Chromatographic studies on trypsin, trypsinogen and the activation process

ARCHIVES

OF

BIOCHEMISTRY

AND

88, 216

BIOPHYSICS

Chromatographic and

the

Studies on Trypsin, Trypsinogen Activation Process’

IRVIN From

the Department

of Agricultural

221 (1960)

E. LIENER

Biochemistry, Received

University

November

of Minnesota,

St. Paul,

Minnesota

6, 1959

Crystalline preparations of trypsin and trypsinogen were chromatographed on carboxymethylcellulose at pH 3.2 using gradient elution of increasing salt concentration. Crystalline trypsin contained an inert component which could be quantitatively precipitated with 0.05 M CaC& at pH 8. This permitted the recovery of trypsin which was chromatographically homogeneous. A chromatographic study of the autocatalytic activation of trypsinogen in the presence and absence of calcium ions suggested that this inert protein may be the result of activation in the absence of calcium ions. It was also demonstrated that this chromatographic technique could be applied to the large-scale preparation of trypsinogen which is relatively low in trypsin. INTRODUCTION

EXPERIMENTAL

Chromatographic studies of trypsin or its precursor, trypsinogen, are complicated by the fact that, at pH values higher than 3, trypsin readily undergoes self-digestion while trypsinogen is autocatalytically transformed into trypsin (1). Recently, Tallan (2) and Keller et al. (3) have reported the successful chromatography of trypsinogen on the Amberlite cationic exchange resin IRC-50 (XE-64) at pH 6. It was not possible, however, to chromatograph trypsin under the same conditions (2). Since, at pH 2-3, trypsin exhibits maximum stability and the autocatalytic activation of trypsinogen is at a minimum (l), chromatography of these proteins at a low pH would be most desirable. This Paper describes a procedure WherebY trypsin, trypsinogen, or a mixture of both can be chromatographed on carboxymethyl cellulose (CMC) at pH 3.2 employing gradient

elution

of increasing

ionic

strength.

1 Paper No. 4277, Scientific Journal Series, Minnesota Agricultural Experiment Station, University of Minnesota, St. Paul, Minn. This work has been supported by grants from the National Institutes of Health, U. S. Public Health Service (RG-4614) and the National Science Foundation (G-5830). 216

Samples of twice-crystallized, salt-free trypsin and once-crystallized trypsinogen (500jo MgSOd) were purchased from the Worthington Biochemical Corporation, Freehold, N. J. A sample of lyophilized, crystalline trypsin (“Trypsilin”) produred by the Mochida Pharmaceutical Company, Tokyo, Japan, was kindly provided by Dr. K. Maekawa. CMC was prepared according to the method described by Peterson and Sober (4). The analytical phase of the chromatographic work was performed on 0.9 X 20.0 cm. columns of CMC equilibrated with 0.005 M citrate buffer, pH 3.2, as the starting buffer. Sufficient pressure for packing the column was provided by the flow of buffer from a separatory funnel located about 40 in. above the top of the column. Solutions containing lo-20 mg. protein, previously dialyzed against the starting buffer, were applied to the column. Gradient elution was immediately started bypassing 0.1 M citrate-O.5 M NaCl buffer, pH 3.2, through a closed mixing chamber containing 250 ml. of the starting buffer. The flow rate was adjusted to 6-9 ml./hr., and 3-ml. fractions were automatically collected with the aid of a drop counter. All of the preceding operations were performed in a cold room where the temperature varied from 4” to 7”. When a comparison between individual runs was critical, the same column was reused simply by re-equilibrating it with the starting buffer between each run. A column was deemed ready for reuse when the effluent no longer

TRYPSIK

AND

contained chloride ions as shown by testing with silver nitrate. The contents of all tubes were examined for protein by measuring their absorbance at 280 rnp. For the determination of activity, duplicate reaction mixtures were prepared consisting of 0.1 ml. sample and 0.5 ml. “activating” buffer. The latter was composed of 0.4 M tris(hydroxymethyl)aminomethane (Tris), pH 8.2, containing 0.05 M CaClz and 4 pg. trypsin/ml. Trypsin activity was determined immediately on one of the mixtures, and, on the other, after standing at 4” for 24 hr. The activity which is measured immediately is due to trypsin, whereas the difference in activit,y before and after activation is attributed to trypsinogen. In each case, activity was measured with benzoyl-L-arginine ethyl ester at 37” employing the spectrophotometric method of Schwert and Takenaka (5). Activities were corrected on the basis of blanks consisting of 0.1 ml. water and 0.5 ml. “activating” buffer. An activity unit is defined as an absorbance shift of 1.0 per min. at 253 rnp. The specific activity refers to the number of activity units/mg. protein, where the latter was obtained by multiplying the absorbance of the protein solution at 280 rnp by a factor of 0.72 (6). The recovery of protein and activity from the chromatographic experiments to be described was essentially quantitative and ranged from 93 to 102y0 of the applied material. RESULTS STUDIES

AND ON

217

TRYPSINOGEN

Volume

of

Effluent

(ml.)

FIG. 1. Chromatography of crystalline trypsin before and after treatment with 0.05 M CaClz . Shaded portions of the curves indicate regions of trypsin activity. A: 15 mg. of twice-crystallized, salt-free trypsin (Worthington). B: Soluble fraction (containing 20 mg. protein) remaining after treatment of trypsin with 0.05 M CaClz . C: Insoluble fraction (8 mg. protein) precipitated by 0.05 M CaCL . D: Rechromatography of pooled tubes (totaling 6 mg. protein) indicated by doubleheaded arrow shown in B. TABLE

I

SPECIFIC ACTIVITIEP OF CRYSTALLINE REFORE AND AFTER TREATMENT 0.05 M CaC12

TRYPSIN WITH

DISCUSSION System

TRYPSIN

Fig. IA shows a chromatogram which is typical of those obtained with the samples of crystalline trypsin available for this study. The most significant feature of this chromatogram is the presence of an inactive peak immediately preceding the active component. As will be noted from Table I, the specific activity of the active peak was about 50 % higher than that of the original sample of trypsin. Of interest also is the presence of what appears to be a second, but minor active component. This point will be considered in greater detail in connection with studies dealing with trypsinogen (see below). It had previously been noted that the addition of CaCL Do a concentrated (0.51.0 %) solution of crystalline trypsin caused the precipitation of inactive protein and an enrichment in the specific activity of the soluble protein (7). It was of interest, therefore, to ascertain whether the inert protein precipitated by CaClz corresponded to the

assayedb

A. B.

Unfractionated trypsin Soluble fraction after treatment with Ca C. Insoluble fraction after treatment with Ca D. Active component of Casoluble fractiond

Before chromatography

Active comag;)e:eent

chromatogrwhy=

14.6 19.7

22.9

0

0

23.8

25.0

26.7

a See text for definition of specific activity. b Capital letters refer to the appropriate chromatographic patterns in Fig. 1. c Specific activity of tube corresponding to major active component. d Tubes pooled as indicated in Fig. 1B and protein precipitated with 0.7 saturated ammonium sulfate (see text for details).

inactive peak noted in the chromatograms of crystalline trypsin. Crystalline, salt-free trypsin solved as completely as possible M borate buffer, pH 8, containing

(30 mg.) was disin 5 ml. of 0.005 0.05 M CaCIZ .

218

LIENER

The resulting suspension was centrifuged, and the pH of t.he supernatant was adjusted to 3.2 and dialyzed against 0.005 M citrate buffer of the same pH. The insoluble protein residue was washed several times with the 0.005 M borate-O.05 M CaClz buffer, dissolved in 3 ml. of 0.1 M citrate buffer, pH 3.2, and finally dialyzed against 0.005 M citrate buffer of the same pH. From absorbance readings at 280 rnp, it was estimated that 30% of the original protein had not become solubilized in the borate-CaClz buffer. All of the activity, however, could be recovered in the soluble fraction (see Table I).

The chromatograms obtained with the soluble and insoluble fractions produced by treatment with CaClz are shown in Figs. 1B

and lC, respectively. It is evident that the soluble fraction no longer contained the inert peak originally present in the untreated enzyme preparation. The inactive, insoluble fraction was eluted as a single, symmetrical peak at a volume which approximated the location of the inert peak of the unfractionated preparation. Tubes corresponding to the active region of the soluble fraction were pooled and brought to 0.7 saturation with solid ammonium sulfate. The precipitated protein was redissolved in water and dialyzed against 0.005 M citrate buffer, pH 3.2. As shown in Fig. lD, a single symmetrical peak was obtained, the specific activity of which was almost twice that of the original enzyme preparation (see Table I>. STUDIES ON TRYPSINOGEN AND THE ACTIVATION PROCESS

‘?;I 0

100

Volume

of Effluent

200

(ml.)

30%

FIG. 2. Activation of trypsinogen in the presence and absence of calcium. Activity due to trypsinogen, . . . . . . . . . trypsin activity shown by shaded portion of curves. A: 25 mg. of crystalline trypsinogen (50’% MgS04) prior to activation. B: Trypsinogen activated in the presence of Ca by dissolving 25 mg. of crystalline trypsinogen (50% MgS04) in 5 ml. of 0.4 M Tris buffer containing 0.05 M CaClz and 161 pg. trypsin. After standing at 4” for 24 hr., the solution was adjusted to pH 3.2 and dialyzed against 0.005 M citrate buffer of the same pH. C: Trypsinogen activated in the absence of Ca under the same conditions as B with the omission of CaClz .

Figure 2A shows the chromatographic pattern of a once-crystallized sample of trypsinogen which had 12 % trypsin activity before activation. Although a single, slightly skewed peak was obtained with respect to the distribution of protein, those portions of the curve having activity due to trypsinogen or trypsin could be delineated. Trypsinogen activity was clearly associated with the main bulk of the protein, whereas the activity due to trypsin occurred at the trailing edge of the main peak with some overlapping of the two types of activities. The close proximity of trypsinogen and trypsin activities is not unexpected in view of their similar isoionic points, 9.3 and 10.1, respectively (8). The slightly higher isoelectric point of trypsin would account for its somewhat slower rate of elution from a cationic exchange adsorbent such as CMC. Clearly discernible in Fig. 2A are three components with trypsin activity. This observation confirms reports that two (9, 10) and possibly three (11) active components can be demonstrated electrophoretically in preparations of crystalline trypsin under certain conditions. When trypsinogen was allowed to undergo autocatalytic activation in the presence of calcium ions, the pattern shown in Fig. 2B was obtained. Trypsinogen activity had

TRYPSIN APiD TRYPSINOGEN

completely disappeared, and there now appeared a peak having trypsin activit’y in a position which coincided with the first (with respect to rate of elut.ion) trypsin component shown in Fig. 2A. The relative magnitude of the other two trypsin components seemed to be unaffected by the activation process. It would appear t.herefore that t,he first trypsin component represents the primary product of the activation process, which, according to Davie and Neurath (12), involves the cleavage of a single pept,ide bond and the release of a hexapeptide from the IX-terminal region of t’he zymogen molecule. The other two minor components having tryptic act,ivit.y may represent. t,he pr0duct.s of secondary reactions involving other cleavage sites, but this is a point which requires furt,her study. The activation of trypsinogen in the absence of calcium ions resulted in the pattern shown in Fig. 2C. The extent of activation in this case was about 40 % of that attainable in the presence of calcium. It is apparent from Fig. 2C that this loss in the efficiency of act’ivation may be attributed to t#he formation of two major inactive components, one of which is closely associated with trypsin itself. These results confirm the observations by Kunitz that, in contrast to the quant,itative conversion of trypsinogen into trypsin that is obtained in the presence of calcium ions (13), activation in the absence of calcium ions leads to t’he formation of “inert protein” as well. Little is known about’ the nature of this inert protein (if indeed it be a single protein) or it,s mode of formation, although Desnuelle and Gabelot,eau (15) noted the appearance of a new K-terminal serine residue during activation without calcium. The technique described here would appear t’o offer a means of isolating these inert protein fractions for further study. Of particular interest here is the inert protein fract’ion which abuts t’he leading edge of the trypsin peak, since a similar component was observed in preparations of crystalline t.rypsin prior t.0 calcium treatment (Fig. 1A). According to informat,ion supplied by the manufacturer (16), the crystalline trypsin had been prepared by the

219

metShodof Kunitz and Northrop (17), which involves the activation of trypsinogen in the absence of calcium ions. Hence the contamination of such preparations with the so-called ‘(inert protein” of Kunitz must be considered a distinct possibility. CHROMATOGRAPHY OF TR~PSINOGES ON A I'RI~PARATIVE SCALE

Difficulty has been frequently experienced in obt,aining preparations of trypsinogen which do not contain appreciable amounts of trypsin (1, 15). Although Tietze (18) used isopropyl phosphorofluoridate as an aid for obtaining trypsinogen which was low in trypsin activity, this technique was not always successful and the author did not recommend his method as a routine preparative procedure. Difficulty in obtaining trypsin-free trypsinogen no doubt arises from the fact that the crystallization of trypsinogen (17) is effected under conditions (pH 8-9) where even traces of trypsin would rapidly accelerat,e the autocatalytic conversion of trypsinogen into trypsin. The chromatographic procedure described here, involving as it does manipulations under exclusively acid conditions where autocatalysis is minimal (14), appeared to offer a simple means for obtaining t’rypsinogen relatively free of trypsin. The starting material was the acid, amorphous filter cake which is precipitated from the filtrate (Tg) remaining after the crystallization of chymotrypsinogen (17) .2Ten grams of this filter cake was dissolved in 50 ml. of 0.1 M citrate buffer, pH 3.2, and exhaustively dialyzed against 1% acetic acid at 4”. Material which precipitated during dialysis was removed b?; centrifugation, and the clear, yellow supernatant solution was applied to a large column (4.5 X 56 cm.) of CMC. The det.ails relating to the operation of this column were essentially the same as t,hose described for the smaller * Filter cake may be purchased from Worthington Biochemical Corp., Freehold, IV. J. If t.he filter cake contains traces of trypsin, the following modification is recommended. Dissolve 10 g. of filter cake in 50 ml. water and adjust pH to 7 with 1 N NaOH. Add 100 ~1. pure isopropyl phosphorofluoridate and allow to st,and at room temperature for 1 hr. Readjust pH to 3.2 with 1 N HCl and dialyze against 1%: acetic acid. The solut,ion is now ready for chromatography.

220

LIENER

6-

a

6-

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2-

c! = 0, $ 0 eD 0.6

500

2* B

,

0.6

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and ultracentrifugal analyses, as well as by finding of a single N-terminal valine residue (8) by Sanger’s technique (19), confirmed the high degree of homogeneity displayed in this chromatogram.3 Attempts to crystallize trypsinogen by the method of Kunita and Northrop (17) were always accompanied by the rapid appearance of the rod-shaped crystals which are characteristic of trypsin. This may have been due to the absence of the pancreatic trypsin inhibitor, since Northrop et al. point out [(I), p. 1251 that, unless this inhibitor is present, it is not possible to crystallize trypsinogen without concomitant activation. REFERENCES 1. NORTHROP,

0

I 0

100 Volume

of Effluen:qOml.)

I 300

FIG.

2.

3. Chromatography of crude trypsinogen. due to trypsinogen: . .. . .. .. . A: Protein derived from 10 g. crude filter cake applied to 4.5 X 56 cm. column. B: Rechromatography of 20 mg. of preparation obtained from fractions which were pooled as indicated by double-headed arrow shown in A.

3.

column with the exception of the following changes: (a) volume of mixing chamber, 1 l., (b) size of fractions collected, 20 ml.; and (c) flow rate, 60 ml./hr.

7.

Activity

Figure 3A shows a typical pattern which was obtained when the crude trypsinogen filter cake was chromatographed on a preparative scale. Tubes exhibiting trypsinogen activity were pooled as indicated, and the protein was precipitated by adding ammonium sulfate to 0.7 saturation. Exhaustive dialysis against 1% acetic acid followed by lyophilization yielded 1.5 g. (per 10 g. of crude filter cake) of a preparation which, after activation, had a specific activity comparable to that of once-crystallized trypsinogen (Fig. 2A). In contrast to the rather high tryptic content of the latter (12 %), however, the chromatographically purified material generally had less than 0.5 % tryptic activity. Rechromatography of this material on a small column gave the pattern shown in Fig. 3B. Electrophoretic

4. 5. 6.

8. 9. 10.

11. 12. 13. 14.

J. H., KIJNITZ, M., AND HERRIOTT, “Crystalline Enzymes.” Columbia R. M., Univ. Press, New York, 1948. TALLAN, H. H., Biochim. et Biophys. Acta 27, 407 (1958). KELLER, P. J., COHEN, E., AND NEURATH, H., J. Biol. Chem. 233,344 (1958). PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot. 78, 751 (1956). SCHWERT, G. W., AND TAKENAKA, Y., Biochim. et Biophys. Acta 16, 570 (1955). DAVIE, E. W., AND NEURATH, H., J. Biol. Chem. 212, 507 (1955). WONG, R., AND LIENER, I. E., Biochim. et Biophys. Acta 36, 80 (1960). NEURATH, H., AND DIXON, G. H., Federation Proc. 16, 791 (1957). NORD, F. F., AND BIER, M., Biochim. et Biophys. Acta 12, 56 (1953). TIMASHEFF, S. N., STURTEVANT, J. M., AND BIER, M., Arch. Biochem. Biophys. 63, 243 (1956). PERRONE, J. C., DISITZER, L. V., AND DOMONT, G., Nature 183, 605 (1959). DAVIE, E. W., AND NEURATH, H., J. Biol. Chem. 212, 515 (1955). MCDONALD, M. R., AND KUNITZ, M., J. Gen. Physiol. 26, 53 (1941). KUNITZ, M., J. Gen. Physiol. 22, 293 (1939).

3 In spite of this evidence of homogeneity, chymotryptic activity (after activation), as measured by the hydrolysis of acetyltyrosine ethyl ester (5), was invariably present to the extent of 0.5-1.0~o. In view of the uncertainty regarding the possibility that even pure trypsin may have some degree of chymotryptic activity (20, 21), any conclusion as to the extent of contamination with chymot,rypsin does not appear to be warranted.

TRYPSIN

15. DESNUELLE, Biochem.

P., AND GABELOTEAU, Biophys. 69, 475 (1957).

C.,

16. “Worthington Descriptive Manual No. p. 34. Worthington Biochemical Corp., hold, N. J., 1959. 17. KUNITZ, M., AND NORTHROP, J. H., J. Physiol. 19, 991 (1936). 18. TIETZE, F., J. Biol. Chem. 204, 1 (1953). 19. FRAENKEL-CONRAT, H., HARRIS, J. I.,

AND

Arch.

IO,” FreeGen.

AND

TRYPSINOGEN

221

A. L., Methods of Biochem. Anal. 2, 359 (1955). 20. MCFADDEN, M. L., AND LASKOWSKI, J., JR., Abstr. p. 71C. 130th Meeting of the American Chemical Society, Atlantic City, N. J., 1956. 21. INAGAMI, T., AND STURTEVANT, J. M., Abstr. p. 60C. 136th Meeting of the American Chemical Society, Atlantic City, N. J., 1959. LEVY,