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Purification of a mammalian peptidase selective for N-terminal arginine and lysine residues: Aminopeptidase B
ARCHIVES
OF
BIOCHEMISTRY
Purification
AND
BIOPHYSICS
114, 557-566 (1966)
of a Mammalian
Arginine
and
V. K. HOPSU, Department of Anatomy, Pathology, National
Lysine
Peptidase Residues:
K. K. MAKINEN,
Selective
for N-Terminal
Aminopeptidase AND
G. G. GLENNER
University of Turku, Turku, Finland; and Laboratory Institute of Arthritis and Metabolic Diseases, National Health, Bethesda, Maryland. Received
August
B
of Experimental Institutes of
4, 1965
A procedure is presented for purification of aminopeptidase B, an aminopeptidase from rat liver tissue hydrolyzing only naphthylamides of the basic amino acids arginine and lysine. A purification of llOO-fold has been obtained by repeated sedimentation with neutral salts, gel filtration, and denaturation. A small amount of contaminating protein was found to be present both by electrophoresis and ultracentrifugation sedimentation analysis. Molecular weight of the enzyme protein was found to be 95,500.
Although data has been reported indicating the .nonspecificity of enzymes hydrolyzing amino acid naphthylamides (1 l), there are now known to exist in mammalian tissues aminopeptidases which can selectively hydrolyze specific amino acid naphthylamides. An aminopeptidase has been described which hydrolyzes only acidic amino acid naphthylamides, i.e., aspartyl and glutamyl derivatives and has been designated a,minopeptidase A (3). Recently another aminopeptidase capable of hydrolyzing only basic amino acid naphthylamides, i.e., those of arginine and lysine has been identified in rat tissues and assigned the name aminopeptidase B (6). This paper presents a procedure for the purification of aminopeptidase B from rat liver and some of the characteristics of the preparation obtained. Further characterization of this enzyme will be described in a subsequent report (7). MATERIALS
AND
METHODS
Substrates and their sources. n-Arginyl-flnaphthylamide (n-Arg-8-NA) was used as the model substrate for basic amino acid naphthylamides. n-leucyl-@-naphthylamide (n-Leu-p-NA) was used throughout this study as the prototype non-basic amino acid naphthylamide substrate in
order to follow the activity of acylamidases hydrolyzing relatively nonpolar substrates of this type. Assay for the hydrolysis of L-Arg-p-NA and L-Leu-&NA. The method is based on measurement of the color intensity produced by diazonium salt coupling of the enzymatically liberated naphthylamine (4, 5). The procedure used was as follows: Buffered stock substrate solution was prepared by mixing 0.1 M tris-HCl buffer pH 7.0 (3 parts), 1 mM n-Arg-fl-NA or n-Leu-p-NA solution (1 part) and distilled water (1 part,) and stored at 0°C. For assay 2.5 ml of this solution and 0.5 ml of the protein (enzyme) fraction to be tested were mixed in a test tube. The test tubes were put in a 37°C water bath for 60 minutes. They were next placed in an ice bath (0°C) and immediately thereafter 1 ml of a solution of the stabilized diazonium salt Garnet GBC (1 mg/ml) in 1 M acetic acid buffer, pH 4.2, containing 10% Tween 20, was added. Color absorbancy was read using a Klett-Summerson calorimeter and filter No. 52 (485-550 rnM wavelength). In control tubes the enzyme was replaced by water or neutral protein (albumin) or protein solution was added after replacing the tubes in an ice bath. The amount of 8-naphthylamine (bM) liberated was calculated from standards prepared identically. The enzymic activity capable of liberating 1 mpM of fl-naphthylamine from n-Arg-@-NA in 1 hour in pH 7.0, 0.1 M tris-HCl buffer at 37” was taken as a unit of enzymic activity. 557
558
HOPSLT. M;iKINEN.
Determination of proteins. Protein concentrations were measured using the Folin-Ciocalteau method as presented by Layne (9). Proteins in electrophoresis slabs were demonstrated using either Amido black or Nigrosin. Preparation of the homogenate. Thirty adult rats of both sexes (Long-Evans strain) were killed and their livers were immediately removed and kept at -15°C for 2 hours after which they were homogenized using a Waring Blendor for 45-60 seconds in 300 ml 0.1 M tris-HCl buffer, pH 7.0, at 4°C. The homogenate was immediately centrifuged (34,000 g; 20 minutes) and the supernatant solution was collected and used in further studies. Studies on the stability of the enzymes with changing pH and temperature. The pH of the homogenate was lowered stepwise by adding into the homogenate appropriate amounts of HCl solution (O.Ol0.5 M). The precipitate formed was collected by centrifugation (20,000 g; 5 minutes) 3-10 minutes later and dissolved in tris-HCl buffel, pH 7.0. The homogenate (l-2 ml) in 0.1 M citric acid-phosphate or tris-HCl buffer of various pH values was placed in 15.ml centrifuge tubes and with continuous agitation was incabat,ed in water baths of varying temperatures for 5-60 minutes. The sediment was centrifuged (20,000 g; 5 minutes) and diluted in 0.1 M tris-HCl buffer, pH 7.0. Hydrolysis of LArg-B-NA was determined both in the sediment and supernatant fractions immediately and after st,orage at several temperatures for various time periods. The same experiments were performed in the presence of the substrate L-Arg-@-NA (1 mM).
Fractionation
Procedures
Neutral salts and organic solvents. Fractional sedimentation was performed using (NH),SO, at pH 7.0. After sedimentation for l-4 hours at various (NH)$Oa concentrations the sediment was collected by centrifugation (20,OOQ g; 20 minutes) and dissolved in 0.1 M tris-HCl buffer, pH 7.0. The organic solvents a,t a temperature of -10°C were added to the homogenate in an ice bath with continuous stirring. The sediment was rapidly centrifuged (15,000 g; 2 minutes) and the solvent was immediately evaporated in vaeuo in the cold. The sediment was dissolved in 0.1 M tris-HCl buffer, pH 7.0, for assay. The heavy metal salts tested are listed in the text. Gel Jiltration. Gel filtration was carried out using Sephadex G25, 100, and 200 gels according to the manufacturer’s instructions (Uppsala, Sweden, Pharmasia: see Ref. 2). Blue dextran was used to test the homogeneit,y of the columns. Column chromatography with substituted celluloses and hydroxyapatite. DEAE-cellulose (Whatman, W. & R. Balston, Ltd., England) and CM-
AND
(;LENNER
cellulose (Carl Schleicher, Schiill, Dassel/Kr. Einbeck, Germany) were used. Elution was carried out at 4°C in 60 X 3.7 cm columns; either pH or salt gradients were used. For details see Results. All fractionations were carried orlt at 0 to 4°C unless otherwise stated.
Evaluation OJthe Purity of the Preparation In order to determine the homogeneity of the enzyme preparation it was subjected to horizontal starch gel and “Oxoid” cellulose acetate electrophoresis and to ultracentrifugation. For quantitation of the enzymic act,ivity in the starch gel slabs, the slabs were cut transversally into 0.5-cm segments, homogenized in buffer, and tested as presented above.
Determination of Molecular Weight The determination of molecular weight was based on gel filtration; Sephadex Cl00 and 200 gels were used as well as the following proteins of known molecular weight as references: thyroglobulin (bovine, Sigma), r-globulin fraction III (bovine, NBC), serum albumin (cattle, KochLight Laborat,ories Ltd.), ovalbumin 3X-crystallized and chymotrypsin 3X-crystallized (NBC). All of these proteins were dissolved in water at a concentration of 5 mg/ml and used immediately. The amount of the enzyme protein used was 4 mg. Calculation of the molecular weight was performed according t,o the methods of Gelott,e (2) and Whitaker (12). RESULTS
Studies on the Stability of the Enzyme and Fractionation Procedures Stability of the enzyme hydrolyzing L-Arg,&NA. Preliminary experiments for fractionation of the enzymes hydrolyzing L-Arg&NA were performed using the supernatant of the centrifuged homogenate which consequently also contained enzymes other than aminopeptidase B capable of hydrolyzing L-Arg-p-NA. A decrease in the hydrolysis rate of the substrate due to various treatments was consequently attributed to all enzymes present. It was possible to keep the enzyme in 0.1 M citric acid-phosphate buffer, pH 5.1-5.5, at 37°C for several hours without loss of enzymic activity. Loss of activity was also not evident in 0.1 M tris-HCl buffer, pH 6.5-8.1, at 47°C during 30 minutes. At 52°C the activity was slowly lost in 0.1 M
PURIFICATION
OF AMINOPEPTIDASE
.u E 2 0.2 5
0i
Saturation
Concentration
of (NH,)?
SO4
(“A)
FIG. 1. Sedimentation of the enzymes hydrolyzing L-Arg-p-NA and L-Leu-&NA from the pH 5.1 supernat,anf fluid using (NH4).$304. The sediment obtained from 1 ml of the starting material at the (NH&SO, concentration was dissolved in 15 ml 0.1 M tri+HCl, pH 7.0, and the activity was n = L-Arg-P-NA and 0 = L-Leudetermined &NA.
tris-HCl buffer, pH 7.0, and after 10 minutes the activit’y was reduced by 20%. Ten minutes at 56°C in t,he same buffer reduced the activity 80%. At 0”-4°C the activity was stable in both buffers at pH 5.1-8.0 for several days. These result’s demonstrate t’he heat lability of the enzyme(s) and exclude the possibility of using fractionation procedures based on graded heat’ denaturation as a means for enzyme purification. However, lowering the pH to 5.0 at 0”-4°C did not destroy the activity. Since this is a practical way to agglutinate mitochondria and microsomes (1) it was adopted as a step in further purification (see Final Purification Procedure) . Fractionation by (NH&S04 and crganic solvents. These experiments were carried out using the supernatant’ aft#er sedimentation of the particles from the homogenate at’ pH 5.1. Very small stepwise concentration increases of (NH&S04 were used (2-5%) in order to determine whether aminopeptidase B could be separated readily by t’his procedure from the other enzymes hydrolyzing L-Arg-P-KA. Hydrolysis of L-Arg-@NA as well as of L-Leu-/3-NA was t’ested
B
559
both in the supernatant and sediment fractions (Fig. I). It appeared that the enzyme hydrolyzing L-Arg-P-NA sedimented earlier than that hydrolyzing L-Leu-/3-NA. Organic solvent fractionations using acetone, methanol, ethanol, propanol and nbutanol were tried. The enzyme preparation was dissolved in 0.033 M tris-HCl buffer, pH 7.0. The solvent was added stepwise in 0.5-ml volumes into 15-ml centrifuge tubes up to 90% (vol), each addition being followed by centrifugation. Only acetone appeared to be suitable, yet it caused some loss of activity (lO-20%) even during a very rapid exposure. In a buffer at pH 5.5 denaturation was very marked. These results demonstrate that fractional sedimentation using (NH& SO* is of value in efforts to purify the enzyme in question while organic solvents are of questionable value in this respect. Fractionation by heavy metal salts. The salts were tested: Zn(Ac)z, following Pb(Ac)2, CUE, U02(A~)2, CaC12, MgCl,, Pb(NO&, CrC&, Hg(NO&. Into 1 ml of homogenate or the pooled fractions obtained aft,er gel filtration with Sephadex G 200 there was added 1 ml of solution of the salts mentioned (containing 0.001-0.1 n-mole) at 2°C. After ten minutes t,he sediment was centrifuged (20,000 g; 5 minutes). The sediment was suspended in 2 ml 0.1 M tris-HCl buffer, pH 7.0. A sample of 100 ~1 of the supernatant and of the solubilized sediment was diluted to 10 ml with the buffer. The enzymic activity toward Arg-P-NA in these solutions and in the supernatant fluids was det,ermined. The effect of the metal ions present in the incubation medium was partly eliminated by adding an equivalent amount of ethylenediaminetetraacetate (EDTA) in control assays. All of the metals caused an irreversible loss of activit’y during sedimentation. Gel Jiltration. Sephadex G 75, 100, and 200 were used to fractionate the homogenate supernatant. It appeared that both Sephadex G 100 and 200 caused some separation of the enzymes hydrolyzing L-Arg-P-NA and L-Leu-P-NA. Gel filtration with G 200 (Fig. 2) gave the best separation and revealed the existence of more than one enzyme peak hydrolyzing L-Arg-/3-NA.
HOPSU,
560
M;IKINEN,
AND
GLENNER . -
GO Fraction
L-Arg-CNA
z E
80 number
FIG. 2. Fractionation of the enzymes hydrolyzing L-Arg-P-NA and L-Leu-&NA using Sephadex G 200. Sample was 2 ml homogenate supernatant fluid: Protein concentration, 20 mg/ml; column, 60 cm X 3.7 cm; Elution, 0.1 M tris-HCl, pH 7.0; flow rate, 0.3 ml/min; fraction volume, 5.8 ml; hydrostatic pressure, 70 cm; temperature, 1°C.
27
Protein
I
5
0.8
E x 2 w
n ”
II
L
I IiI LO
1
50 Fraction
GO number
70
nLL
”
FIG. 3. Fractionation of the enzymes hydrolyzing L-Arg-@-NA and L-Leu-p-NA using gel filtration with Sephadex G 100. Sample was 2 ml of an (NH&SOI sediment, between 32 and 407, saturation; elution with 0.1 M tris-HCl, pH 7.0; flow rate, 0.43 ml/min; hydrostatic pressure, 20 cm; temperature, 3°C; fraction volume, 6 ml.
The same experim.ent was performed using an enzyme fract’ion obtained by sedimentation with (NH&CO4 (Figs. 3 and 4). It appeared that the sediment obtained between salt concentrations of 32-40% contained high enzyme activity toward L-Argp-NA and significant separation from that acting on L-Leu-P-NA was achieved. A partial separation, though less complete, was also obtained when fractionating by gel fil-
Fraction
number
FIG. 4. The same conditions as in Fig. 7 except that the sample used was obtained by (NH&S04 sedimentation between 40 and 507; saturation.
tration the sediment obtained between (NH&SO4 concentrations of 40-50 %. This suggested that the former concentration limits are preferable in purification of the enzyme. These experiments demonstrated that. Sephadex gel filtration was the most likely means for the purification of aminopeptidase B. Gel filtration was therefore included in the purification procedure. Sephadex-DEAE as well as CM-Sephadex produced some fractional enzyme separation, but due to the considerable shrinkage of these dextrans,
PURIFICATION
OF AMINOPEPTIDASE
---
L-Leu-O-NA
-
L-Arg-fl-NA Protein
561
200
150 Fraction
B
number
FIG. 5. DEAE-cellulose chromatography of the pH 5.2 supernatant fluid of liver tissue homogenate. Column 65 X 4.0 cm; sample 1.75 ml; elution with NaCl gradient 0.00%0.3 M in 0.005 &I tris-HCI buffer; flow rate, 0.5 ml/min; fraction volume, 7.5 ml.
their physical. lability as well as the rather poor reproducibility of the experiments, they were not included in the final purification procedure. Chromatography using substituted celluloses. DEAE-cellulose and CM-cellulose chromatography also produced some fracbional eneymic separation as reported earlier (6). DEAE-chromatography with a NaCl gradient was found to produce some purification. Elution of the enzyme from the column was effected by a concentration of 0.25 Ri (Fig. 5). A continuous pH gradient from 6.5 bo 9.0 failed to give a satisfactory separation. CM-cellulose produced best purification at pH 5.5 at which pH no binding of aminopept#idase B took place. Final purification procedure. The preliminary experiments presented revealed several methods which could afford some separation of the enzymes. On the basis of these studies the final purification procedure adopted was that presented in Table I. By this scheme purification took 40 days, 30 days of which involved storage in the cold in order to facilitate denaturation of inactive proteins. The precipitation produced after gel filtration with Sephadex G 25 was most likely due to a sharp decrease in the ionic strength of the preparation. The enzyme
preparation was never frozen since that was found to produce inactivation of the enzyme. During the 30 days of storage both the hydrolytic activity per milligram of protein of the enzyme preparation as well as the activity per milliliter were increased. This indicated that the increase was due to sedimentation of protein as well as activation of the enzyme. The sedimented material was not soluble in 0.1 M tris-HCl, pH 7.0, showing that this protein had been denaturated. When the sedimented material was added back to the enzyme preparation no change in the enzymic activity took place. The increase of enzymic activity during storage is shown in Table II. A summary of the changes in the enzymic activity during the purification is presented in Table III and indicates that a purification of about 1,100 fold has been achieved. The hydrolysis of L-Arg-/3-NA by the supernatant of the whole homogenate is due at least to two distinct enzymes which are separated by G 200 column chromatography (see Fig. 2). The peak of aminopeptidase B is about equal to the sum of the other peaks in which hydrolysis of L-Arg-/3-NA occurs, i.e., about half of the hydrolysis of L-Arg-@-NA is due to this enzyme. This was taken into account when estimating t’he activity of aminopep-
562
HOPSU,
Homogenization fresh rat liver
(1 kg of tissue)
MdKINEN,
AXI>
GLENNEI:
TABLE I PURIFICATION PROCEDURE (Carried out at, O”4”C.) Homogenate, 2150 ml, in 0.1 JI tris-HCI buffer, pH 7.0 Centrifugation 20,000 g; 30 min
I Srlpernat,ant fluid pH lowered to 5.1 with 0.2 N HCl. Left to stand for 16 hours. Centrifugation, 20,000 g; 30 min. I Supernatant fluid (NHJZ SO4 added up to 35y0 saturation. Volume 1575 ml; (NH& SO, 327.5 gm. Left to stand for 2.5 hours, centrifugation 20,000 g; 10 min
I Sediment
1
Sediment
(discarded)
Sediment
(discarded)
Supernitant fluid (NH& SO, added up to 45y0 saturation Volume, 1700 ml; (NH,)&‘04 102 gm. Left to stand for 3 hours, centrifugation, 20,000 g; 10 min Super&ant
fluid
(discarded)
Sediment. Solubilized in 0.1 M tris-HCl buffer, pH 7.0, to make 92 ml. Gel filtration using Sephadex G 200 (column 100 x 6.7 cm, elution with 0.1 J1 tris-HCl,
SO, was added up to 30% saturation
Super&ant fluid Volume 260 ml, (NH&SO* added up to 50% saturation (32.5 pm). Left to stand 2 hours, centrifugation 30,000 g; 10 min. Supernatant
fluid
I Supernatant
fluid
I Supernatant
(discarded)
fluid
(52.5 gm). Left
to stand
Sediment
1.5 hours,
(discarded)
Sediment (discarded) Solubilized in 50 ml 0.1 M tris-HCl, pH 7.0. Transferred to 0.005 M trisHCl, pH 7.0, using Sephadex G 25 (column 37 x 3.7 cm). Opalescent preparation was centrifuged at 2000 g; 30 min and (NH& SOa sediment between 25 and 400/, collected, again transferred into dilute buffer. DEAE-cellulose chromatography (column 45 x 2 cm), Whatman DE II, elution linear pH 7.0, flow rate 0.5 ml/min). gradient 0.0-0.25 M NaCl; 0.005 M tris-HCl, The most active fractions pooled (150 ml) and (NH& SO, was added to 45y0 saturation. I (discarded) Sediment pH 7.0 (25 ml), and transferred to 1:lO Solubilized in 0.1 M tris-HCl, diluted McIlvaine’s buffer. Left to stand 30 days, the seditient centrifuged off every second day (10,000 g; 10 min). (NH&SO., was added up to 60% saturation. I (discarded) Sediment Solubilized in 17 ml 0.1 M tris-HCl buffer, pH 7.0.
PURIFICATION
OF AMINOPEPTIDASE
tidase B in the homogenate in the calculations for Table III. Physical Studies on the Preparation Molecular weight. To obtain an approximate estimate of the molecular weight of the enzyme a sample of the preparation prior to the 30 days’ storage phase was used. The Sephadex G 100 column used was 130 X 2.4 cm. The sample was mixed with the reference proteins in a volume of 2 ml. The total amount of protein was 30 mg, flow rate 1 ml/6 minutes, elution with 0.1 M tris-HCl, pH 7.0, using a hydrostatic pressure of 20-30 cm. Results of the gel filtration are presented in Fig. 6 and a plot of part of the data obtained is shown in Fig. 7. The molecular weight as estimated from t’he TABLE
;
.-c ;
Ii 0
Percentage increase in activity
6 10 14 30
80
90
100
number
FIG. 6. Distribution of the enzyme hydrolyzing L-Argg-NA with several reference proteins in gel filtration using Sephadex G 190. Proteins were measured using the Folin-Ciocalteau method and the enzymic activity by testing the hydrolysis of L-Arg-&NA. (1) Thyroglobulin, (2) r-globulin, (3) sample, (4) serum albumin, (5) ovalbumin, (6) enzymic activity toward n-Arg-&NA.
TABLE
III
SUMMARY OF THE PURIFICATION
Homogenate supernatant fluid (NH&SOa 3545% sedlment Sephadex G 200 pool (NH&SO4 5096 sediment Sephadex G 25 pool Before DEAE chromatography (NH),SO( 45yb sediment Before storage 2 days storage 6 days storage 10 days storage 14 days storage 30 days storage Final
70 Fraction
0 33 48 104 107 192
step
0.15 ,” Cl ”
II
Initial 2
Purification
563
data according to Whitaker (12) was 95,500. For calculation of the molecular weight according to Gelotte (2) the KD value was calculated for albumin and found to be 0.27. The K, value for the enzyme was found to
CHANGE IN THE ENZYMIC ACTIVITY (UNITS/MG) OF THE PREPARATION DURING STORAGE OF 30 DAYS Storage period. (days)
B
Volume (ml)
Units,ml
Total amount of units
2150
1
2150
92
21
300 50 109 45 25 32 32 31 31 30 30 17
PROCEDURE Protein h.dml)
Units/mg
Percentage yield
130
0.003
1OQ
1940
105
0.02
93
4.3 17 6 10
1290 850 600 450
24 63 11 11
0.18 0.27 0.55 0.91
60 40 28 21
9 4.7 4.7 4.8 4.5 4.3 4.3 7
225 159 159 150 140 130 130 120
1.92 2.94 3.92 4.36 6.0 6.1 8.6 8.75
10 7 7 7 6.5 6 G 5.5
4.7 1.6 1.2 1.1 0.75 0.7 0.5 0.8
Purification coefficient
1 2.5 22 34 70 114 240 370 490 540 750 760 1100 1100
HOPSU,
MdKINEN,
AND
GLENNER
cm 5 L 3 2 1 start
FIG. 7. Determination of the molecular weight of the enzyme by gel filtration in Sephadex G 100. (1) r-globulin, (2) serum albumin, (3) ovalbumin.
2 1 start
cm 5 4 3 2 1 start
m {
]
H
FIG. 9. Eleetrophoresis of the enzyme preparation in starch gel at various pH values. Voltage 6 V/cm, 0.1 M tris-HCl buffer pH 6.5-9.3. Duration of electrophoresis up to 12 hours. Sample, 30 ~1 in a Whatman paper strip. Dark bands demonstrate protein, and light bands show those pieces of the sectioned electrophoresis slab in which enzymic activity was demonstrated using L-Arg-@-NA.
9.0 8.0 7.0 PH FIG. 8. Electrophoresis of the enzyme preparation in cellulose acetate “Oxoid” strips at various pH values. Voltage 10 V/cm, 0.1 M tris-HCl buffer, pH 7.0-9.0. Sample 4-5 ~1 in a Whatman No. 1 paper strip, duration of electrophoresis 180 minutes. Dark bands demonstrate the location of protein.
be 0.20. The value calculated for the molecular weight of the enzyme with the aid of these figures was 95,000. The molecular weight of albumin was taken to be 70,300 in these calculations (8). Homogeneity. Starch gel electrophoresis as well as electrophoresis in “Oxoid” cellulose acetate was carried out at a number of pH values in order to test the homogeneity of the final preparation as well as to determine the isoelectric point of the enzymically active protein. Results are given in Figs. 8 and 9. Following cellulose acetate electrophoresis, only one protein band was found. Two protein bands were observed following starch gel electrophoresis, only one, the
FIG. 10. Ultracentrifugal pattern of the enzyme preparation. Sedimentation from left to right, exposures at 54 and 78 minutes. For details see text.
more intensely stained, being enzymically active. The isoelectric point was about pH 7.0. In order to determine homogeneity by sedimentation velocity patterns in the ultracentrifuge a portion of the final preparation was lyophilized and dissolved in 0.1 M
PURIFICATION
OF AMINOPEPTIDASE
tris-HCl buffer, pH 7.0, so that the final protein concentration was 5 mg/ml. Sedimentation was carried out in 12-mm cells with a Spinco model E ultracentrifuge operating at 59,780 rpm at 21.6”C. The phase plate angle was 50” and photographs were taken at 54, 62, 70, and 78 minutes. Figure 10 shows the sedimentation pattern of the preparation. The preparation appeared to behave homogeneously. Some slight curling of the base line, however, can be seen, indicating the possible presence of impurities at a low concentration.
B
565
M tris-HCl buffer, pH 7.0, also was found to cause a total loss of activity. These findings, therefore, demonstrated that the enzyme was very easily inactivated by several physical procedures, while the range of optimal preparative conditions was limited. On the other hand, storing of frozen intact liver tissue over several months was found not to cause any loss of activity. Attempts to use various organic solvents and heavy metal ions in the precipitation of the proteins revealed that the enzyme was fairly sensitive to most of these treatments. These reagents might be considered to cause DISCUSSION an irreversible change in the structure of the The preliminary fractionation studies per- molecule or to react with some chemical formed demonstrated that the enzymes hy- groups essential to the function of the endrolyzing L-Arg-P-NA are fairly stable under zyme, e.g., sulfhydryl groups. This finding certain experimental conditions and, there- was in agreement with the earlier evidence fore, a purificat,ion was possible. Studies of concerning the effect of some sulfhydryl the individual steps commonly used in en- reagents on the activity (6). zyme purification revealed that a partial The most unexpected step required in the purification of the enzyme hydrolyzing L- purification procedure was the denaturation Arg+NA, but not n-Leu-/3-NA, could be of an inactive protein from the preparation obtained by several means. Results of simi- during 30 days of storage. The most likely lar efforts using DEAE-celluIose have been explanation for the denaturation was the published earlier by Hopsu et al. (6) and low ionic concentration of the solution. The formed the basis for the discovery of the protein removed was either a contaminating present enzyme. This latter investigation protein or an actual part of the enzyme showed that a simple fractional sedimentamolecule, possibly an enzyme inhibitor. The tion using (NH&SO4 could also be used, as fact that the total enzymic activity of the well as Sephadex gel filtration. preparation increased over threefold during The purification procedure finally em- the precipitation process shows that the ployed in the present study was based on protein removed was actually inhibitory to these preliminary experiments and the result the enzyme. The fact that it was not possible obtained was a highly purified enzyme prep- to dissolve the precipitated protein in buffer aration. Total purification from contamisolutions, showed that an irreversible denating proteins was, however, not achieved, naturation had taken place. as shown most clearly by starch gel electroThe molecular weight of the enzymically phoresis. The proportional amount of con- active protein was found to be fairly large. taminating proteins was, however, small Both methods of calculation gave about and, as far as tested, these were found to be equal results. These techniques for molecucompletely inert enzymically. lar weight estimation are commonly conThe purified enzyme preparation was very sidered to have an accuracy of about f stable and could be stored in the cold as a 10,000. A prerequis’te is that the elution concentrated solution without loss of activvolumes are accurately measured. A possible ity for several months. Excessive heat as error due to the use of a drop counter in the well as freezing of the preparation were, fraction collection was estimated by Whitahowever, destructive. Freezing overnight ker (12) as small (0.5-1.0%) while Leach was found to destroy about two-thirds of and O’Shea (10) found a greater variation the activity of the pure preparation as well (15-20 %). The error was due to variation as of the preparation obtained by gel filtrain drop size as the result of changes in the tion. Freezing in starch gel containing 0.1 surface tension of the liquid in the presence
566
HOPSU,
MilKINEN,
of proteins. Small variations in drop size possibly cause some loss in accuracy in exact measurements, but do not overshadow the estimation of the weight range of the molecule. These differences as well as variations in pretreatment of the gel [decanting, packing, hydrostatic pressure (smaller under high pressure) and buffer] render it likely that small differences are to be found in the calculation of KB values and thus in molecular weight. The value reported earlier for the Xn of albumin is 0.2 (2) while that found by us was 0.27. The latter value as measured in our experimental conditions, was used in the present calculations. The results of this investigation extend our earlier report on the existence in mammalian tissues of an enzyme selectively hydrolyzing n-Arg-/I-NA (as well as L-Lysp-NA) without effect on n-Leu-p-NA or several other amino acid naphthylamides (6). A more complete characterization of the present enzyme follows in a subsequent paper (7). ACKNOWLEDGMENT This work was aided by grants from Sigrid Juselius Foundation, The Finnish State Medical Research Council, and The National Institutes of Health grant TW-00103 to VIinB K. Hopsu.
AND
GLENNER REFERENCES
1. CLAUDE, A., Advan. Protein Chem. 5, 423 (1949). 2. GELOTTE, B., in “New Biochemical Separations” (A. T. Barnes and L. S. Morris, eds.), p. 105. van Nostrand, Princeton, London (1964). 3. GLENNER, G. G., MCMILLAN, P. J., AND FOLK, J. E., fVature 194, 867 (1962). 4. GLENNER, G. G., HOPSU, V. K., AND COHEN, L. A., J. Histochem. Cytochem. 10, 109 (1962). 5. GLENNER, G. G., HOPSU, V. K., AND COHEN, L. A., J. Histochem. Cytochem. 12, 545 (1964). 6. HOPSU, V. K., KANTONEN, U.-M., AND GLENNER, G. G., Life Sci. 3, 1449 (1964). 7. HOPSU, V. K., M~KINEN, K., AND GLENNER, G. G., Arch. Biochem. Biophys. 11.4,567 (1966). 8. KLEINER, S. M., AND KEGELES, G., Arch. Biochem. Biophys. 63, 247 (1956). 9. LAYNE, E., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 447. Academic Press, New York (1957). 10. LEACH, A. A., AND O’SHEA, P. C., J. Chromatog. 16, 306 (1964). 11. NACHLAS, M. M., GOLDSTEIN, T. P., AND SELIGMAN, A. M., Arch. Biochem. Biophys. 97, 223 (1962). 13 WHITAKER, J. R., Anal. _-. Chem. 36, 1950 (1963).
OF
BIOCHEMISTRY
Purification
AND
BIOPHYSICS
114, 557-566 (1966)
of a Mammalian
Arginine
and
V. K. HOPSU, Department of Anatomy, Pathology, National
Lysine
Peptidase Residues:
K. K. MAKINEN,
Selective
for N-Terminal
Aminopeptidase AND
G. G. GLENNER
University of Turku, Turku, Finland; and Laboratory Institute of Arthritis and Metabolic Diseases, National Health, Bethesda, Maryland. Received
August
B
of Experimental Institutes of
4, 1965
A procedure is presented for purification of aminopeptidase B, an aminopeptidase from rat liver tissue hydrolyzing only naphthylamides of the basic amino acids arginine and lysine. A purification of llOO-fold has been obtained by repeated sedimentation with neutral salts, gel filtration, and denaturation. A small amount of contaminating protein was found to be present both by electrophoresis and ultracentrifugation sedimentation analysis. Molecular weight of the enzyme protein was found to be 95,500.
Although data has been reported indicating the .nonspecificity of enzymes hydrolyzing amino acid naphthylamides (1 l), there are now known to exist in mammalian tissues aminopeptidases which can selectively hydrolyze specific amino acid naphthylamides. An aminopeptidase has been described which hydrolyzes only acidic amino acid naphthylamides, i.e., aspartyl and glutamyl derivatives and has been designated a,minopeptidase A (3). Recently another aminopeptidase capable of hydrolyzing only basic amino acid naphthylamides, i.e., those of arginine and lysine has been identified in rat tissues and assigned the name aminopeptidase B (6). This paper presents a procedure for the purification of aminopeptidase B from rat liver and some of the characteristics of the preparation obtained. Further characterization of this enzyme will be described in a subsequent report (7). MATERIALS
AND
METHODS
Substrates and their sources. n-Arginyl-flnaphthylamide (n-Arg-8-NA) was used as the model substrate for basic amino acid naphthylamides. n-leucyl-@-naphthylamide (n-Leu-p-NA) was used throughout this study as the prototype non-basic amino acid naphthylamide substrate in
order to follow the activity of acylamidases hydrolyzing relatively nonpolar substrates of this type. Assay for the hydrolysis of L-Arg-p-NA and L-Leu-&NA. The method is based on measurement of the color intensity produced by diazonium salt coupling of the enzymatically liberated naphthylamine (4, 5). The procedure used was as follows: Buffered stock substrate solution was prepared by mixing 0.1 M tris-HCl buffer pH 7.0 (3 parts), 1 mM n-Arg-fl-NA or n-Leu-p-NA solution (1 part) and distilled water (1 part,) and stored at 0°C. For assay 2.5 ml of this solution and 0.5 ml of the protein (enzyme) fraction to be tested were mixed in a test tube. The test tubes were put in a 37°C water bath for 60 minutes. They were next placed in an ice bath (0°C) and immediately thereafter 1 ml of a solution of the stabilized diazonium salt Garnet GBC (1 mg/ml) in 1 M acetic acid buffer, pH 4.2, containing 10% Tween 20, was added. Color absorbancy was read using a Klett-Summerson calorimeter and filter No. 52 (485-550 rnM wavelength). In control tubes the enzyme was replaced by water or neutral protein (albumin) or protein solution was added after replacing the tubes in an ice bath. The amount of 8-naphthylamine (bM) liberated was calculated from standards prepared identically. The enzymic activity capable of liberating 1 mpM of fl-naphthylamine from n-Arg-@-NA in 1 hour in pH 7.0, 0.1 M tris-HCl buffer at 37” was taken as a unit of enzymic activity. 557
558
HOPSLT. M;iKINEN.
Determination of proteins. Protein concentrations were measured using the Folin-Ciocalteau method as presented by Layne (9). Proteins in electrophoresis slabs were demonstrated using either Amido black or Nigrosin. Preparation of the homogenate. Thirty adult rats of both sexes (Long-Evans strain) were killed and their livers were immediately removed and kept at -15°C for 2 hours after which they were homogenized using a Waring Blendor for 45-60 seconds in 300 ml 0.1 M tris-HCl buffer, pH 7.0, at 4°C. The homogenate was immediately centrifuged (34,000 g; 20 minutes) and the supernatant solution was collected and used in further studies. Studies on the stability of the enzymes with changing pH and temperature. The pH of the homogenate was lowered stepwise by adding into the homogenate appropriate amounts of HCl solution (O.Ol0.5 M). The precipitate formed was collected by centrifugation (20,000 g; 5 minutes) 3-10 minutes later and dissolved in tris-HCl buffel, pH 7.0. The homogenate (l-2 ml) in 0.1 M citric acid-phosphate or tris-HCl buffer of various pH values was placed in 15.ml centrifuge tubes and with continuous agitation was incabat,ed in water baths of varying temperatures for 5-60 minutes. The sediment was centrifuged (20,000 g; 5 minutes) and diluted in 0.1 M tris-HCl buffer, pH 7.0. Hydrolysis of LArg-B-NA was determined both in the sediment and supernatant fractions immediately and after st,orage at several temperatures for various time periods. The same experiments were performed in the presence of the substrate L-Arg-@-NA (1 mM).
Fractionation
Procedures
Neutral salts and organic solvents. Fractional sedimentation was performed using (NH),SO, at pH 7.0. After sedimentation for l-4 hours at various (NH)$Oa concentrations the sediment was collected by centrifugation (20,OOQ g; 20 minutes) and dissolved in 0.1 M tris-HCl buffer, pH 7.0. The organic solvents a,t a temperature of -10°C were added to the homogenate in an ice bath with continuous stirring. The sediment was rapidly centrifuged (15,000 g; 2 minutes) and the solvent was immediately evaporated in vaeuo in the cold. The sediment was dissolved in 0.1 M tris-HCl buffer, pH 7.0, for assay. The heavy metal salts tested are listed in the text. Gel Jiltration. Gel filtration was carried out using Sephadex G25, 100, and 200 gels according to the manufacturer’s instructions (Uppsala, Sweden, Pharmasia: see Ref. 2). Blue dextran was used to test the homogeneit,y of the columns. Column chromatography with substituted celluloses and hydroxyapatite. DEAE-cellulose (Whatman, W. & R. Balston, Ltd., England) and CM-
AND
(;LENNER
cellulose (Carl Schleicher, Schiill, Dassel/Kr. Einbeck, Germany) were used. Elution was carried out at 4°C in 60 X 3.7 cm columns; either pH or salt gradients were used. For details see Results. All fractionations were carried orlt at 0 to 4°C unless otherwise stated.
Evaluation OJthe Purity of the Preparation In order to determine the homogeneity of the enzyme preparation it was subjected to horizontal starch gel and “Oxoid” cellulose acetate electrophoresis and to ultracentrifugation. For quantitation of the enzymic act,ivity in the starch gel slabs, the slabs were cut transversally into 0.5-cm segments, homogenized in buffer, and tested as presented above.
Determination of Molecular Weight The determination of molecular weight was based on gel filtration; Sephadex Cl00 and 200 gels were used as well as the following proteins of known molecular weight as references: thyroglobulin (bovine, Sigma), r-globulin fraction III (bovine, NBC), serum albumin (cattle, KochLight Laborat,ories Ltd.), ovalbumin 3X-crystallized and chymotrypsin 3X-crystallized (NBC). All of these proteins were dissolved in water at a concentration of 5 mg/ml and used immediately. The amount of the enzyme protein used was 4 mg. Calculation of the molecular weight was performed according t,o the methods of Gelott,e (2) and Whitaker (12). RESULTS
Studies on the Stability of the Enzyme and Fractionation Procedures Stability of the enzyme hydrolyzing L-Arg,&NA. Preliminary experiments for fractionation of the enzymes hydrolyzing L-Arg&NA were performed using the supernatant of the centrifuged homogenate which consequently also contained enzymes other than aminopeptidase B capable of hydrolyzing L-Arg-p-NA. A decrease in the hydrolysis rate of the substrate due to various treatments was consequently attributed to all enzymes present. It was possible to keep the enzyme in 0.1 M citric acid-phosphate buffer, pH 5.1-5.5, at 37°C for several hours without loss of enzymic activity. Loss of activity was also not evident in 0.1 M tris-HCl buffer, pH 6.5-8.1, at 47°C during 30 minutes. At 52°C the activity was slowly lost in 0.1 M
PURIFICATION
OF AMINOPEPTIDASE
.u E 2 0.2 5
0i
Saturation
Concentration
of (NH,)?
SO4
(“A)
FIG. 1. Sedimentation of the enzymes hydrolyzing L-Arg-p-NA and L-Leu-&NA from the pH 5.1 supernat,anf fluid using (NH4).$304. The sediment obtained from 1 ml of the starting material at the (NH&SO, concentration was dissolved in 15 ml 0.1 M tri+HCl, pH 7.0, and the activity was n = L-Arg-P-NA and 0 = L-Leudetermined &NA.
tris-HCl buffer, pH 7.0, and after 10 minutes the activit’y was reduced by 20%. Ten minutes at 56°C in t,he same buffer reduced the activity 80%. At 0”-4°C the activity was stable in both buffers at pH 5.1-8.0 for several days. These result’s demonstrate t’he heat lability of the enzyme(s) and exclude the possibility of using fractionation procedures based on graded heat’ denaturation as a means for enzyme purification. However, lowering the pH to 5.0 at 0”-4°C did not destroy the activity. Since this is a practical way to agglutinate mitochondria and microsomes (1) it was adopted as a step in further purification (see Final Purification Procedure) . Fractionation by (NH&S04 and crganic solvents. These experiments were carried out using the supernatant’ aft#er sedimentation of the particles from the homogenate at’ pH 5.1. Very small stepwise concentration increases of (NH&S04 were used (2-5%) in order to determine whether aminopeptidase B could be separated readily by t’his procedure from the other enzymes hydrolyzing L-Arg-P-KA. Hydrolysis of L-Arg-@NA as well as of L-Leu-/3-NA was t’ested
B
559
both in the supernatant and sediment fractions (Fig. I). It appeared that the enzyme hydrolyzing L-Arg-P-NA sedimented earlier than that hydrolyzing L-Leu-/3-NA. Organic solvent fractionations using acetone, methanol, ethanol, propanol and nbutanol were tried. The enzyme preparation was dissolved in 0.033 M tris-HCl buffer, pH 7.0. The solvent was added stepwise in 0.5-ml volumes into 15-ml centrifuge tubes up to 90% (vol), each addition being followed by centrifugation. Only acetone appeared to be suitable, yet it caused some loss of activity (lO-20%) even during a very rapid exposure. In a buffer at pH 5.5 denaturation was very marked. These results demonstrate that fractional sedimentation using (NH& SO* is of value in efforts to purify the enzyme in question while organic solvents are of questionable value in this respect. Fractionation by heavy metal salts. The salts were tested: Zn(Ac)z, following Pb(Ac)2, CUE, U02(A~)2, CaC12, MgCl,, Pb(NO&, CrC&, Hg(NO&. Into 1 ml of homogenate or the pooled fractions obtained aft,er gel filtration with Sephadex G 200 there was added 1 ml of solution of the salts mentioned (containing 0.001-0.1 n-mole) at 2°C. After ten minutes t,he sediment was centrifuged (20,000 g; 5 minutes). The sediment was suspended in 2 ml 0.1 M tris-HCl buffer, pH 7.0. A sample of 100 ~1 of the supernatant and of the solubilized sediment was diluted to 10 ml with the buffer. The enzymic activity toward Arg-P-NA in these solutions and in the supernatant fluids was det,ermined. The effect of the metal ions present in the incubation medium was partly eliminated by adding an equivalent amount of ethylenediaminetetraacetate (EDTA) in control assays. All of the metals caused an irreversible loss of activit’y during sedimentation. Gel Jiltration. Sephadex G 75, 100, and 200 were used to fractionate the homogenate supernatant. It appeared that both Sephadex G 100 and 200 caused some separation of the enzymes hydrolyzing L-Arg-P-NA and L-Leu-P-NA. Gel filtration with G 200 (Fig. 2) gave the best separation and revealed the existence of more than one enzyme peak hydrolyzing L-Arg-/3-NA.
HOPSU,
560
M;IKINEN,
AND
GLENNER . -
GO Fraction
L-Arg-CNA
z E
80 number
FIG. 2. Fractionation of the enzymes hydrolyzing L-Arg-P-NA and L-Leu-&NA using Sephadex G 200. Sample was 2 ml homogenate supernatant fluid: Protein concentration, 20 mg/ml; column, 60 cm X 3.7 cm; Elution, 0.1 M tris-HCl, pH 7.0; flow rate, 0.3 ml/min; fraction volume, 5.8 ml; hydrostatic pressure, 70 cm; temperature, 1°C.
27
Protein
I
5
0.8
E x 2 w
n ”
II
L
I IiI LO
1
50 Fraction
GO number
70
nLL
”
FIG. 3. Fractionation of the enzymes hydrolyzing L-Arg-@-NA and L-Leu-p-NA using gel filtration with Sephadex G 100. Sample was 2 ml of an (NH&SOI sediment, between 32 and 407, saturation; elution with 0.1 M tris-HCl, pH 7.0; flow rate, 0.43 ml/min; hydrostatic pressure, 20 cm; temperature, 3°C; fraction volume, 6 ml.
The same experim.ent was performed using an enzyme fract’ion obtained by sedimentation with (NH&CO4 (Figs. 3 and 4). It appeared that the sediment obtained between salt concentrations of 32-40% contained high enzyme activity toward L-Argp-NA and significant separation from that acting on L-Leu-P-NA was achieved. A partial separation, though less complete, was also obtained when fractionating by gel fil-
Fraction
number
FIG. 4. The same conditions as in Fig. 7 except that the sample used was obtained by (NH&S04 sedimentation between 40 and 507; saturation.
tration the sediment obtained between (NH&SO4 concentrations of 40-50 %. This suggested that the former concentration limits are preferable in purification of the enzyme. These experiments demonstrated that. Sephadex gel filtration was the most likely means for the purification of aminopeptidase B. Gel filtration was therefore included in the purification procedure. Sephadex-DEAE as well as CM-Sephadex produced some fractional enzyme separation, but due to the considerable shrinkage of these dextrans,
PURIFICATION
OF AMINOPEPTIDASE
---
L-Leu-O-NA
-
L-Arg-fl-NA Protein
561
200
150 Fraction
B
number
FIG. 5. DEAE-cellulose chromatography of the pH 5.2 supernatant fluid of liver tissue homogenate. Column 65 X 4.0 cm; sample 1.75 ml; elution with NaCl gradient 0.00%0.3 M in 0.005 &I tris-HCI buffer; flow rate, 0.5 ml/min; fraction volume, 7.5 ml.
their physical. lability as well as the rather poor reproducibility of the experiments, they were not included in the final purification procedure. Chromatography using substituted celluloses. DEAE-cellulose and CM-cellulose chromatography also produced some fracbional eneymic separation as reported earlier (6). DEAE-chromatography with a NaCl gradient was found to produce some purification. Elution of the enzyme from the column was effected by a concentration of 0.25 Ri (Fig. 5). A continuous pH gradient from 6.5 bo 9.0 failed to give a satisfactory separation. CM-cellulose produced best purification at pH 5.5 at which pH no binding of aminopept#idase B took place. Final purification procedure. The preliminary experiments presented revealed several methods which could afford some separation of the enzymes. On the basis of these studies the final purification procedure adopted was that presented in Table I. By this scheme purification took 40 days, 30 days of which involved storage in the cold in order to facilitate denaturation of inactive proteins. The precipitation produced after gel filtration with Sephadex G 25 was most likely due to a sharp decrease in the ionic strength of the preparation. The enzyme
preparation was never frozen since that was found to produce inactivation of the enzyme. During the 30 days of storage both the hydrolytic activity per milligram of protein of the enzyme preparation as well as the activity per milliliter were increased. This indicated that the increase was due to sedimentation of protein as well as activation of the enzyme. The sedimented material was not soluble in 0.1 M tris-HCl, pH 7.0, showing that this protein had been denaturated. When the sedimented material was added back to the enzyme preparation no change in the enzymic activity took place. The increase of enzymic activity during storage is shown in Table II. A summary of the changes in the enzymic activity during the purification is presented in Table III and indicates that a purification of about 1,100 fold has been achieved. The hydrolysis of L-Arg-/3-NA by the supernatant of the whole homogenate is due at least to two distinct enzymes which are separated by G 200 column chromatography (see Fig. 2). The peak of aminopeptidase B is about equal to the sum of the other peaks in which hydrolysis of L-Arg-/3-NA occurs, i.e., about half of the hydrolysis of L-Arg-@-NA is due to this enzyme. This was taken into account when estimating t’he activity of aminopep-
562
HOPSU,
Homogenization fresh rat liver
(1 kg of tissue)
MdKINEN,
AXI>
GLENNEI:
TABLE I PURIFICATION PROCEDURE (Carried out at, O”4”C.) Homogenate, 2150 ml, in 0.1 JI tris-HCI buffer, pH 7.0 Centrifugation 20,000 g; 30 min
I Srlpernat,ant fluid pH lowered to 5.1 with 0.2 N HCl. Left to stand for 16 hours. Centrifugation, 20,000 g; 30 min. I Supernatant fluid (NHJZ SO4 added up to 35y0 saturation. Volume 1575 ml; (NH& SO, 327.5 gm. Left to stand for 2.5 hours, centrifugation 20,000 g; 10 min
I Sediment
1
Sediment
(discarded)
Sediment
(discarded)
Supernitant fluid (NH& SO, added up to 45y0 saturation Volume, 1700 ml; (NH,)&‘04 102 gm. Left to stand for 3 hours, centrifugation, 20,000 g; 10 min Super&ant
fluid
(discarded)
Sediment. Solubilized in 0.1 M tris-HCl buffer, pH 7.0, to make 92 ml. Gel filtration using Sephadex G 200 (column 100 x 6.7 cm, elution with 0.1 J1 tris-HCl,
SO, was added up to 30% saturation
Super&ant fluid Volume 260 ml, (NH&SO* added up to 50% saturation (32.5 pm). Left to stand 2 hours, centrifugation 30,000 g; 10 min. Supernatant
fluid
I Supernatant
fluid
I Supernatant
(discarded)
fluid
(52.5 gm). Left
to stand
Sediment
1.5 hours,
(discarded)
Sediment (discarded) Solubilized in 50 ml 0.1 M tris-HCl, pH 7.0. Transferred to 0.005 M trisHCl, pH 7.0, using Sephadex G 25 (column 37 x 3.7 cm). Opalescent preparation was centrifuged at 2000 g; 30 min and (NH& SOa sediment between 25 and 400/, collected, again transferred into dilute buffer. DEAE-cellulose chromatography (column 45 x 2 cm), Whatman DE II, elution linear pH 7.0, flow rate 0.5 ml/min). gradient 0.0-0.25 M NaCl; 0.005 M tris-HCl, The most active fractions pooled (150 ml) and (NH& SO, was added to 45y0 saturation. I (discarded) Sediment pH 7.0 (25 ml), and transferred to 1:lO Solubilized in 0.1 M tris-HCl, diluted McIlvaine’s buffer. Left to stand 30 days, the seditient centrifuged off every second day (10,000 g; 10 min). (NH&SO., was added up to 60% saturation. I (discarded) Sediment Solubilized in 17 ml 0.1 M tris-HCl buffer, pH 7.0.
PURIFICATION
OF AMINOPEPTIDASE
tidase B in the homogenate in the calculations for Table III. Physical Studies on the Preparation Molecular weight. To obtain an approximate estimate of the molecular weight of the enzyme a sample of the preparation prior to the 30 days’ storage phase was used. The Sephadex G 100 column used was 130 X 2.4 cm. The sample was mixed with the reference proteins in a volume of 2 ml. The total amount of protein was 30 mg, flow rate 1 ml/6 minutes, elution with 0.1 M tris-HCl, pH 7.0, using a hydrostatic pressure of 20-30 cm. Results of the gel filtration are presented in Fig. 6 and a plot of part of the data obtained is shown in Fig. 7. The molecular weight as estimated from t’he TABLE
;
.-c ;
Ii 0
Percentage increase in activity
6 10 14 30
80
90
100
number
FIG. 6. Distribution of the enzyme hydrolyzing L-Argg-NA with several reference proteins in gel filtration using Sephadex G 190. Proteins were measured using the Folin-Ciocalteau method and the enzymic activity by testing the hydrolysis of L-Arg-&NA. (1) Thyroglobulin, (2) r-globulin, (3) sample, (4) serum albumin, (5) ovalbumin, (6) enzymic activity toward n-Arg-&NA.
TABLE
III
SUMMARY OF THE PURIFICATION
Homogenate supernatant fluid (NH&SOa 3545% sedlment Sephadex G 200 pool (NH&SO4 5096 sediment Sephadex G 25 pool Before DEAE chromatography (NH),SO( 45yb sediment Before storage 2 days storage 6 days storage 10 days storage 14 days storage 30 days storage Final
70 Fraction
0 33 48 104 107 192
step
0.15 ,” Cl ”
II
Initial 2
Purification
563
data according to Whitaker (12) was 95,500. For calculation of the molecular weight according to Gelotte (2) the KD value was calculated for albumin and found to be 0.27. The K, value for the enzyme was found to
CHANGE IN THE ENZYMIC ACTIVITY (UNITS/MG) OF THE PREPARATION DURING STORAGE OF 30 DAYS Storage period. (days)
B
Volume (ml)
Units,ml
Total amount of units
2150
1
2150
92
21
300 50 109 45 25 32 32 31 31 30 30 17
PROCEDURE Protein h.dml)
Units/mg
Percentage yield
130
0.003
1OQ
1940
105
0.02
93
4.3 17 6 10
1290 850 600 450
24 63 11 11
0.18 0.27 0.55 0.91
60 40 28 21
9 4.7 4.7 4.8 4.5 4.3 4.3 7
225 159 159 150 140 130 130 120
1.92 2.94 3.92 4.36 6.0 6.1 8.6 8.75
10 7 7 7 6.5 6 G 5.5
4.7 1.6 1.2 1.1 0.75 0.7 0.5 0.8
Purification coefficient
1 2.5 22 34 70 114 240 370 490 540 750 760 1100 1100
HOPSU,
MdKINEN,
AND
GLENNER
cm 5 L 3 2 1 start
FIG. 7. Determination of the molecular weight of the enzyme by gel filtration in Sephadex G 100. (1) r-globulin, (2) serum albumin, (3) ovalbumin.
2 1 start
cm 5 4 3 2 1 start
m {
]
H
FIG. 9. Eleetrophoresis of the enzyme preparation in starch gel at various pH values. Voltage 6 V/cm, 0.1 M tris-HCl buffer pH 6.5-9.3. Duration of electrophoresis up to 12 hours. Sample, 30 ~1 in a Whatman paper strip. Dark bands demonstrate protein, and light bands show those pieces of the sectioned electrophoresis slab in which enzymic activity was demonstrated using L-Arg-@-NA.
9.0 8.0 7.0 PH FIG. 8. Electrophoresis of the enzyme preparation in cellulose acetate “Oxoid” strips at various pH values. Voltage 10 V/cm, 0.1 M tris-HCl buffer, pH 7.0-9.0. Sample 4-5 ~1 in a Whatman No. 1 paper strip, duration of electrophoresis 180 minutes. Dark bands demonstrate the location of protein.
be 0.20. The value calculated for the molecular weight of the enzyme with the aid of these figures was 95,000. The molecular weight of albumin was taken to be 70,300 in these calculations (8). Homogeneity. Starch gel electrophoresis as well as electrophoresis in “Oxoid” cellulose acetate was carried out at a number of pH values in order to test the homogeneity of the final preparation as well as to determine the isoelectric point of the enzymically active protein. Results are given in Figs. 8 and 9. Following cellulose acetate electrophoresis, only one protein band was found. Two protein bands were observed following starch gel electrophoresis, only one, the
FIG. 10. Ultracentrifugal pattern of the enzyme preparation. Sedimentation from left to right, exposures at 54 and 78 minutes. For details see text.
more intensely stained, being enzymically active. The isoelectric point was about pH 7.0. In order to determine homogeneity by sedimentation velocity patterns in the ultracentrifuge a portion of the final preparation was lyophilized and dissolved in 0.1 M
PURIFICATION
OF AMINOPEPTIDASE
tris-HCl buffer, pH 7.0, so that the final protein concentration was 5 mg/ml. Sedimentation was carried out in 12-mm cells with a Spinco model E ultracentrifuge operating at 59,780 rpm at 21.6”C. The phase plate angle was 50” and photographs were taken at 54, 62, 70, and 78 minutes. Figure 10 shows the sedimentation pattern of the preparation. The preparation appeared to behave homogeneously. Some slight curling of the base line, however, can be seen, indicating the possible presence of impurities at a low concentration.
B
565
M tris-HCl buffer, pH 7.0, also was found to cause a total loss of activity. These findings, therefore, demonstrated that the enzyme was very easily inactivated by several physical procedures, while the range of optimal preparative conditions was limited. On the other hand, storing of frozen intact liver tissue over several months was found not to cause any loss of activity. Attempts to use various organic solvents and heavy metal ions in the precipitation of the proteins revealed that the enzyme was fairly sensitive to most of these treatments. These reagents might be considered to cause DISCUSSION an irreversible change in the structure of the The preliminary fractionation studies per- molecule or to react with some chemical formed demonstrated that the enzymes hy- groups essential to the function of the endrolyzing L-Arg-P-NA are fairly stable under zyme, e.g., sulfhydryl groups. This finding certain experimental conditions and, there- was in agreement with the earlier evidence fore, a purificat,ion was possible. Studies of concerning the effect of some sulfhydryl the individual steps commonly used in en- reagents on the activity (6). zyme purification revealed that a partial The most unexpected step required in the purification of the enzyme hydrolyzing L- purification procedure was the denaturation Arg+NA, but not n-Leu-/3-NA, could be of an inactive protein from the preparation obtained by several means. Results of simi- during 30 days of storage. The most likely lar efforts using DEAE-celluIose have been explanation for the denaturation was the published earlier by Hopsu et al. (6) and low ionic concentration of the solution. The formed the basis for the discovery of the protein removed was either a contaminating present enzyme. This latter investigation protein or an actual part of the enzyme showed that a simple fractional sedimentamolecule, possibly an enzyme inhibitor. The tion using (NH&SO4 could also be used, as fact that the total enzymic activity of the well as Sephadex gel filtration. preparation increased over threefold during The purification procedure finally em- the precipitation process shows that the ployed in the present study was based on protein removed was actually inhibitory to these preliminary experiments and the result the enzyme. The fact that it was not possible obtained was a highly purified enzyme prep- to dissolve the precipitated protein in buffer aration. Total purification from contamisolutions, showed that an irreversible denating proteins was, however, not achieved, naturation had taken place. as shown most clearly by starch gel electroThe molecular weight of the enzymically phoresis. The proportional amount of con- active protein was found to be fairly large. taminating proteins was, however, small Both methods of calculation gave about and, as far as tested, these were found to be equal results. These techniques for molecucompletely inert enzymically. lar weight estimation are commonly conThe purified enzyme preparation was very sidered to have an accuracy of about f stable and could be stored in the cold as a 10,000. A prerequis’te is that the elution concentrated solution without loss of activvolumes are accurately measured. A possible ity for several months. Excessive heat as error due to the use of a drop counter in the well as freezing of the preparation were, fraction collection was estimated by Whitahowever, destructive. Freezing overnight ker (12) as small (0.5-1.0%) while Leach was found to destroy about two-thirds of and O’Shea (10) found a greater variation the activity of the pure preparation as well (15-20 %). The error was due to variation as of the preparation obtained by gel filtrain drop size as the result of changes in the tion. Freezing in starch gel containing 0.1 surface tension of the liquid in the presence
566
HOPSU,
MilKINEN,
of proteins. Small variations in drop size possibly cause some loss in accuracy in exact measurements, but do not overshadow the estimation of the weight range of the molecule. These differences as well as variations in pretreatment of the gel [decanting, packing, hydrostatic pressure (smaller under high pressure) and buffer] render it likely that small differences are to be found in the calculation of KB values and thus in molecular weight. The value reported earlier for the Xn of albumin is 0.2 (2) while that found by us was 0.27. The latter value as measured in our experimental conditions, was used in the present calculations. The results of this investigation extend our earlier report on the existence in mammalian tissues of an enzyme selectively hydrolyzing n-Arg-/I-NA (as well as L-Lysp-NA) without effect on n-Leu-p-NA or several other amino acid naphthylamides (6). A more complete characterization of the present enzyme follows in a subsequent paper (7). ACKNOWLEDGMENT This work was aided by grants from Sigrid Juselius Foundation, The Finnish State Medical Research Council, and The National Institutes of Health grant TW-00103 to VIinB K. Hopsu.
AND
GLENNER REFERENCES
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