded. The acid filtrates were combined, and solid ammonium sulfate (114 gm per liter) was added t,o give 0.2 saturation. The mixture was filtered overnight by gravity filtrat)ion. The precipitat,e was discarded. The clear supernabant, was adjusted to pH 7.0 using 2 N nTaOH. A second protein fract,ion was removed after addition of solid ammonium sulfate (121 gm per liter) t’o give 0.4 saturation. This fraction was isolated by filt,ration with suction and with Celite as a filt’er aid, dissolved in water, and reprecipitated with ammonium sulfat,e at 0.4 sat)uration. A wat’er solution of t’he twice-precipitated prot)ein (about 1%) was dialyzed against1 distilled water in the cold for 24 hours with conhinual agitation of t)he dialysis t,ubing and frequent changes of water. A heavy protein precipit’ate which formed during dialysis was removed by filtration and the clear solut’ion was lyophilized. Similarly, another protein fraction was precipt’ated at 0.6 saturat)ion of ammonium sulfat’e, isolated, dialyzed, and lyophilized. The two fractions thus isolated were tested for enzyme precursors by the addition of bovine trypsin or chicken intestinal extracts and assayed for active enzymes. Table II is a summary of the extractions and fractionations. Table II shows that’ extraction of chicken pancreata with acid preferentially solubilized chymotrypsin but almost: completely cx-

eluded t,rypsin, and this selective extraction was completely reversed in the case of turkey pancreas, which yielded trypsin and very little chymotrypsin. Table II also shows that, all of t’he chymotrypsin or t’rypsin activity recovered from either chicken or turkey pancreas was found in 1he fraction precipitated at 0.2-0.4 saturation of ammonium sulfate; none was recovered in a fraction isolated between 0.4 and 0.6 saturation. Trypsin or chymotrypsin inhibitors were not detectable in the fractions t’hat precipit,ated at higher salt concentrations. In similar ext)ractions of mammalian pancreatic tissue, chymotrypsinogen and trypsinogen are usually recovered from t,he fractions that precipitated at higher salt concentrations (11) rat’her than from t)he fractions precipitat.ed at low concentrat,ions as reported here. Chromatography of Chicken Chylmotrypsin and Turkey Trypsin with DEAECellulose and Amberlite CG 50 The lyophilized fractions contJaining the zymogens from either chicken or t’urkey pancreas were dissolved in 0.005 ill trischloride buffer at a final pH of 8.0. This solution (about 1 % protein) was passed t.hrough a 2.5 X IO-cm column of DEAF, previously equilibrated with t’he same buffer. DEAE absorbed neither chymotrypsin, trypsin, nor their precursors under these condit,ions, but the column held back one

172

RYAN

h PH

0.8

0.6

0.4 0.2

Jo

0 0

10

20 TUBE

30

40

50

Y

60

NUMBER

PH 6

5 4

TUBE

NUMBER

FIG. 1. Chromatography

of chicken chymotrypsin (top) and turkey t,rypsin (bottom); Amerlite CG 50 used. The conditions are described in the text. The effluent pH is indicated above each elution profile.

half t’o two thirds of the total protein. The breakthrough peak was collected and adjusted to pH 3.0. The solution was dialyzed overnight against 0.001 N HCl, lyophilized, and stored at 0”. During these operations almost all of the turkey trypsinogen and nearly 50 % of the chicken chymot,rypsinogen became active, probably through autocatalysis by trypsin itself. Complete activation of the chymotrypsinogen in t#he lyophilized fractions took place in 4 hours in 1% solutions of the protein in water at pH 7.0 and 25”; 1 mg beef trypsin was used for each 500 mg protein. Preparations containing turkey trypsin usually required IIO further

activat’ion. In either case solutions of activated enzymes were adjusted to pH 3.0 and lyophilized. Chromatography with Amberlit)e CG 50, Type I resin was at 25”. Columns of resin l.fj X 10 cm were prepared by standard procedures (9). The resin was equilibrated with 0.2 M sodium phosphate buffer at pH 4.80 for chromatography of turkey trypsin, or at pH Fj.60 for chromatography of chicken chymotrypsin. Lyophilized preparations of the pancreatic enzymes partially purified on DEAE (40-50 mg) dissolved in a minimal quantity of the equilibrating buffer were applied to the columns. Elution of prot’eins was carried out at appropriate pH gradients with a flow rate of 40-60 ml per hour as 4-ml samples and monitored at 280 mp with a Beckman DU spectrophotomet,er. Suitable aliquots were removed for assays of chymotrypsin and t,rypsin. A pH gradient was established to elute t’urkey trypsin by introducing 0.2 M sodium phosphate, pH .5.74, into a 200.nil constant volume reservoir initially containing 0.2 M sodium phosphate, pH 4.80. Turkey trypsin eluted at pH of about 5.3 (Fig. 1, bottom). The peak was neither sharp nor symmetrical, but’ the profile was reproducible. The protein in the achive peak represented about 70% of the protein put into the column. Chicken chymotrypsin elut’ed between pH 5.60 and 6.90 on a pH gradient established in a similar way with 0.2 M sodium phosphat’e (Fig. 1, top). Chymot’rypsin eluted as a sharp, symmet,rical peak at approximately pH 6.0. The protein in the active peak represented approximately 80% of the total protein put into the column and almost 100% of the enzymic activity. The specific activity of both enzymes remained constant, throughout the elution profiles. Larger columns, 25 X 15 cm, were employed for preparatory work, and the enzymes were eluted step by step with the buffers previously used to establish gradients. Samples of protein of approximately 300-400 mg in 2 or 3 ml of equilibrating buffer were applied to the columns. The breakthrough peaks were completely eluted from the columns with the equilibrating buffer before elution of the enzymes was

AT’IAN

CHYMOTRYPRIN

init,iated. The eluates were collected in 25 ml samples and the enzyme fractions were pooled. The volume of the active fractions was reduced to about 150 ml by lyophilization. The pH was lowered to 3.0 and the solutions were dialyzed against 0.001 N HCI with frequent changes of the dilute acid. The protein was recovered by lyophilization and st’ored at 0”. Crystallization

of Turkey Trypsin

Turkey trypsin was crystallized in either of t,wo ways from t’he active protein preparations that had been partially purified 011 DEAE. In the first method a 10% solut)ion of protein in 0.4 M sodium borate, pH 9.3, was cooled to 5”. Cold saturated magnesium sulfate was added slowly t’o give a 0.5 saturated solution at a final pH of 8.0. After this mixture stood at O”-2” for 3 days, many tiny pointed rods appeared throughout t)he amorphous material. Sat,urated sodium borate was added slowly to dissolve away the amorphous material. The small rods remained insoluble and were isolated by ten trifugation. The cryst’als were dissolved at pH 4 and recrystallized by repeating the above procedure. The following method proved more satisfactory in both facility and yield: To a 10 o/o solution of the prot’ein just] described, in 0.4 M sodium borate, pH 9.3, was added enough saturated ammonium sulfate solution t,o give 0.25 saturation. The solution was adjusted to pH 8.0 and allowed to stand

FIG. 2. Crystals

of turkey

trypsin.

AND TRYPSIN.

173

I TABLE

III

QUMM.WY OF PIWFIED AVI.\N ENZYMES

P.INCRE.ITIC

Enzyme preparations Chicken chymoTurkey trypsitl trypsin

-

mg Protein recovered per gm whole pancreas Specific activity, wit.h TEE TAME

-Chromatog- Chromatog- Twice-crystallized wh Mv ___ ~__

0.9

0.067 0.001

1.2

ca. 0.5

Trace 0.284

Trace 0.268

at, O”-2” for 4-5 days or until crystallization was complet,e. These crystals were thin hexagonal plates rather than t’he small rods obt’ained from magnesium sulfate. The crystals were soluble in dilute acid and could be recryst,allized by repeat,ing the procedure just, described. Figure 2 shows the second crystals. They were extremely fragile and fragmented easily. lcace views were difhcult, to see in the light microscope and even more difficult to photograph clearly, but edge views were easy to see and looked much like the rods obtained from magnesium sulfat,e. Table III summarizes the recoveries and act,ivities of turkey t rypsin, purified by both chromatography and cryst,allization, and includes a summary of purified chicken chymotrypsin. The contaminat,ion of either enzyme with chymot,rypsin or trypsin from the same species is small (less t,han 2 04 in either cast). Extraction of pancreatic enzymes from chicken and t’urkey wit,h acid differed from mammals. The organization of zymogens in the avian pancreas may have influenced t,he way in which acid extracts them, but t.he nearly complete absence of chicken trypsin or turkey chynlotrypsin in the acid-soluble proteins more likely suggests a lability of t.hc two zymogens to acid. Further purification of these latter proteins should be performed using more neutral conditions for solubilizatJion. The crude preparations of chicken chymotrypsin and turkey trypsin after DEAL’

174

RYAN

elution and activation were almost pure enzymes. Chromatography of either chicken or turkey preparations on Amberlite CG 50 gave 70-80% recovery of protein as actfive enzyme. The ease with which trypsin crystallized from the crude preparations further indicates its purity after DEAE treatment. Turkey trypsin and chicken chymotrypsin as well as their zymogens behaved like cationic proteins when eluted from DEAE at pH 8.0. Under identical conditions DEAE absorbs bovine chymotrypsinogen B, but it allows chymotrypsinogen A to elut,e along with trypsinogen (1, 2). Thus, by this comparison chicken chymotrypsinogen is similar to bovine chymotrypsinogen A rather t’han bovine chymotrypsinogen B. A study of the physical and enzymic properties of chicken chymotrypsin in the following communication supports this suggestion (10). The conditions of crystallization of trypsin were similar to those of both bovine (11) and porcine (12) trypsin. Small pointed rods were crystallized from magnesium sulfate solutions, and hexagonal plates (cf. Fig. 2) were obtained from ammonium sulfate solutions. It is possible that the small rods from magnesium sulfate were actually small plates, visible only from an edge view. Birefringence was too weak t’o ident’ify face views of the small rods, if present’. The elution profile of trypsin from Amberlite CG 50 was not symmetrical and gave the appearance of a two-component syst’em. The activity profile gave t’he same double peak. Whether the two peaks are isozymes or a result of autolysis is not’ known. Buck et al. (5) obtained a similar elution profile for both bovine and ovine trypsin by chromatography from Ch3 cellulose. The isolation of chyrnot,rypsin and trypsin from avian pancreata, the evidence that . . these enzymes are present as t’hen” zymogens

and are activated by bovine t,rypsin and by chicken intestinal cont,ents, and the quantities of enzyme found in the pancreat’ic tissue,

support the hypothesis that these enzymes contribute to the digest’ion of protein4 in the avian intestines in much the same way as in mammalian intestines. ACKNOWLEDGMENT The author wishes to thank Dr. Francis Jones for photographing the crystals, and Dr. Hans Lineweaver for his interest and encouragement. REFERENCES P. J., COHEN, E., .IND NEURATH, H., J. Biol. Chem. 233, 344 (1958). GREENE, L. J., HIRS, C. H. W., AND PALADE, G. E., J. Bid. Chem. 238, 2054 (1963). VONK, H. J., in “Comparative Biochemistry” (M. FLORKIN .~ND H. MASON, eds.) Vol. VI, p. 347. Academic Press, New York (1964). HEKRIOTT, R. M., BARTZ, Q. R., AND NORTHROP, J. H., J. Gen. Phylsiol. 21, 575 (1938). ZEIGEL, R. F., J. Ultrastructure Res. 7, 286 (1962). WHITESIDE, C. H., .~ND PRESCOTT, J. M., Proc. Sot. Ezptl. Biol. Med. 110, 741 (1962). ALDRICH, F. L., JR., AND BALLS, A. K., J. Biol. Chem. 233, 1355 (1958). GORN.\LL, A. G., BARDAWILL, G. S., AND I)AVID, A. M., J. Biol. Chem. 177, 751 (1949). HIM, C. H. W., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), \‘ol. I, p. 113, Academic Press, New York (1955). RY.IN, C. A., CLARY, J. J., .\SD TOMIMATSU, Y., Arch. Biochem. Ri0phy.u. 110, 175 (1965). NORTHROP, J. H., KC.NITZ, M., BND HEHKIOTT, Enzymes.” Columbia 1~. M., “Crystalline University Press, New York (1948). VAN MELLE, P. J., LEWIS, S. H., SAMSA, E. G., .\ND WESTFALL, R. J., Enzymologia XXVI, 133 (1963). BUCK, F. F., YITHSYATHIL, A. J., BIER, M., .~IND NORD, F. F., Arch. Biochem. Biophys. 97, 417 (1962). RYAN, C. A., -END CLARY, J. J., Arch. Biochem. Biophys. 108, 169 (1964). KELLER,

4. 5. 6. 7. 8. 9.

10. 11.

12. 13. 14.

1 Both enzymes are highly active proteases that use casein as substrate (10, 14). Chicken chymotrypsin will also clot milk, but turkey trypsin will

not.