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Pyrrolidonecarboxylyl peptidase: Stabilization and purification
ARCHIVES
OF
BIOCHEMISTRY
AND
Pyrrolidonecarboxylyi RICHARD Departments
RIOPHYSICS
132, 8(r90 (1969)
Peptidase:
Stabilization
W. ARMENTROUT
of Biology
and Chemistry,
Received December
RUSSELL
AND
University
and
of
California,
Purification’32
F. DOOLITTLE La Jolla,
14, 1968; accepted February
California
$2037
3, 1969
Pyrrolidonecarboxylyl peptidase, an enzyme which specifically hydrolyzes aminoterminal pyrroIidoneearboxyly1 residues from peptides, has been stabilized and purified. The enzymatic activity, derived from a strain of Pseudomonas $uorescens, was protected during purification by the addition of 2-pyrrolidone to all solutions. The final preparation exhibited two close bands on acrylamide gel and was 200 times more active than the crude material. The enzyme demonstrated the ability to specifically remove pyrrolidonecarboxylyl groups from a high molecular weight protein. naturally occurring proteins and from a lvide variety of sources have pyrrolidonecarboxylyl residues at their amino-terminals. Furthermore, this kind of blocked group is frequently produced artifactually in the fragments from enzymatic digests of proteins. Any such molecule terminating in a pyrrolidonecarboxylyl residue lacks a reactive amino-terminal group and, as a result,, presents a major problem for protein sequencing that is difficult to overcome by chemical methods. It is possible, however, to free the amino-terminal successfully by enzymatic means. Earlier, we reported the discovery of an enzyme, pyrrolidonyl peptidase, (now called pyrrolidonecarboxylyl peptidase) which is capable of removing these residues from the aminoterminal of peptides while maintaining the integrity of the remainder of the molecule
specifically remove pyrrolidonecarboxylyl residues from an intact high molecular weight protein. The further purification of the enzyme depended on a means of stabilization during both storage and purification. Various combinations of ammonium sulfate precipitation and freezing were found capable of maintaining activity during storage. The enzyme was very unstable in dilute solution, however, and activity declined during the manipulations of purification. The introduction of relatively high concentrations of the compound 2-pyrrolidone was found to stabilize the enzyme and facilitated further purification. The enzyme was purified from a crude sonicate by high speed centrifugation, protamine sulfate removal of nucleic acids, an
0).
zyme, followed by G-200 Sephadex and DEAE-Sephadex chromatography. This proeedure produced an approximate 70-100 fold increase in specific activity, and an enzyme preparation of sufficient purity for many of the experiments described in this paper. The final stage in purification employed preparative gel electrophoresis. Polyacrylamide gel containing 2-pyrrolidone was used with a Tris-borate buffer-system. The resulting enzyme exhibits two very close bands on analytical aerylamide gels and has about
Many peptides
Although the enzyme was partially purified, a number of problems associated with stability were noted. In this paper, we report the stabilization and further purification of the enzyme and demonstrate its ability to 1 This work was supported by National Institutes of Health Grant AI-07781. p The enzyme was referred t.o in a previous paper (1) as pyrrolidonyl peptidase. It was brought to our attention, and we fully agree, that a more accurat.e designation for the enzyme is pyrrolidonecarboxylyl peptidase. 80
ammonium
sulfate
precipitation
of the en-
PURIFICATION
OF
PYRROLIDONECARBOXYLYL
200 times the specific activity of the starting material. MATERIALS
AND
METHODS
Materials used were the same as previously reported (1) with the following details and additions. 2-Pyrrolidone (practical) was purchased from Matheson, Coleman, and Bell. Protamine sulfate was purchased from Nutritional Biochemicals Corporation. Ammonium sulfate (special enzyme grade) used for protein precipitations was purchased from ?rIann Research Laboratories. Sodium tetrathionate was prepared by the method of Gilman et al. (2)) and stored at 4”. Fibrinogen. Bovine fibrinogen was prepared by the met.hod of Laki (3) and was 95y0 clottable. Bovine fibrinogen was stored in 0.3 M NaCl solution at -20” prior t.0 use. Culture Conditions. The enzyme was prepared from extracts of a strain of fluorescent Pseudomoms which was cultured as previously described (1). Stock solutions used in the maintenance and growth of these cells are as follows. All solutions were stored at 4” unless otherwise noted. Salts and trace metals for the pyrrolidonecarboxylic acid (PCA)” minimal medium (solution A) contained KHzPO~ (2.38 g), KzHPOJ. 3Hz0 (5.66 g), MgS04.7H20 (1.00 g), and trace metals (Solution B) (6.4 ml) in a total volume of 1.0 liter. Trace metals (Solution B) contained CuS04.5HzO (1.02 g), FeS04.7HsO (1.76 g), MnC12.4H20 (1.26 g), ZnSO~.?H~O (0.24 g) in 1 liter. This solution was stored frozen. A PCA solution was made with 50 g of PCA per 300 ml of water and sterilized by filtration through a Millipore filter. The glucose minimal medium contained K2HP04.3H20 (46.0 g), KHsP04 (10.0 g), (NH4)nSOa (5.0 g), NaB citrate.2HzO (3.0 g), MgS04.7HzO (0.5 g), and trace metals (Solution B) (31.25 ml) in a total volume of 5.0 liters. This solution was autoclaved and allowed to cool prior to the addition of 250 ml of sterile S”/;! glucose solution. Slants for the maintenance of the Pseudomonas 3 This straiu of Pseudovaonas is now being carried by the Cycle Chemical Corp., Los Angeles. Freeze-dried cells prepared according to our procedure are currently available from them. 4 Abbreviat,ions used: PCA, free pyrrolidonecarboxylic acid; Pyr: t’he sequence designation for pyrrolidoneearboxylyl residues in peptides or proteins, as in Pyr-Ala. PTH-amino acid, the phenylthiohydantoin derivative of the amino acid produring Edman degradations. TCA, duced trichloroacetic acid.
PEPTIDASE
81
strain cont,ained minimal medium with PCA as the sole source of carbon and nitrogen. The slants were made from 2.1 g of special Noble agar (Difco) added to 100 ml of salts and trace metals (Solution A) and autoclaved. 3.0 ml of sterile PCA solution and 3.0 ml of sterile 5L;Ic NaOH were added after the agar solution had cooled, but while still liquid. Slants were poured from this solution; the pH was close to neutrality. Slant cultures were transferred to PCA liquid medium and this culture, in turn, was used to inoculate larger volumes of glucose minimal medium. PC.4 liquid medium was made by the addition of 6 mls of sterile PCA solution and 6 ml of sterile 5V0 NaOH to 200 ml of sterile salts aud trace metals (Solution A). When the PCA liquid culture reached an OD,,, greater than 1.0, it was used to inoculate 5.0 liters of glucose medium, which was then used as the inoculum for 100 liters of glucose medium. The large scale fermentation was carried out in a Fermacell Fermentor, Model F-130 (New Brunswick Scientific Company) at 30” for approximately 48 hr. Cells were harvested directly from the fermentor by continuous-flow centrifugation in a Sharples refrigerated centrifuge (type AS-16). The average yield of bacteria from 100 liters was 420 g wet weight. The cell paste was frozen and lyophilized (average yield from 100 liters of cells: 117 g dry weight), and the pulverized dry material stored at -20”. Assay and units. Pyrrolidonecarboxylyl peptidase activity was assayed by the method previously reported (1) using L-pyrrolidonecarboxylylL-alanine (Pyr-Ala) as a substrate. The reaction was carried out in a total volume of 75 ~1 at 30”. 50 bl of buffered enzyme solution (pH 7.3) was added to 25 ~1 of a 0.57, Pyr-Ala solution (0.63 &moles). One unit of enzyme activity was defined as the amount of enzyme which produces 1 mpmole of free alanine per minute. Relative protein concentration was measured as the opt,ical density at 280 millimicrons of a solution appropriately diluted with 0.15 M NaCl. Specific activity of the enzyme is expressed as 1000 times the millimicromoles of alanine produced per minute under the assay conditions defined above, divided by the ODssa of the enzyme solution. Bujkrs. Enzyme preparations containing either ammonium sulfate or 2-pyrrolidone were dialyzed against 0.05 M potassium phosphate buffer, pH 7.3, containing 0.01 1~ 2-mercaptoethanol and 0.001 M EDTA prior to assay. This buffer is referred to as “Buffer A” throughout this paper. All preparative steps through G-200 Sephadex column chromatography were carried out in this phosphate buffer with 0.1 M 2-pyrrolidone added. The latter
82
ARMENTROUT
AND
buffer with 2-pyrrolidone added is referred to as “Buffer B”. DEAE-Sephadex (A-25) columns were equilibrated in 0.05 M sodium phosphate buffer, pH 8.0 with 0.01 M 2-mercaptoethanol, 0.1 M 2-pyrrolidone, and 0.001 M EDTA (“Buffer C”). Analytical and preparative polyacrylamide gel electrophoresis was conducted using a Tris-borate buffer system. This buffer (referred to as “Trisborate buffer”) contained 0.025 M Tris (pH 8.4), 0.20 M boric acid, 0.1 M NaCl, 0.005 M sodium thioglycolate, 0.001 M EDTA, and 0.10 M 2-pyrrolidone. Preparation of sephadex. G-200 and G-25 Sephadex (coarse) were prepared by boiling for 4-6 hr and were stored in Buffer A. “Fines” were not removed; G-200 columns were used only once. A-25 was prepared by extensive washing with 0.5 N NaOH, water, and 0.5 N HCL. A-25 columns were equilibrated first at room temperature with Buffer C which was 0.25 M in phosphate, then at 4” with the normal 0.05 M phosphate Buffer C. Equilibration was continued until constant conductivity and pH were attained. Dialysis tubing. Dialysis tubing was prepared by boiling for 2 hr in 0.001 M EDTA, followed by rinsing in glass-distilled water and boiling again in water. Amino-terminal analysis. The amino-terminal amino acids were determined by the quantitative Edman method (4) with the following conditions and variations. PTH-amino acids were identified
DOOLITTLE
qualitatively by thin layer chromatography on Eastman Chromagram Silica Gel sheets with fluorescent indicator. Two ascending solvent systern3 were used for each qualitative analysis: System “D” and system “E” of Edman and SjGquist (5). Quantitation of PTH-amino acids was carried out on descending paper chromatography in Solvents one and three or two and three for each sample (4). Each of the PTH-amino acids and the appropriate background control were eluted from the chromatogram in 2.0 mls of 95% ethanol. Spectra from 235 to 330 rnp gave positive identification of the eluted material as being PTH-derivatives. The optical density at 269 w was used to calculat’e the amount of the PTH-amino acid, using the molar extinction coefficients of 15,900 for PTH-glu, 15,600 for PTH-tyr, 15,500 for PTH-phe, 16,100 for PTH-asp, and 14,900 for PTH-gly. Electrophoresis. Analytical acrylamide gel electrophoresis was carried out by the method described by Davis (6). A running gel of 60-mm length was poured in glass tubes of 6-mm diameter. A 13-mm concentrating gel was poured directly on to the running gel without a spacer. The running gel was 5% acrylamide and was polymerized with ammonium persulfate. Prior to polymerization, the gel mixture was brought to 0.1 M 2-pyrrolidone and 0.001 M EDTA. A standard 2.5% acrylamide concentrating gel with sucrose was used; it also contained 0.1 M 2-pyrrolidone and 0.001 M EDTA,
2.0
-
0
100 EFFLUENT
200
200 VOLUME
(Mlsl
FIG. 1. A-25 column chromatography of pyrrolidonecarboxylyl peptidase. The ammonium precipitates from five G-200 column pools (90 ODm units, 103 enzyme units) were applied to column (2.5 X 21 cm) after desalting on a G-25 column. The A-25 column was washed with Buffer C followed by elution with a O-3y0 NaCl gradient (see Methods). Effluent samples (0.10 assayed for 12 hr; activity (broken line) is expressed as millimicromoles of alanine produced
sulfate the A-25 80 ml of ml) were in 12 hr.
PURIFICATION
OF
PYRROLIDONECARBOXYLYL
but was photo-polymerized with riboflavin as catalyst. A few grains of solid sucrose were added to the samples prior to application in order to prevent diffusion. Samples were run at 3 mA per tube for approximately 3 hr at 4”. Protein in the gels was fixed for 20 min in 10% TCA and then stained in 176 (w/v) solution of Amido Schwartz dye in 70/b acetic acid. The gels were destained with extensive washing in 10yO acetic acid. Preparative gel electrophoresis. A Buchler preparative polyacrylamide gel electrophoresis apparatus was employed. A 6% acrylamide running gel, containing 0.1 M 2-pyrrolidone and 0.001 M EDTA was used without a concentrating gel. Ammonium persulfate was the catalyst used in the polymerization of the gel; the gel volume was 100 ml. The apparatus was run at 50 mA for 2 hr at 4” prior to applying the sample in order to allow the thioglycolate to migrate into the gel and reduce any residual ammonium persulfate. The sample was applied in a total volume of 2.0 ml and contained sucrose. Approximately 18 hr electrophoresis at 50 mA (4”) was required to collect the enzyme from the gel. Purijkation. All manipulations were carried out in the cold, at 0” where possible, but at 4” otherwise. 12.5 g of lyophilized bacteria were suspended in 60 ml of ice cold Buffer B (containing P-pyrrolidone). The cells were placed in a glass “flowaround” vessel on ice and Rere disrupted with ten to fifteen 30.set blasts from a Branson Sonifier at maximal power setting. The sonicated material was centrifuged twice, first at 39,000g for 30 min,
I
I
0
lo
I
PEPTIDASE
53
and then the supernatant material was centrifuged again at the same speed for 10 min. The final supernate was diluted with Buffer B to an OD280 of 50. This material is referred to as “crude enzyme” here after. Nucleic acids were precipitat)ed by the slow addition of freshly prepared 1’7, protamine sulfate solution to a final concentration of 0.14yc. After the addition, the enzyme preparation was stirred on ice for 20 min and t,hen centrifuged at 15,000g for 20 min. The pellet was discarded; the supernate is referred to as “protamine sulfate enzyme.” The enzyme activity was precipitated by the cautious addition of saturated ammonium sulfate to 42% saturation. The solution was stirred at 0” for 1 hr after this addition, followed by centrifugation for 30 min at 39,000g. The resulting precipitates were resuspended in Buffer B to an approximate OD,,, of 150 and quick-frozen in an isopropanol-dry ice freezing mixture for storage at -70”. This material is referred to as “4274 ammonium sulfate enzyme.” On G-200 Sephadex gel columns, the enzyme activity eluted between the excluded protein and the included salt fraction (1). The 42yc ammonium sulfate enzyme was thawed and 2 ml (300 ODQ8,, units) were applied to each of two G-200 columns (2.5 X 36 cm) equilibrated with Buffer B. The active fractions were pooled and precipitated by the slow addition of 0.45 g solid ammonium sulfate per milliliter of enzyme solution. These ammonium sulfate suspensions were allowed to stand overnight at 0” and were subsequently centrifuged 30 min at, 39,000g. The supernatant fluid was carefldly
I
I
20 30 40 mm from Origin
I
I
50
60
FIG. 2. Location of enzymatic activity on an analytical polya.crylamide gel. Dialyzed G-200 enzyme (100 ~1) was applied to each 5% acrylamide gel and electrophoresed as per usual (see text). The enaymatic activity was located by assay of gel slices and is represented as millimicromoles of alanine produced in 10 hr. The photograph is of one of the stained gels that was run simultaneously with the assayed gel.
84
ARMENTROUT
and completely decanted pellets were stored at -20” “G-200 enzyme.” The pellets from four to redissolved in Buffer C to a This material was applied
and discarded. and are referred
AND The to as
DOOLITTLE
(2.5 X 38 cm) equilibrated in ing. The enzyme was recovered and approximately 100 OD~W very slowly to an A-25 column, application of the enzyme, the with approximately a column
five G-200 pools were volume of about 5 ml. to a G-25 column
Buffer in unit,s 2.5 X column volume
C for desaltabout 23 ml, were applied 24 cm. After was washed of Buffer C.
125 1.200
‘;’ P s
;‘\ ’ ‘1 I \ \ I \ I \ I \ \ I \
0.800
; 2
I
de 0 ii 0.400
I I I :i A
100
\
\
\ I I \ \
75 c 5 F 9 50
\
\
\-,w--
--
--_#*--*
‘-‘.I.-
-co--.--
--
1
25
‘-.
4
0
100
200 EFFLUENT
FIG. 3. Preparative carboxylyl pept)idase and electrophoresed with 25 ~1 of Pyr-Ala n 12 hr (solid line).
0
300 VOLUME
400
500
500
(MIS)
acrylamide gel electrophoresis: Elution patterns of ODzso and pyrrolidoneactivity. An A-25 enzyme preparation was applied to a By0 polyacrylamide gel using the Tris-borate running buffer. Samples of the effluent (0.1 ml) were assayed solution for 12 hr. Activity is presented as millimicromoles of alanine produced
FIG. 4. Polyacrylamide gels of enzyme preparations at various stages in purification. The samples were run on 5% acrylamide gels with a concentrating gel for approximately 210 min using the Trisborate buffer system at 3 mA per tube at 4” (see Methods). Gel B 1 was run with 200 ~1 of electrophoretically prepared enzyme; gel #2 was run with 100 ~1 of A-25 enzyme; and gel #3 was run with 25 ~1 of G-200 enzyme.
PURIFICATION
OF
PYRROLIDONECARBOXYLY
The activity was eluted by application of a salt gradient made from equal volumes (50 ml) of Buffer C without NaCl and the same buffer containing 3% NaCI. The column was allowed to flow at the rate of 18 ml per hr; activity was eluted early in the salt gradient. Effluent tubes were placed on ice as they came off the fraction collector and were allowed to stand overnight, during which time, O.l-ml aliquots from fractions were incubating for assay (Fig. 1). The peak tubes were pooled and precipitated by the addition of 0.6 g of solid ammonium sulfate per milliliter and allowed to stand overnight on ice. The slurry was spun down at 39,OOOg, and stored as a pellet at -20”. These precipitates are referred to as “A-25 enzyme.” Experiments using analytical polyacrylamide gel electrophoresis demonstrated the feasibility of using preparative electrophoresis as a further purification step. By using suitable stabilizing agents in the buffer system, it was possible to recover enzymatic activity from the analytical gels and correlate this activity with a specific set of bands. The recovery of activity depended on the use of a Tris-borate buffer, which did not interfere with the ninhydrin reagent in the enzyme assay. The 2-pyrrolidone and EDTA were included in the gel prior t,o polymerization. A reducing agent was not included at this point since it would interfere with the polymerization process. To provide a reducing milieu within the gel, thioglycolate was included in the tray buffer; it preceded the protein during electrophoresis. Figure 2 shows the position of the enzymatic activity relative to the major protein bands in the gel. In these experiments, 100 pl of the dialyzed G-200 enzyme was electrophoresed in each gel. One of the gels was stained and another was cut into approximately 1.5.mm slices. The gel slices were placed in tubes containing 0.2 ml of phosphate buffer (Buffer A) and 25 ~1 of Pyr-Ala solution. The slices were allowed to react for 10 hours and were assayed in the usual fashion. It is clear that the enzyme is one of the fastest migrating proteins under these conditions (Fig. 2). For preparative electrophoresis, the ammonium sulfate precipitates from one or two A-25 columns were redissolved to a volume of 2.0 ml in diluted Tris-borate buffer (0.01 M Tris, 0.02 M borate, 0.02 M NaCl, 0.004 M thioglycolate, 0.004 M EDTA, 0.04 M 2-pyrrolidone) and then briefly dialyzed versus two changes of this same buffer (total 3 hr dialysis). A small amount of solid sucrose was added to the sample before application to the surface of the acrylamide gel. In the elution profile of the electrophoresis (Fig. 3), the major OD2m is due to a non-protein contaminant in the buffer
85
PEPTIDASE
system and corresponds to the leading band on the analytical gel. Because of the small amount of protein applied, no distinct ODzso profile was obtained. The enzyme activity, however, was present close to the front as expected, and appeared after 16-18 hr of electrophoresis. The pooled active fractions were concentrated under nitrogen using a Diaflow pressure dialysis apparatus with a 10,000 molecular weight cut-off filter. The enzyme was stored frozen. The relative purity of this preparation can be seen in Fig. 4. Analytical gels of G-200, A-25, and
TABLE
I
SUMMARY OFTHEPURIFICATION CARBOXYLYLPEPTIDASEFROM Pseudomonad
OFPYRROLIDONEAFLUORESCENT
Total proteinb
Fraction”
Crude Protamine sulfate 42y0 Ammonium sulfate G-200
4360 3024
4634 4621
46 75
(100) 99.7
1311
2767
104
59.7
137
2888
1054
62.3
G-200, applied to A-25e A-25
93
879
475
(100)
9
604
3342
68
5.2
200
1925
0.7
121
8788
A-25, applied electrophoresis” Electrophoretic
to
(1~)
60
0 See Methods for a description of fractions. b (ODm X Vol). c (Millimicromoles of alanine produced per minute per milliliter) X (Vol). d Millimicromoles of alanine produced per minute per ODZW of 1.0. B The data presented are for a single preparation purified through G-200 column chromatography and give the activity recovered from ammonium sulfate precipitation of the peak fraction of the column effluent. The A-25 and electrophoretic steps each used preparations that were not peak fractions, but were larger pools of effluent. As a result, the specific activity of the material applied in these two steps was less than the peak activity of the preceding step. There is some variation in the specific activity of the crude sonicate; a specific activity of 25 is average. The data presented for the purification through G-266 chromatography are in the upper range of activities obtained.
86
ARMENTROUT
AND
electrophoretic enzyme preparations are compared. In the G-200 sample, the leading protein band (the second band visible in the gels; the first is the non-protein artifact referred to above) is faint when compared to the trailing bands. However, in the A-25 sample, the leading protein band has become a major band, and some of the trailing bands have disappeared. This leading band is also the one corresponding to the position of enzymatic activity. The product after preparative electrophoresis exhibits two major bands, again at the leading position,
DOOLITTLE
enzyme stability produced irregular yields and did not permit the lengthy procedures required for greater purification. Before further purification of this enzyme was possible, the problem of instability in solution had to be solved. While activity declines rapidly in solution, it was observed that enzymatic hydrolysis of the substrate PyrAla continued at a linear rate for up to 18 hr in some cases. This indication of substrate stabiliiation led us to search for a substrateanalogue which might afford protection. Z-pyrrolidone was found suitable in this regard. This substance acted as an unusual inhibitor of the enzyme (Fig. 5). An enzyme preparation (through the G-200 step) was assayed at a constant level of substrate (8 X lOA M) while the concentration of 2-pyrrolidone was varied. While there was clearly inhibition, suggesting that the enzyme was binding 2pyrrolidone, very high concentrations of 2-pyrrolidone (100 times the amount producing 50% inhibition) did not produce complete inhibition. The kinetics
RESULTS
The purification of pyrrolidonecarboxylyl peptidase is summarized in Table I. The procedure produces an approximate 200 fold increase in specific activity. The final enzyme preparation exhibits two bands on acrylamide gel (Fig. 4). Stabilization. We have previously reported a routine procedure for purification of the enzyme including G-200 gel filtration (1). Although we had successfully recovered active enzyme from A-25 columns, lack of
0.5
0.4
1
iI-1 0.1
0
o
-
I 0
.02
r
I
.04
.06
t .lO
.08
2 - PYRROLIDONE
.12
.14
M/L
FIG. 5. Inhibition of pyrrolidonecarboxylyl peptidase by 2-pyrrolidone. Dialyzed G-200 enzyme with a specific activity of 454 was used. Reaction was carried out in a total volume of 125 pl containing the usual volume of enzyme solution, 50 ~1. The concentration of the substrate, Pyr-Ala, was 0.8 X 10-a M and the concentration of the inhibitor 2-pyrrolidone was varied from 0 to 0.1 M. The reaction time was 2 hr at 30”; the concentration of 2-pyrrolidone is presented as molarity; activity is presented as millimicromoles of alanine produced per minute.
PURIFICATION
OF
PYRROLIDONECARBOXYLYL
of inhibition resemble a noncompetitive type (Fig. 6). In this experiment, G-200 enzyme was assayed in the presence of sufficient inhibitor to produce 50% reduction in the rate of hydrolysis of Pyr-Ala (2.6 X 1O-3 M). An increase in the concentration of the substrate from none to saturation and beyond did not reverse the effects of 2-pyrrolidone. Enzyme preparations to which 0.1 M 2-pyrrolidone had been added showed 100 % recovery of activity when 2-pyrrolidone was removed by rapid dialysis (2 changes of Buffer A, 3 hr total of dialysis). Pyrrolidonecarboxylyl peptidase was dramatically stabilized by the addition of 2-pyrrolidone at 0.1 M concentration (Fig. 7). An enzyme preparation from a G-200 Sephadex column run with buffer A (without 2-pyrrolidone) was divided into two portions, one of which was brought to 0.1 dcT2-pyrrolidone. Both were stored at 0” and at intervals, aliquots were removed from
l/V
2.8
--
2.4
--
2.0
--
1.6
--
PEPTIDASE
87
each, dialyzed against Buffer A, and assayed. After 4 days storage at 0”, the solution without 2-pyrrolidone had declined 80% in specific activity, while the activity of the solution protected by 2-pyrrolidone was virtually unchanged. Storage. Stability tests were conducted to find at which stages of purification the enzyme preparation could be conveniently stored. Our normal procedure was to store freeze-dried cells at -20°, 42 % ammonium sulfate enzyme at -7O”, and the ammonium sulfate precipitates from the active pools of the G-200 and A-25 columns at -20”. The G-200 enzyme remained unchanged in activity for at least 6 weeks; the A-25 material suffered some decline in activity, but temporary storage for several days was possible (Fig. 8). Protection of the active sulfyhydryl. Pyrrolidonecarboxylyl peptidase was found to be a “sulfyhydryl” enzyme (1); as such, EDTA
FIG. 6. Reciprocal plots of pyrrolidonecarboxylyl peptidase activity. Dialyzed G-266 enzyme with a specific activity of 475 was used in a total volume of 100 pl containing the usual 50 pl of the enzyme solution. The concentration of substrate Pyr-Ala was varied from 0 to 5 X 1O-3 M at two levels of the inhibitor, 2-pyrrolidone, 0 (X) and 2.6 X lOMa M (0). Reaction time was 1.75 hours at 30”. The substrate is presented as the reciprocal of the molarity; velocity as the reciprocal of millimicromoles alanine produced per minute. The approximate K, indicated for Pyr-Ala is 2 X 10e3 M.
88
ARMENTROUT
AND DOOLITTLE
units were recovered by this method after the powder had been stored for 3 days at room temperature. Removal of the pyrrolidonecarboxylyl residue from bovine jibrinogen. The usual means of dealing with a pyrrolidonecarboxylyl blocked amino-terminal in a protein involve isolation of an amino-terminal fragment from an enzyme digest. It would be more convenient if the blocking residue could be removed from the intact protein molecule. Accordingly, we wished to seeif pyrrolidonecarboxylyl peptidase might act on whole 01 I 0 I 2 4 proteins as well as small peptides. For this 3 TIME (Days1 purpose, bovine fibrinogen was chosen as the test protein since it is known to have a FIG. 7. Stabilization of pyrrolidonecarboxylyl residue as one of peptidase by 2-pyrrolidone. Enzyme prepared as pyrrolidonecarboxylyl its three amino-terminal amino acids (8). usual through the 42y0 ammonium sulfate precipitation was applied to a G-200 Sephadex column Phenylalanine is the residue adjacent to the prepared with Buffer A (without 2-pyrrolidone). terminal pyrrolidonecarboxylyl residue in The ammonium sulfate precipitate of the resulting the /3 chain. The relative rates at which the G-200 enzyme pool was divided into two parts. enzyme splits pyrrolidonecarboxylyl residues One part was dialyzed against buffer A (A) and from phenylalanine and other penultimate the other against Buffer B (with 2-pyrrolidone) amino acids have been studied (9). The (0). Both dialysates were stored on ice. Aliquots presence of two unblocked amino-terminals of each were removed and dialyzed against Buffer A prior to assay. Specific activity is presented as (glutamic acid and tyrosine) in the same molecule which contained the blocked endmillimicromoles of alanine produced per minute per 1.0 ODzso unit of protein (X 100). group offered a convenient internal control, permitting us to measure the efficiency of the Edman method for each determination. and 2-mercaptoethanol were used as protectFor this experiment “A-25 enzyme” was ing agents in solution. It is possible to dialyzed against buffer A containing 0.1 effectively protect the active sulfhydryl M NaCl. The enzyme had a specific activity group of such an enzyme by reversibly of about 1,000 and an OD2g0 of 5. One derivatizing it to an inactive form with milliliter of this solution (10.4 enzyme units) certain -SH blocking reagents. A recent was added to 8 ml of a solution of bovine example of such a procedure is the protection fibrinogen (ca 1.0 micromoles) dialyzed of ficin during purification by blocking with against buffer A containing 0.1 M NaCl. The tetrathionate (7). This method is highly NaCl was necessary to keep the fibrinogen suitable for protecting pyrrolidonecarboxylyl peptidase as a lyophilized powder, since in solution. The final reaction volume was 9.0 ml. An equal amount of enzyme was once the derivative is formed, all salts and added to a control preparation. Immediately protecting reagents can be removed. after addition of the enzyme, 3.0 ml were An enzyme preparation purified through removed from both the control and the G-200 chromatography was dialyzed against two changes of 0.0015 M tetrathionate solu- digestion tubes. Each sample was added to 3.0 ml of pyridine and frozen at -20” tion, and then against water (1 hr each). until subjected to amino-terminal analysis. This treatment completely inactivated the enzyme. The dialysate was then quick frozen Digestion was carried out at 30”. The amino-terminals of bovine fibrinogen and lyophilized. When the dry powder was which had been digested in this manner for resuspended in Buffer A, the 2-mercaptofor 0, 13, and 21 hr were qualitatively idenethanol reversed the inhibition by tetrathionate. About 50% of the original enzyme tified on thin-layer chromatography and 600
PURIFICATION
OF
0
PYRROLIDONECARBOXYLYL
10
30
20
89
PEPTIDASE
40
50
TIME (Days)
FIG. 8. Stability of pyrrolidonecarboxylyl tates of G-260 and A-25 preparations. The column were divided into equal parts and lyzed against Buffer A and assayed. The produced per minute per 1.0 ODm unit of TABLE
peptidase during storage ammonium sulfate precipitates stored at -20”. At intervals, specific activity is presented protein (X 1000).
II
SPECIFIC REMOVAL OF THE PYRROLIDONECARBOXYLYL RESIDUES FROM THE AMINO-TERMINAL BOVINE FIBRINOGEN BY PYRROLIDONECARBOXYLYL
PEPTIDASE” Hours
FTH
OF
Amino
200.1c 197.7 None
regardless of digestion time. As anticipated, phenylalanine was the only PTH-amino acid unmasked during the course of the digestion; no other residue increased significantly (Table II).
of digestionb
acid 0
Glutamic acid Tyrosine Phenylalanine
as ammonium sulfate precipifrom one G-200 and one A-25 a sample precipitate was diaas millimicromoles of alanine
13
187.8 171.6 45.3
a See text for details. b Reaction time at 30”. 0 Millimicromoles of PTH-amino ered from 60 mg of bovine fibrinogen @ 330,000 MW).
21
184.2 210.6 170.1
acid recov(180 mpmoles
then quantitated using a paper chromatographic system. The enzyme controls (no substrate) did not produce any identifiable PTH-amino acid. Our fibrinogen samples, however, always exhibited small amounts of PTH-glycine and PTH-aspartic acid,
DISCUSSION
We have attempted to provide a convenient laboratory reagent which will specifically remove pyrrolidonecarboxylyl residues from proteins or peptides. In order to accomplish this end, we searched for and found an enzyme activity present in a soil microorganism which specifically removes pyrrolidonecarboxylyl residues. It was necessary to purify this enzyme to the point where it was free of contaminating proteolytic activities and was sufliciently active that small quantities of enzyme would be effective. That we were successful in these efforts was demonstrated by the ability of the enzyme to remove 80% of the pyrrolidone-
90
ARMENTROUT
carboxylyl residues from as large a protein as bovine fibrinogen without detectably splitting any other peptide bonds. In that particular experiment an A-25 preparation, representing an overall purification of about 60-fold, was used. In general, however, G-200 enzyme preparations, ranging from 20 to 40 times purified relative to crude extracts, are sufficient for use on small peptides where there is less risk of indiscriminate proteolysis (1, 9). The enzyme itself appears to be relatively small and acidic from its high mobility in 5% acrylamide gel at pH 8.4 and inclusion in G-200 and G-150 Sephadex. It is stabilized against loss of activity in solution at 0” by high concentrations of 2-pyrrolidone. This substance is a noncompetitive inhibitor of the enzyme, whose action is reversible and does not produce total inhibition even at high concentrations. The physiological role of the enzyme is still unknown. The particular strain of Pseudomonas used as a source of enzyme was isolated from the soil by enrichment with PCA and selected for its ability to use PCA as the sole source of both carbon and nitrogen. While the enzyme activity of pyrrolidonecarboxylyl peptidase appears to be irrelevent to this degradation of PCA, we cannot exclude the possibility that the enzyme is involved in some nutritional process. On the other hand, there is recent evidence implicating PCA in the initiation of protein synthesis in rabbit lymphocytes (10). One is tempted to draw a parallel between mammalian chain initiation and the mechanism known to exist in E. coli. In the latter case, the initiating formylated methionyl residue is specifically removed from the protein by
AND
DOOLITTLE
an enzyme after synthesis is completed (11). It is conceivable that pyrrolidonecarboxylyl peptidase might have a similar function in the mammalian system. We have found an activity similar to that of pyrrolidonecarboxylyl peptidase in rat liver. However, the fact that pyrrolidonecarboxylyl residues exist at the amino-terminal of some proteins, and that bacterial pyrrolidonecarboxylyl peptidase, at any rate, is active in removing these residues, would tend to argue against such a role for the enzyme. ACKNOWLEDGMENTS We are pleased to acknowledge the assistance of Miss Malinda H&in, Schmidt, and Mr. Francis Lau. We wish Dr. G. Fuller for help and instruction techniques of gel electrophoresis.
technical Mrs. L. to thank in the
REFERENCES 1. DOOLITTLE, R. F. AND ARMENTROUT, R. W., Biochem. 7, 516 (1968). 2. GILMAN, A., PHILIPS, F. S., KOELLE, E. S., ALLEN, R. P., AND ST. JOHN, E., Am. J. Physiol. 147, 115 (1946). 3. LAKI, K., Arch. Biochem. Biophys. 33, 317 (1951). 4. SJGQUIST, J., Biochim. Biophys. Acta 41, 20 (1960). 5. EDMAN, P., AND SJ~QUIST, J., Acta Chem. Stand. 10, 1507 (1956). 6. DAVIS, B. J., Ann. N.Y. Accd Sci. 121, 405 (1964) . 7. ENGLUND, P. T., KING, T. P., CRAIG, L. C., AND WALTI, A., Biochem. 7, 163 (1968). 8. BLOMBXCK, B. AND DOOLITTLE, R. F., Ada Chem. Stand. 17, 1816 (1963). 9. ULIANA, J. A. AND DOOLITTLE, R. F., Arch. Biochem. Biophys. 131, 561 (1969). 10. MOAV, B. AND HARRIS, T. N., Biochem. Biophys. Res. Commun. 29, 773 (1967). 11. ADAMS, J. M., J. Mol. Bid. 33, 571 (1968).
OF
BIOCHEMISTRY
AND
Pyrrolidonecarboxylyi RICHARD Departments
RIOPHYSICS
132, 8(r90 (1969)
Peptidase:
Stabilization
W. ARMENTROUT
of Biology
and Chemistry,
Received December
RUSSELL
AND
University
and
of
California,
Purification’32
F. DOOLITTLE La Jolla,
14, 1968; accepted February
California
$2037
3, 1969
Pyrrolidonecarboxylyl peptidase, an enzyme which specifically hydrolyzes aminoterminal pyrroIidoneearboxyly1 residues from peptides, has been stabilized and purified. The enzymatic activity, derived from a strain of Pseudomonas $uorescens, was protected during purification by the addition of 2-pyrrolidone to all solutions. The final preparation exhibited two close bands on acrylamide gel and was 200 times more active than the crude material. The enzyme demonstrated the ability to specifically remove pyrrolidonecarboxylyl groups from a high molecular weight protein. naturally occurring proteins and from a lvide variety of sources have pyrrolidonecarboxylyl residues at their amino-terminals. Furthermore, this kind of blocked group is frequently produced artifactually in the fragments from enzymatic digests of proteins. Any such molecule terminating in a pyrrolidonecarboxylyl residue lacks a reactive amino-terminal group and, as a result,, presents a major problem for protein sequencing that is difficult to overcome by chemical methods. It is possible, however, to free the amino-terminal successfully by enzymatic means. Earlier, we reported the discovery of an enzyme, pyrrolidonyl peptidase, (now called pyrrolidonecarboxylyl peptidase) which is capable of removing these residues from the aminoterminal of peptides while maintaining the integrity of the remainder of the molecule
specifically remove pyrrolidonecarboxylyl residues from an intact high molecular weight protein. The further purification of the enzyme depended on a means of stabilization during both storage and purification. Various combinations of ammonium sulfate precipitation and freezing were found capable of maintaining activity during storage. The enzyme was very unstable in dilute solution, however, and activity declined during the manipulations of purification. The introduction of relatively high concentrations of the compound 2-pyrrolidone was found to stabilize the enzyme and facilitated further purification. The enzyme was purified from a crude sonicate by high speed centrifugation, protamine sulfate removal of nucleic acids, an
0).
zyme, followed by G-200 Sephadex and DEAE-Sephadex chromatography. This proeedure produced an approximate 70-100 fold increase in specific activity, and an enzyme preparation of sufficient purity for many of the experiments described in this paper. The final stage in purification employed preparative gel electrophoresis. Polyacrylamide gel containing 2-pyrrolidone was used with a Tris-borate buffer-system. The resulting enzyme exhibits two very close bands on analytical aerylamide gels and has about
Many peptides
Although the enzyme was partially purified, a number of problems associated with stability were noted. In this paper, we report the stabilization and further purification of the enzyme and demonstrate its ability to 1 This work was supported by National Institutes of Health Grant AI-07781. p The enzyme was referred t.o in a previous paper (1) as pyrrolidonyl peptidase. It was brought to our attention, and we fully agree, that a more accurat.e designation for the enzyme is pyrrolidonecarboxylyl peptidase. 80
ammonium
sulfate
precipitation
of the en-
PURIFICATION
OF
PYRROLIDONECARBOXYLYL
200 times the specific activity of the starting material. MATERIALS
AND
METHODS
Materials used were the same as previously reported (1) with the following details and additions. 2-Pyrrolidone (practical) was purchased from Matheson, Coleman, and Bell. Protamine sulfate was purchased from Nutritional Biochemicals Corporation. Ammonium sulfate (special enzyme grade) used for protein precipitations was purchased from ?rIann Research Laboratories. Sodium tetrathionate was prepared by the method of Gilman et al. (2)) and stored at 4”. Fibrinogen. Bovine fibrinogen was prepared by the met.hod of Laki (3) and was 95y0 clottable. Bovine fibrinogen was stored in 0.3 M NaCl solution at -20” prior t.0 use. Culture Conditions. The enzyme was prepared from extracts of a strain of fluorescent Pseudomoms which was cultured as previously described (1). Stock solutions used in the maintenance and growth of these cells are as follows. All solutions were stored at 4” unless otherwise noted. Salts and trace metals for the pyrrolidonecarboxylic acid (PCA)” minimal medium (solution A) contained KHzPO~ (2.38 g), KzHPOJ. 3Hz0 (5.66 g), MgS04.7H20 (1.00 g), and trace metals (Solution B) (6.4 ml) in a total volume of 1.0 liter. Trace metals (Solution B) contained CuS04.5HzO (1.02 g), FeS04.7HsO (1.76 g), MnC12.4H20 (1.26 g), ZnSO~.?H~O (0.24 g) in 1 liter. This solution was stored frozen. A PCA solution was made with 50 g of PCA per 300 ml of water and sterilized by filtration through a Millipore filter. The glucose minimal medium contained K2HP04.3H20 (46.0 g), KHsP04 (10.0 g), (NH4)nSOa (5.0 g), NaB citrate.2HzO (3.0 g), MgS04.7HzO (0.5 g), and trace metals (Solution B) (31.25 ml) in a total volume of 5.0 liters. This solution was autoclaved and allowed to cool prior to the addition of 250 ml of sterile S”/;! glucose solution. Slants for the maintenance of the Pseudomonas 3 This straiu of Pseudovaonas is now being carried by the Cycle Chemical Corp., Los Angeles. Freeze-dried cells prepared according to our procedure are currently available from them. 4 Abbreviat,ions used: PCA, free pyrrolidonecarboxylic acid; Pyr: t’he sequence designation for pyrrolidoneearboxylyl residues in peptides or proteins, as in Pyr-Ala. PTH-amino acid, the phenylthiohydantoin derivative of the amino acid produring Edman degradations. TCA, duced trichloroacetic acid.
PEPTIDASE
81
strain cont,ained minimal medium with PCA as the sole source of carbon and nitrogen. The slants were made from 2.1 g of special Noble agar (Difco) added to 100 ml of salts and trace metals (Solution A) and autoclaved. 3.0 ml of sterile PCA solution and 3.0 ml of sterile 5L;Ic NaOH were added after the agar solution had cooled, but while still liquid. Slants were poured from this solution; the pH was close to neutrality. Slant cultures were transferred to PCA liquid medium and this culture, in turn, was used to inoculate larger volumes of glucose minimal medium. PC.4 liquid medium was made by the addition of 6 mls of sterile PCA solution and 6 ml of sterile 5V0 NaOH to 200 ml of sterile salts aud trace metals (Solution A). When the PCA liquid culture reached an OD,,, greater than 1.0, it was used to inoculate 5.0 liters of glucose medium, which was then used as the inoculum for 100 liters of glucose medium. The large scale fermentation was carried out in a Fermacell Fermentor, Model F-130 (New Brunswick Scientific Company) at 30” for approximately 48 hr. Cells were harvested directly from the fermentor by continuous-flow centrifugation in a Sharples refrigerated centrifuge (type AS-16). The average yield of bacteria from 100 liters was 420 g wet weight. The cell paste was frozen and lyophilized (average yield from 100 liters of cells: 117 g dry weight), and the pulverized dry material stored at -20”. Assay and units. Pyrrolidonecarboxylyl peptidase activity was assayed by the method previously reported (1) using L-pyrrolidonecarboxylylL-alanine (Pyr-Ala) as a substrate. The reaction was carried out in a total volume of 75 ~1 at 30”. 50 bl of buffered enzyme solution (pH 7.3) was added to 25 ~1 of a 0.57, Pyr-Ala solution (0.63 &moles). One unit of enzyme activity was defined as the amount of enzyme which produces 1 mpmole of free alanine per minute. Relative protein concentration was measured as the opt,ical density at 280 millimicrons of a solution appropriately diluted with 0.15 M NaCl. Specific activity of the enzyme is expressed as 1000 times the millimicromoles of alanine produced per minute under the assay conditions defined above, divided by the ODssa of the enzyme solution. Bujkrs. Enzyme preparations containing either ammonium sulfate or 2-pyrrolidone were dialyzed against 0.05 M potassium phosphate buffer, pH 7.3, containing 0.01 1~ 2-mercaptoethanol and 0.001 M EDTA prior to assay. This buffer is referred to as “Buffer A” throughout this paper. All preparative steps through G-200 Sephadex column chromatography were carried out in this phosphate buffer with 0.1 M 2-pyrrolidone added. The latter
82
ARMENTROUT
AND
buffer with 2-pyrrolidone added is referred to as “Buffer B”. DEAE-Sephadex (A-25) columns were equilibrated in 0.05 M sodium phosphate buffer, pH 8.0 with 0.01 M 2-mercaptoethanol, 0.1 M 2-pyrrolidone, and 0.001 M EDTA (“Buffer C”). Analytical and preparative polyacrylamide gel electrophoresis was conducted using a Tris-borate buffer system. This buffer (referred to as “Trisborate buffer”) contained 0.025 M Tris (pH 8.4), 0.20 M boric acid, 0.1 M NaCl, 0.005 M sodium thioglycolate, 0.001 M EDTA, and 0.10 M 2-pyrrolidone. Preparation of sephadex. G-200 and G-25 Sephadex (coarse) were prepared by boiling for 4-6 hr and were stored in Buffer A. “Fines” were not removed; G-200 columns were used only once. A-25 was prepared by extensive washing with 0.5 N NaOH, water, and 0.5 N HCL. A-25 columns were equilibrated first at room temperature with Buffer C which was 0.25 M in phosphate, then at 4” with the normal 0.05 M phosphate Buffer C. Equilibration was continued until constant conductivity and pH were attained. Dialysis tubing. Dialysis tubing was prepared by boiling for 2 hr in 0.001 M EDTA, followed by rinsing in glass-distilled water and boiling again in water. Amino-terminal analysis. The amino-terminal amino acids were determined by the quantitative Edman method (4) with the following conditions and variations. PTH-amino acids were identified
DOOLITTLE
qualitatively by thin layer chromatography on Eastman Chromagram Silica Gel sheets with fluorescent indicator. Two ascending solvent systern3 were used for each qualitative analysis: System “D” and system “E” of Edman and SjGquist (5). Quantitation of PTH-amino acids was carried out on descending paper chromatography in Solvents one and three or two and three for each sample (4). Each of the PTH-amino acids and the appropriate background control were eluted from the chromatogram in 2.0 mls of 95% ethanol. Spectra from 235 to 330 rnp gave positive identification of the eluted material as being PTH-derivatives. The optical density at 269 w was used to calculat’e the amount of the PTH-amino acid, using the molar extinction coefficients of 15,900 for PTH-glu, 15,600 for PTH-tyr, 15,500 for PTH-phe, 16,100 for PTH-asp, and 14,900 for PTH-gly. Electrophoresis. Analytical acrylamide gel electrophoresis was carried out by the method described by Davis (6). A running gel of 60-mm length was poured in glass tubes of 6-mm diameter. A 13-mm concentrating gel was poured directly on to the running gel without a spacer. The running gel was 5% acrylamide and was polymerized with ammonium persulfate. Prior to polymerization, the gel mixture was brought to 0.1 M 2-pyrrolidone and 0.001 M EDTA. A standard 2.5% acrylamide concentrating gel with sucrose was used; it also contained 0.1 M 2-pyrrolidone and 0.001 M EDTA,
2.0
-
0
100 EFFLUENT
200
200 VOLUME
(Mlsl
FIG. 1. A-25 column chromatography of pyrrolidonecarboxylyl peptidase. The ammonium precipitates from five G-200 column pools (90 ODm units, 103 enzyme units) were applied to column (2.5 X 21 cm) after desalting on a G-25 column. The A-25 column was washed with Buffer C followed by elution with a O-3y0 NaCl gradient (see Methods). Effluent samples (0.10 assayed for 12 hr; activity (broken line) is expressed as millimicromoles of alanine produced
sulfate the A-25 80 ml of ml) were in 12 hr.
PURIFICATION
OF
PYRROLIDONECARBOXYLYL
but was photo-polymerized with riboflavin as catalyst. A few grains of solid sucrose were added to the samples prior to application in order to prevent diffusion. Samples were run at 3 mA per tube for approximately 3 hr at 4”. Protein in the gels was fixed for 20 min in 10% TCA and then stained in 176 (w/v) solution of Amido Schwartz dye in 70/b acetic acid. The gels were destained with extensive washing in 10yO acetic acid. Preparative gel electrophoresis. A Buchler preparative polyacrylamide gel electrophoresis apparatus was employed. A 6% acrylamide running gel, containing 0.1 M 2-pyrrolidone and 0.001 M EDTA was used without a concentrating gel. Ammonium persulfate was the catalyst used in the polymerization of the gel; the gel volume was 100 ml. The apparatus was run at 50 mA for 2 hr at 4” prior to applying the sample in order to allow the thioglycolate to migrate into the gel and reduce any residual ammonium persulfate. The sample was applied in a total volume of 2.0 ml and contained sucrose. Approximately 18 hr electrophoresis at 50 mA (4”) was required to collect the enzyme from the gel. Purijkation. All manipulations were carried out in the cold, at 0” where possible, but at 4” otherwise. 12.5 g of lyophilized bacteria were suspended in 60 ml of ice cold Buffer B (containing P-pyrrolidone). The cells were placed in a glass “flowaround” vessel on ice and Rere disrupted with ten to fifteen 30.set blasts from a Branson Sonifier at maximal power setting. The sonicated material was centrifuged twice, first at 39,000g for 30 min,
I
I
0
lo
I
PEPTIDASE
53
and then the supernatant material was centrifuged again at the same speed for 10 min. The final supernate was diluted with Buffer B to an OD280 of 50. This material is referred to as “crude enzyme” here after. Nucleic acids were precipitat)ed by the slow addition of freshly prepared 1’7, protamine sulfate solution to a final concentration of 0.14yc. After the addition, the enzyme preparation was stirred on ice for 20 min and t,hen centrifuged at 15,000g for 20 min. The pellet was discarded; the supernate is referred to as “protamine sulfate enzyme.” The enzyme activity was precipitated by the cautious addition of saturated ammonium sulfate to 42% saturation. The solution was stirred at 0” for 1 hr after this addition, followed by centrifugation for 30 min at 39,000g. The resulting precipitates were resuspended in Buffer B to an approximate OD,,, of 150 and quick-frozen in an isopropanol-dry ice freezing mixture for storage at -70”. This material is referred to as “4274 ammonium sulfate enzyme.” On G-200 Sephadex gel columns, the enzyme activity eluted between the excluded protein and the included salt fraction (1). The 42yc ammonium sulfate enzyme was thawed and 2 ml (300 ODQ8,, units) were applied to each of two G-200 columns (2.5 X 36 cm) equilibrated with Buffer B. The active fractions were pooled and precipitated by the slow addition of 0.45 g solid ammonium sulfate per milliliter of enzyme solution. These ammonium sulfate suspensions were allowed to stand overnight at 0” and were subsequently centrifuged 30 min at, 39,000g. The supernatant fluid was carefldly
I
I
20 30 40 mm from Origin
I
I
50
60
FIG. 2. Location of enzymatic activity on an analytical polya.crylamide gel. Dialyzed G-200 enzyme (100 ~1) was applied to each 5% acrylamide gel and electrophoresed as per usual (see text). The enaymatic activity was located by assay of gel slices and is represented as millimicromoles of alanine produced in 10 hr. The photograph is of one of the stained gels that was run simultaneously with the assayed gel.
84
ARMENTROUT
and completely decanted pellets were stored at -20” “G-200 enzyme.” The pellets from four to redissolved in Buffer C to a This material was applied
and discarded. and are referred
AND The to as
DOOLITTLE
(2.5 X 38 cm) equilibrated in ing. The enzyme was recovered and approximately 100 OD~W very slowly to an A-25 column, application of the enzyme, the with approximately a column
five G-200 pools were volume of about 5 ml. to a G-25 column
Buffer in unit,s 2.5 X column volume
C for desaltabout 23 ml, were applied 24 cm. After was washed of Buffer C.
125 1.200
‘;’ P s
;‘\ ’ ‘1 I \ \ I \ I \ I \ \ I \
0.800
; 2
I
de 0 ii 0.400
I I I :i A
100
\
\
\ I I \ \
75 c 5 F 9 50
\
\
\-,w--
--
--_#*--*
‘-‘.I.-
-co--.--
--
1
25
‘-.
4
0
100
200 EFFLUENT
FIG. 3. Preparative carboxylyl pept)idase and electrophoresed with 25 ~1 of Pyr-Ala n 12 hr (solid line).
0
300 VOLUME
400
500
500
(MIS)
acrylamide gel electrophoresis: Elution patterns of ODzso and pyrrolidoneactivity. An A-25 enzyme preparation was applied to a By0 polyacrylamide gel using the Tris-borate running buffer. Samples of the effluent (0.1 ml) were assayed solution for 12 hr. Activity is presented as millimicromoles of alanine produced
FIG. 4. Polyacrylamide gels of enzyme preparations at various stages in purification. The samples were run on 5% acrylamide gels with a concentrating gel for approximately 210 min using the Trisborate buffer system at 3 mA per tube at 4” (see Methods). Gel B 1 was run with 200 ~1 of electrophoretically prepared enzyme; gel #2 was run with 100 ~1 of A-25 enzyme; and gel #3 was run with 25 ~1 of G-200 enzyme.
PURIFICATION
OF
PYRROLIDONECARBOXYLY
The activity was eluted by application of a salt gradient made from equal volumes (50 ml) of Buffer C without NaCl and the same buffer containing 3% NaCI. The column was allowed to flow at the rate of 18 ml per hr; activity was eluted early in the salt gradient. Effluent tubes were placed on ice as they came off the fraction collector and were allowed to stand overnight, during which time, O.l-ml aliquots from fractions were incubating for assay (Fig. 1). The peak tubes were pooled and precipitated by the addition of 0.6 g of solid ammonium sulfate per milliliter and allowed to stand overnight on ice. The slurry was spun down at 39,OOOg, and stored as a pellet at -20”. These precipitates are referred to as “A-25 enzyme.” Experiments using analytical polyacrylamide gel electrophoresis demonstrated the feasibility of using preparative electrophoresis as a further purification step. By using suitable stabilizing agents in the buffer system, it was possible to recover enzymatic activity from the analytical gels and correlate this activity with a specific set of bands. The recovery of activity depended on the use of a Tris-borate buffer, which did not interfere with the ninhydrin reagent in the enzyme assay. The 2-pyrrolidone and EDTA were included in the gel prior t,o polymerization. A reducing agent was not included at this point since it would interfere with the polymerization process. To provide a reducing milieu within the gel, thioglycolate was included in the tray buffer; it preceded the protein during electrophoresis. Figure 2 shows the position of the enzymatic activity relative to the major protein bands in the gel. In these experiments, 100 pl of the dialyzed G-200 enzyme was electrophoresed in each gel. One of the gels was stained and another was cut into approximately 1.5.mm slices. The gel slices were placed in tubes containing 0.2 ml of phosphate buffer (Buffer A) and 25 ~1 of Pyr-Ala solution. The slices were allowed to react for 10 hours and were assayed in the usual fashion. It is clear that the enzyme is one of the fastest migrating proteins under these conditions (Fig. 2). For preparative electrophoresis, the ammonium sulfate precipitates from one or two A-25 columns were redissolved to a volume of 2.0 ml in diluted Tris-borate buffer (0.01 M Tris, 0.02 M borate, 0.02 M NaCl, 0.004 M thioglycolate, 0.004 M EDTA, 0.04 M 2-pyrrolidone) and then briefly dialyzed versus two changes of this same buffer (total 3 hr dialysis). A small amount of solid sucrose was added to the sample before application to the surface of the acrylamide gel. In the elution profile of the electrophoresis (Fig. 3), the major OD2m is due to a non-protein contaminant in the buffer
85
PEPTIDASE
system and corresponds to the leading band on the analytical gel. Because of the small amount of protein applied, no distinct ODzso profile was obtained. The enzyme activity, however, was present close to the front as expected, and appeared after 16-18 hr of electrophoresis. The pooled active fractions were concentrated under nitrogen using a Diaflow pressure dialysis apparatus with a 10,000 molecular weight cut-off filter. The enzyme was stored frozen. The relative purity of this preparation can be seen in Fig. 4. Analytical gels of G-200, A-25, and
TABLE
I
SUMMARY OFTHEPURIFICATION CARBOXYLYLPEPTIDASEFROM Pseudomonad
OFPYRROLIDONEAFLUORESCENT
Total proteinb
Fraction”
Crude Protamine sulfate 42y0 Ammonium sulfate G-200
4360 3024
4634 4621
46 75
(100) 99.7
1311
2767
104
59.7
137
2888
1054
62.3
G-200, applied to A-25e A-25
93
879
475
(100)
9
604
3342
68
5.2
200
1925
0.7
121
8788
A-25, applied electrophoresis” Electrophoretic
to
(1~)
60
0 See Methods for a description of fractions. b (ODm X Vol). c (Millimicromoles of alanine produced per minute per milliliter) X (Vol). d Millimicromoles of alanine produced per minute per ODZW of 1.0. B The data presented are for a single preparation purified through G-200 column chromatography and give the activity recovered from ammonium sulfate precipitation of the peak fraction of the column effluent. The A-25 and electrophoretic steps each used preparations that were not peak fractions, but were larger pools of effluent. As a result, the specific activity of the material applied in these two steps was less than the peak activity of the preceding step. There is some variation in the specific activity of the crude sonicate; a specific activity of 25 is average. The data presented for the purification through G-266 chromatography are in the upper range of activities obtained.
86
ARMENTROUT
AND
electrophoretic enzyme preparations are compared. In the G-200 sample, the leading protein band (the second band visible in the gels; the first is the non-protein artifact referred to above) is faint when compared to the trailing bands. However, in the A-25 sample, the leading protein band has become a major band, and some of the trailing bands have disappeared. This leading band is also the one corresponding to the position of enzymatic activity. The product after preparative electrophoresis exhibits two major bands, again at the leading position,
DOOLITTLE
enzyme stability produced irregular yields and did not permit the lengthy procedures required for greater purification. Before further purification of this enzyme was possible, the problem of instability in solution had to be solved. While activity declines rapidly in solution, it was observed that enzymatic hydrolysis of the substrate PyrAla continued at a linear rate for up to 18 hr in some cases. This indication of substrate stabiliiation led us to search for a substrateanalogue which might afford protection. Z-pyrrolidone was found suitable in this regard. This substance acted as an unusual inhibitor of the enzyme (Fig. 5). An enzyme preparation (through the G-200 step) was assayed at a constant level of substrate (8 X lOA M) while the concentration of 2-pyrrolidone was varied. While there was clearly inhibition, suggesting that the enzyme was binding 2pyrrolidone, very high concentrations of 2-pyrrolidone (100 times the amount producing 50% inhibition) did not produce complete inhibition. The kinetics
RESULTS
The purification of pyrrolidonecarboxylyl peptidase is summarized in Table I. The procedure produces an approximate 200 fold increase in specific activity. The final enzyme preparation exhibits two bands on acrylamide gel (Fig. 4). Stabilization. We have previously reported a routine procedure for purification of the enzyme including G-200 gel filtration (1). Although we had successfully recovered active enzyme from A-25 columns, lack of
0.5
0.4
1
iI-1 0.1
0
o
-
I 0
.02
r
I
.04
.06
t .lO
.08
2 - PYRROLIDONE
.12
.14
M/L
FIG. 5. Inhibition of pyrrolidonecarboxylyl peptidase by 2-pyrrolidone. Dialyzed G-200 enzyme with a specific activity of 454 was used. Reaction was carried out in a total volume of 125 pl containing the usual volume of enzyme solution, 50 ~1. The concentration of the substrate, Pyr-Ala, was 0.8 X 10-a M and the concentration of the inhibitor 2-pyrrolidone was varied from 0 to 0.1 M. The reaction time was 2 hr at 30”; the concentration of 2-pyrrolidone is presented as molarity; activity is presented as millimicromoles of alanine produced per minute.
PURIFICATION
OF
PYRROLIDONECARBOXYLYL
of inhibition resemble a noncompetitive type (Fig. 6). In this experiment, G-200 enzyme was assayed in the presence of sufficient inhibitor to produce 50% reduction in the rate of hydrolysis of Pyr-Ala (2.6 X 1O-3 M). An increase in the concentration of the substrate from none to saturation and beyond did not reverse the effects of 2-pyrrolidone. Enzyme preparations to which 0.1 M 2-pyrrolidone had been added showed 100 % recovery of activity when 2-pyrrolidone was removed by rapid dialysis (2 changes of Buffer A, 3 hr total of dialysis). Pyrrolidonecarboxylyl peptidase was dramatically stabilized by the addition of 2-pyrrolidone at 0.1 M concentration (Fig. 7). An enzyme preparation from a G-200 Sephadex column run with buffer A (without 2-pyrrolidone) was divided into two portions, one of which was brought to 0.1 dcT2-pyrrolidone. Both were stored at 0” and at intervals, aliquots were removed from
l/V
2.8
--
2.4
--
2.0
--
1.6
--
PEPTIDASE
87
each, dialyzed against Buffer A, and assayed. After 4 days storage at 0”, the solution without 2-pyrrolidone had declined 80% in specific activity, while the activity of the solution protected by 2-pyrrolidone was virtually unchanged. Storage. Stability tests were conducted to find at which stages of purification the enzyme preparation could be conveniently stored. Our normal procedure was to store freeze-dried cells at -20°, 42 % ammonium sulfate enzyme at -7O”, and the ammonium sulfate precipitates from the active pools of the G-200 and A-25 columns at -20”. The G-200 enzyme remained unchanged in activity for at least 6 weeks; the A-25 material suffered some decline in activity, but temporary storage for several days was possible (Fig. 8). Protection of the active sulfyhydryl. Pyrrolidonecarboxylyl peptidase was found to be a “sulfyhydryl” enzyme (1); as such, EDTA
FIG. 6. Reciprocal plots of pyrrolidonecarboxylyl peptidase activity. Dialyzed G-266 enzyme with a specific activity of 475 was used in a total volume of 100 pl containing the usual 50 pl of the enzyme solution. The concentration of substrate Pyr-Ala was varied from 0 to 5 X 1O-3 M at two levels of the inhibitor, 2-pyrrolidone, 0 (X) and 2.6 X lOMa M (0). Reaction time was 1.75 hours at 30”. The substrate is presented as the reciprocal of the molarity; velocity as the reciprocal of millimicromoles alanine produced per minute. The approximate K, indicated for Pyr-Ala is 2 X 10e3 M.
88
ARMENTROUT
AND DOOLITTLE
units were recovered by this method after the powder had been stored for 3 days at room temperature. Removal of the pyrrolidonecarboxylyl residue from bovine jibrinogen. The usual means of dealing with a pyrrolidonecarboxylyl blocked amino-terminal in a protein involve isolation of an amino-terminal fragment from an enzyme digest. It would be more convenient if the blocking residue could be removed from the intact protein molecule. Accordingly, we wished to seeif pyrrolidonecarboxylyl peptidase might act on whole 01 I 0 I 2 4 proteins as well as small peptides. For this 3 TIME (Days1 purpose, bovine fibrinogen was chosen as the test protein since it is known to have a FIG. 7. Stabilization of pyrrolidonecarboxylyl residue as one of peptidase by 2-pyrrolidone. Enzyme prepared as pyrrolidonecarboxylyl its three amino-terminal amino acids (8). usual through the 42y0 ammonium sulfate precipitation was applied to a G-200 Sephadex column Phenylalanine is the residue adjacent to the prepared with Buffer A (without 2-pyrrolidone). terminal pyrrolidonecarboxylyl residue in The ammonium sulfate precipitate of the resulting the /3 chain. The relative rates at which the G-200 enzyme pool was divided into two parts. enzyme splits pyrrolidonecarboxylyl residues One part was dialyzed against buffer A (A) and from phenylalanine and other penultimate the other against Buffer B (with 2-pyrrolidone) amino acids have been studied (9). The (0). Both dialysates were stored on ice. Aliquots presence of two unblocked amino-terminals of each were removed and dialyzed against Buffer A prior to assay. Specific activity is presented as (glutamic acid and tyrosine) in the same molecule which contained the blocked endmillimicromoles of alanine produced per minute per 1.0 ODzso unit of protein (X 100). group offered a convenient internal control, permitting us to measure the efficiency of the Edman method for each determination. and 2-mercaptoethanol were used as protectFor this experiment “A-25 enzyme” was ing agents in solution. It is possible to dialyzed against buffer A containing 0.1 effectively protect the active sulfhydryl M NaCl. The enzyme had a specific activity group of such an enzyme by reversibly of about 1,000 and an OD2g0 of 5. One derivatizing it to an inactive form with milliliter of this solution (10.4 enzyme units) certain -SH blocking reagents. A recent was added to 8 ml of a solution of bovine example of such a procedure is the protection fibrinogen (ca 1.0 micromoles) dialyzed of ficin during purification by blocking with against buffer A containing 0.1 M NaCl. The tetrathionate (7). This method is highly NaCl was necessary to keep the fibrinogen suitable for protecting pyrrolidonecarboxylyl peptidase as a lyophilized powder, since in solution. The final reaction volume was 9.0 ml. An equal amount of enzyme was once the derivative is formed, all salts and added to a control preparation. Immediately protecting reagents can be removed. after addition of the enzyme, 3.0 ml were An enzyme preparation purified through removed from both the control and the G-200 chromatography was dialyzed against two changes of 0.0015 M tetrathionate solu- digestion tubes. Each sample was added to 3.0 ml of pyridine and frozen at -20” tion, and then against water (1 hr each). until subjected to amino-terminal analysis. This treatment completely inactivated the enzyme. The dialysate was then quick frozen Digestion was carried out at 30”. The amino-terminals of bovine fibrinogen and lyophilized. When the dry powder was which had been digested in this manner for resuspended in Buffer A, the 2-mercaptofor 0, 13, and 21 hr were qualitatively idenethanol reversed the inhibition by tetrathionate. About 50% of the original enzyme tified on thin-layer chromatography and 600
PURIFICATION
OF
0
PYRROLIDONECARBOXYLYL
10
30
20
89
PEPTIDASE
40
50
TIME (Days)
FIG. 8. Stability of pyrrolidonecarboxylyl tates of G-260 and A-25 preparations. The column were divided into equal parts and lyzed against Buffer A and assayed. The produced per minute per 1.0 ODm unit of TABLE
peptidase during storage ammonium sulfate precipitates stored at -20”. At intervals, specific activity is presented protein (X 1000).
II
SPECIFIC REMOVAL OF THE PYRROLIDONECARBOXYLYL RESIDUES FROM THE AMINO-TERMINAL BOVINE FIBRINOGEN BY PYRROLIDONECARBOXYLYL
PEPTIDASE” Hours
FTH
OF
Amino
200.1c 197.7 None
regardless of digestion time. As anticipated, phenylalanine was the only PTH-amino acid unmasked during the course of the digestion; no other residue increased significantly (Table II).
of digestionb
acid 0
Glutamic acid Tyrosine Phenylalanine
as ammonium sulfate precipifrom one G-200 and one A-25 a sample precipitate was diaas millimicromoles of alanine
13
187.8 171.6 45.3
a See text for details. b Reaction time at 30”. 0 Millimicromoles of PTH-amino ered from 60 mg of bovine fibrinogen @ 330,000 MW).
21
184.2 210.6 170.1
acid recov(180 mpmoles
then quantitated using a paper chromatographic system. The enzyme controls (no substrate) did not produce any identifiable PTH-amino acid. Our fibrinogen samples, however, always exhibited small amounts of PTH-glycine and PTH-aspartic acid,
DISCUSSION
We have attempted to provide a convenient laboratory reagent which will specifically remove pyrrolidonecarboxylyl residues from proteins or peptides. In order to accomplish this end, we searched for and found an enzyme activity present in a soil microorganism which specifically removes pyrrolidonecarboxylyl residues. It was necessary to purify this enzyme to the point where it was free of contaminating proteolytic activities and was sufliciently active that small quantities of enzyme would be effective. That we were successful in these efforts was demonstrated by the ability of the enzyme to remove 80% of the pyrrolidone-
90
ARMENTROUT
carboxylyl residues from as large a protein as bovine fibrinogen without detectably splitting any other peptide bonds. In that particular experiment an A-25 preparation, representing an overall purification of about 60-fold, was used. In general, however, G-200 enzyme preparations, ranging from 20 to 40 times purified relative to crude extracts, are sufficient for use on small peptides where there is less risk of indiscriminate proteolysis (1, 9). The enzyme itself appears to be relatively small and acidic from its high mobility in 5% acrylamide gel at pH 8.4 and inclusion in G-200 and G-150 Sephadex. It is stabilized against loss of activity in solution at 0” by high concentrations of 2-pyrrolidone. This substance is a noncompetitive inhibitor of the enzyme, whose action is reversible and does not produce total inhibition even at high concentrations. The physiological role of the enzyme is still unknown. The particular strain of Pseudomonas used as a source of enzyme was isolated from the soil by enrichment with PCA and selected for its ability to use PCA as the sole source of both carbon and nitrogen. While the enzyme activity of pyrrolidonecarboxylyl peptidase appears to be irrelevent to this degradation of PCA, we cannot exclude the possibility that the enzyme is involved in some nutritional process. On the other hand, there is recent evidence implicating PCA in the initiation of protein synthesis in rabbit lymphocytes (10). One is tempted to draw a parallel between mammalian chain initiation and the mechanism known to exist in E. coli. In the latter case, the initiating formylated methionyl residue is specifically removed from the protein by
AND
DOOLITTLE
an enzyme after synthesis is completed (11). It is conceivable that pyrrolidonecarboxylyl peptidase might have a similar function in the mammalian system. We have found an activity similar to that of pyrrolidonecarboxylyl peptidase in rat liver. However, the fact that pyrrolidonecarboxylyl residues exist at the amino-terminal of some proteins, and that bacterial pyrrolidonecarboxylyl peptidase, at any rate, is active in removing these residues, would tend to argue against such a role for the enzyme. ACKNOWLEDGMENTS We are pleased to acknowledge the assistance of Miss Malinda H&in, Schmidt, and Mr. Francis Lau. We wish Dr. G. Fuller for help and instruction techniques of gel electrophoresis.
technical Mrs. L. to thank in the
REFERENCES 1. DOOLITTLE, R. F. AND ARMENTROUT, R. W., Biochem. 7, 516 (1968). 2. GILMAN, A., PHILIPS, F. S., KOELLE, E. S., ALLEN, R. P., AND ST. JOHN, E., Am. J. Physiol. 147, 115 (1946). 3. LAKI, K., Arch. Biochem. Biophys. 33, 317 (1951). 4. SJGQUIST, J., Biochim. Biophys. Acta 41, 20 (1960). 5. EDMAN, P., AND SJ~QUIST, J., Acta Chem. Stand. 10, 1507 (1956). 6. DAVIS, B. J., Ann. N.Y. Accd Sci. 121, 405 (1964) . 7. ENGLUND, P. T., KING, T. P., CRAIG, L. C., AND WALTI, A., Biochem. 7, 163 (1968). 8. BLOMBXCK, B. AND DOOLITTLE, R. F., Ada Chem. Stand. 17, 1816 (1963). 9. ULIANA, J. A. AND DOOLITTLE, R. F., Arch. Biochem. Biophys. 131, 561 (1969). 10. MOAV, B. AND HARRIS, T. N., Biochem. Biophys. Res. Commun. 29, 773 (1967). 11. ADAMS, J. M., J. Mol. Bid. 33, 571 (1968).