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A simple method of isolating tonin using chromatofocusing
ANALYTICAL
BIOCHEMISTRY
126, 295-300 (1982)
A Simple Method of Isolating Tonin Using Chromatofocusing’ ELVINS. Department
of Physiology,
P. CHENG ANDBRIAN
The University
of Sydney,
Sydney,
J. MORRIS’ New South
Wales 2006,
Australia
Received March I 1, 1982 A simple procedure for the purification of the serine protease tonin from rat submandibular glands was developed. The method involves steps of ammonium sulfate precipitation (50-80s) and gel filtration on Sephadex G-100 before complete purification by chromatofocusing on Polybuffer exchanger PBE 94 with a pH 7.4-5.0 gradient. Tonin eluted with high resolution at its isoelectric point of 6.2. After separation of Polybuffer 74 on a Bio-Gel column, the final product ran as a single IU, 3 1,500 band on sodium dodecyl sulfate gel electrophoresis. Digestion aureus VA protease yielded peptides of Mr 16,700, 10,000, and 9000. with Staphylococcus Pure tonin was used to produce affinity-purified antitonin having a binding capacity of 11
Tonin is a serine protease capable of hydrolyzing angiotensin II directly from angiotensinogen, 1- 14 tetradecapeptide renin substrate, and angiotensin I (1). By far the richest source of tonin is the submandibular3 gland of the adult rat, which has a tonin concentration of 4 mg/g (2). In fact, 8% of the protein in gland homogenates is tonin. Tonin was first purified by steps of differential centrifugation, ammonium sulfate precipitation, gel filtration on Sephadex G-l 50, and ionexchange chromatography on DEAE-cellulose, phosphocellulose, SP-Sephadex C25, and SP-Sephadex C50 (3). The purified tonin was shown to be homogeneous by analytical gel electrophoresis, and ultracentrifugation and was remarkably stable. A more rapid purification procedure was later developed (2). This involved differential centrifugation, followed by ion-exchange chromatography on DEAE-Bio-Gel A, phosphocellulose, DEAE-Sephacel, and finally preparative isoelectric focusing on Sephadex IEF.4 A tech-
nique involving affinity chromatography on Sephadex 4B coupled to antitonin, followed by chromatography on DEAE-cellulose and Sephadex G-100, has also been described (4). However, although this method involves few steps, it requires antitonin or pure tonin for raising antibodies for the affinity chromatography. To circumvent this, tonin has been isolated recently by chromatography on a CH-Sepharose 4B column to which soybean trypsin inhibitor had been coupled, followed by chromatography on DEAE-cellulose and Sephadex G-100 (5). The present paper describes a quick, simple, and reliable method of purifying tonin from the rat submandibular gland using the new technique of chromatofocusing, which is a column chromatographic technique in which proteins are separated according to their isoelectric point (6,7). METHODS
Isolation procedure. Submandibular glands were removed from male Wistar rats, greater than 400 g in weight, and frozen immediately in liquid N2. Tissue was stored at -20°C and thawed on the day of use. All subsequent procedures were performed at 0 to 4°C. Tissue was weighed, cut into small pieces, and homogenized in 0.0 1 M potassium phosphate
r This work was supported by grants from the National Heart Foundation of Australia, the National Health and Medical Research Council of Australia, and the Children’s Assistance Fund. ’ To whom correspondence should be addressed. 3 Often less correctly termed “submaxillary.” 4 Abbreviations used IEF, isoelectric focusing; BSA, bovine serum albumin: SDS, sodium dodecyl sulfate. 295
0003-2697/82/160295-06$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
296
CHENG
AND
buffer, pH 6.8, containing 0.25 M sucrose and 0.1% Tween 20 (5 ml/g tissue) using a motordriven Potter-Elvehjem homogenizer. The resulting suspension was centrifuged in a Sorvall RC-5B refrigerated centrifuge at 30,OOOg for 20 min. The supernatant was decanted and adjusted to pH 6.2, which is the isoelectric point of tonin (4). Finely crushed solid ammonium sulfate (special enzyme grade) was then added slowly with constant stirring to gradually increase the salt concentration to 50% of saturation. After further stirring for 20 min the sample was centrifuged at 10,OOOg for 10 min. The supernatant was decanted, made to 80% saturation, stirred, and centrifuged as before. The pellet was resuspended to 10 ml with distilled water, dialyzed against 2 changes of 2 liters distilled water overnight, centrifuged as before to remove debris, and either stored at -20°C or applied to a 2.5 X 92-cm column of Sephadex G-100 (Pharmacia) equilibrated in 0.025 M potassium phosphate buffer, pH 6.8, containing 0.1 M NaCl. Fractions of 13.8 ml were collected with a LKB 2070 Ultrorac at a flow rate of 32 ml/h, maintained by a Gilson peristaltic pump, and absorbance at 280 nm was monitored by a LISB Uvicord S spectrophotometer. Fractions with the highest tonin activity were pooled, dialyzed exhaustively for 24 h against 3 changes of distilled water, and lyophilized in a Dynavac FD-1 freeze dryer. Chromatofocusing was performed on a 1.5 X 30-cm column packed with Polybuffer exchanger PBE 94 (Pharmacia) and equilibrated with start buffer (0.025 M imidazoleHCl, pH 7.4) at a high constant flow rate (> 100 ml/h) until the gel bed had settled completely; this required about 10 to 15 bed volumes of start buffer. About 5 ml of eluant buffer (l/8 Polybuffer 74/HCl, pH 5.0) was run into the column before application of 5 to 10 ml of sample redissolved in start or eluant buffer. The column was then eluted with eluant buffer at a constant flow rate of 32 ml/h, and fractions of 13.8 ml were collected while monitoring absorbance at 280 nm as before. The pH was measured by an Anax digital pH meter. Fractions with the
MORRIS
highest tonin activity were pooled and dialyzed overnight against distilled water. To separate Polybuffer from tonin, protein was precipitated with ammonium sulfate at 85% of saturation, and the pellet obtained by centrifugation at 10,OOOg for 10 min was resuspended in a minimal volume (approx 0.4 ml) of 0.01 M potassium phosphate buffer, pH 6.8, before application to a 0.8 X 20-cm BioGel column which was eluted at 8 ml/h. One major peak of protein was obtained. These fractions were pooled, dialyzed exhaustively against distilled water for 24 h, and lyophilized. Tonin assay. Tonin activity in column fractions was detected by a quick and simple procedure based on the hydrolysis of ‘251-labeled angiotensin I under conditions in which proteolytic activities other than tonin were eliminated (1,3). Serine proteases other than tonin were inhibited by using phenylmethanesulfonyl fluoride at a concentration of 1 mM, which is insufficient to inhibit tonin; e.g., a molar ratio exceeding 10,000 results in only 40% inhibition (2). Samples of 10 and 100 ~1 were made up to 350 ~1 with incubation buffer (0.1 M potassium phosphate buffer, pH 6.8, containing 1 mM phenylmethanesulphonyl fluoride, 5 mM EDTA, 8.5 mM 2,2’-bipyridyl, 3.4 mM 8-quinolinol, and 0.1% bovine serum albumin). Two blanks were included in which buffer instead of sample was used. To each tube was added 50 ~1 12’1labeled angiotensin I (New England Nuclear, Boston, Mass., specific activity 900- 1100 &i/pg, approx 10,000 cpm) and the mixtures were incubated at 37°C for 15 min. Tubes were then placed in a boiling-water bath for 30 min to stop the reaction of tonin, which has a high thermal stabihty (3). After cooling on ice, 400 ~1 of angiotensin I antiserum was added to give a final titer of 1:20,000. Samples were incubated overnight at 4°C before addition of 400 ~10.1 M TrisHCl, pH 8, containing 0.5% Dextran T-10 and 2.5% charcoal (8). This rapidly absorbs free radioactivity, which was then separated from antibody-bound lz51-labeled angiotensin I by centrifugation at 1OOOg for I5 min.
ISOLATION
OF TONIN BY CHROMATOFOCUSING
After decanting the supernatant, radioactivity in the charcoal pellets was measured using a LKB 1282 Compugamma counter. The percentage of radioactivity absorbed by the charcoal (“free” radioactivity) for blanks was generally close to 45. Values >45, for samples, were most likely due to absorption of ‘251-labeled angiotensin II octapeptide (which does not bind to the angiotensin I antibody) since the single tyrosine residue in decapeptide angiotensin I is at position four and fragments smaller than angiotensin II are not formed under the reaction conditions employed (2). The tonin activity of the samples was then expressed on a 0 to 100 scale by using the formula Tonin activity (units) =
100
x
(t &&m,le
-
(d+blank) +
(1
-
(Y/~)blank),
where x = cpm lz51-labeled angiotensin I added and y = cpm in charcoal pellet. The results obtained, although semiquantitative, quickly pinpointed column fractions having the highest tonin activity. Protein measurement. Protein was quantified by the method of Lowry et al. (9) using bovine serum albumin (Commonwealth Serum Laboratories, Melbourne) as standard. SDS’ gel electrophoresis. Electrophoresis was performed on SDS-polyacrylamide gels in which the monomer concentration of the running gel was 12.5% (w/v) (10). Protein was visualized by staining with Coomassie blue. Peptide mapping. This was performed by the method of Cleveland et al. (11) which uses Staphylococcus aureus VA protease. The latter is useful because it is not denatured by SDS, so that the SDS (used to ensure that proteins migrate as single polypeptide chains during electrophoresis in a charge-independent fashion) does not have to be removed before digestion of the protein (11). In fact, no elution is needed, as the gel slice is applied directly on to the resolving gel and is then overlaid with S. aureus VA protease. More-
297
over, S. aureus VA protease usually generates relatively large peptide fragments rather than small ones. The site of cleavage is at the COOH-terminal side of aspartic and glutamic acid residues. Individual bands of 5 pg pure tonin were excised from the lanes of a stained SDS gel using a razor blade, trimmed to 2-4 mm wide, and then soaked for 30 min with occasional swirling in 1 ml of 0.125 M Tris-HCl, pH 6.8, containing 0.1% SDS and 1 mM EDTA. The gel slices were pushed to the bottom of the sample wells of a 15% acrylamide gel containing 0.1% SDS. Each slice was then overlaid with 0.01 ml of the buffer containing, in addition, 20% glycerol followed by 0.01 ml buffer/glycerol containing 0.04, 0.4, or 4 pg S. aureus VA protease. Electrophoresis was then performed normally, except that the current was turned off for 30 min when the bromphenol blue dye (which had been added to sample to track the electrophoretic front) neared the bottom of the stacking gel to allow digestion of tonin by VA protease. Immunological procedures. Antitonin antisera were raised in Castle Hill white rabbits by SCinjection of 1, 0.5, and 0.1 mg pure tonin in Freund’s complete adjuvant at 0, 3, and 7 weeks, respectively, with serum being collected on week 9. Antitonin was then isolated by affinity chromatography of a 3350% ammonium sulfate cut of serum on CHSepharose 4B to which tonin had been coupled according to the directions supplied by Pharmacia. The column was equilibrated in 20 mM sodium phosphate buffer, pH 7.2, containing 0.5% BSA and 0.03% NaN+ The rabbit antitonin immunoglobulin fraction was mixed with BSA to 0.5% and applied to the column, which was then washed with 10 ml 20 mM sodium phosphate buffer, pH 7.2, followed by extensive washing with the same buffer containing 1 M NaCl. When the absorbance of the eluate reached zero, antitonin was eluted with 20 mM citrate buffer, pH 3.0. After adjusting to neutral pH, antitonin was dialyzed against distilled water overnight, lyophilized, and then redissolved in 0.1 M sodium phosphate buffer, pH 7.4, containing
298
CHENG AND MORRIS TABLE
. loo-
-3
E 2
s 2 3 :
PURIFICATIONOFTONINFFCOM SUBMANDIBULARGLANDS
B
I =
1
j
3.8 g OFRAT
-2; 50-
,:
:
P 0 2
: ; ::: :
s I-" 0
1 100
.:
Protein (%I
190 155 78 4.9 4.2
100 82 34 2.6 2.2”
'1
: : i
Protein (a4
..' . . . ...' J 200
vohm
t , 300
of &ate
,.'..._.. .,,.... 400
.._ '.
...'
'..,
4 4
500
(ml)
FIG. I. Elution of tonin from Sephadex G- 100. Sample applied was a 50-80% (NH4)rS04 cut of an extract of rat submandibular glands. The nine fractions having highest tonin activity were pooled and prepared for the next column step. 0, Tonin activity; . * . + . , absorbance at 280 nm.
0.9% NaCl to a volume equal to the volume of the original sample of antiserum. Antisera and affinity-purified antitonin were tested by the Ouchterlony double-immunodiffusion method (12) and by titration with 1251-labeled tonin ( lo6 cpm/pg), prepared using a lactoperoxidase-glucose oxidase radioiodination kit (New England Nuclear). Portions of 2 ~1 (2 pg) tonin were mixed with various dilutions of affinity-purified antitonin and 10 ~1 ‘251-lab-eled tonin (20,000 cpm) in a final total volume of 0.3 ml with 0.025 M sodium phosphate buffer, pH 7.6, containing 0.5% BSA, 0.1 M NaCl, and 0.05% Nonidet-P-40. After an overnight incubation at 4”C, the immunoprecipitates were sedimented by centrifugation in an Eppendorf microfuge, washed in 0.025 M sodium phosphate buffer, pH 7.6,O. 1 M NaC1,
Volume of ekrate
Homogenate (NH4)$04 precipitate Sephadex G- 100 peak Chromatofocusing peak Bio-Gel peak a Electrophoretically
pure tonin.
and 0.25% NP-40, sured.
and radioactivity
mea-
RESULTS
The elution of tonin from Sephadex G- 100 is shown in Fig. 1. Chromatofocusing of this peak separated tonin from other proteins (Fig. 2). The peaks in tonin activity on these two columns occurred at positions on the columns equivalent to M, 3 1,000 and ~16.2 in each case, identical with values reported for tonin (3,4). The protein recovered from step to step is shown in Table 1. After sep aration of Polybuffer on Bio-Gel the tonin ran as a single band during SDS gel electrophoresis (Fig. 3). By comparison with the relative mobility of protein standards, the M, of this band was estimated as 3 1,500 (Fig. 4). Although the final preparation had high tonin activity, specific activity could not be
(ml)
FIG. 2. Elution of tonin from chromatofocusing column. The pooled Sephadex fractions were applied to the column of Polybuffer exchanger PBE 94 and eluted over the pH range 7.4-5.0. 0, Tonin activity; 0,pH; ..*‘., absorbance at 280 nm.
ISOLATION
299
OF TONIN BY CHROMATOFOCUSING
0.8 -
92.5 0.6 -
69
0.5 u!
‘;) 0 7 x r”
46
0.4 0.3 0.2 O.l-
1
I
25
I I Illll
2
1
3 4
Molecular
6
weight
810
I
x 10m4
FIG. 4. Calibration curve of protein standards on SDS gel shown in Fig. 3, showing estimation of M, of the pure tonin. Abbreviations of protein standards are apparent from the legend to Fig. 3.
13 FIG. 3. SDS gel electrophoresis of purified tonin (left lane) and crude extract of rat submandibular gland (right lane). Protein standards run simultaneously in an adjacent lane included, from top to bottom, phosphorylase b, bovine serum albumin, ovalbumin, cu-chymotrypsinogen, and ribonuclease A.
determined because the assay was only semiquantitative. Peptide mapping showed that S, aureus VA protease could produce three major polypeptide fragments of tonin (Fig. 5). Little digestion took place with 0.04 pg VA protease. With 0.4 pg a band appeared at M, 16,700 and with 4 pg additional bands appeared at M, 10,000 and 9000 accompanied by a decreased intensity of the tonin band. Tonin was highly antigenic in rabbits. The Ouchterlony immunodiffusion of crude antiserum or affinity-purified antitonin against pure tonin or total submandibular gland proteins revealed a single precepitin line, indicating monospecificity. Titration with rz51-labeled tonin demonstrated that 2 pg tonin was precipitated optimally (86%) by 180 ~1 of the affinity purified antitonin, with 50 ~1 required to precipitate 50%.
1
92.5, 69-
c;) 0 ‘;;
2
” ^,,^, :
3
, (~
46-
6 -a 25-
r”
1s‘ -b 13-;
_‘
FIG. 5. Peptide map of tonin obtained by digestion with 0.04 (lane l), 0.4 (lane 2), and 4 jig (lane 3) of S. aureu~ VA protease and electrophoresis. (a) Undigested tonin; (b, c, and d), the major peptides obtained after digestion.
300
CHENG AND MORRIS
DISCUSSION
The present method of using chromatofocusing for the purification of tonin would appear to be the easiest procedure yet described. The advantage of chromatofocusing is that it gives high resolution such as is obtained with separation techniques based on differences in p1 (isoelectric focusing) together with the high capacity of ion-exchange procedures (7). Moreover, the self-generating pH gradient eliminated the need for a gradient-making device. Furthermore, this simple chromatographic technique is relatively cheap, unlike preparative isoelectric focusing. The final yield in the method described was 28%, which is comparable to that obtained by methods involving affinity chromatography on CH-Sepharose 4B coupled to antitonin, 33% (4), or soybean trypsin inhibitor, 2 1% (5). Higher yields of 55% have been obtained by more cumbersome methods (2). Despite this, the rapidity and simplicity of the present procedure must outweigh considerations based on yield since rat submandibular glands are readily obtainable. Thus, the present method can quickly isolate 4 mg tonin from 4 g tissue and provides an avenue for the isolation of even larger amounts of tonin, which is receiving increasing attention as a putative pathogenic factor in hypertension (13).
ACKNOWLEDGMENT We thank David Tomlinson of Pharmacia and Dr. Joseph F. Y. Hoh for helpful advice.
REFERENCES 1. Boucher, R., Saidi, M., and Genest, J. (1972) in Hypertension 1972 (Genest, J., and Koiw, E., eds.), pp. 5 12-523, Springer-Verlag, Berlin. 2. Thibault, G., and Genest, J. (1981) Biochim. Biophys. Actu 660, 23-29.
3. Demassieux, S., Boucher, R., Grise, C., and Genest, J. (1976) Canad. J. Biochem. 54,788-795. 4. Ikeda, M., Gutkowska, J., Thibault, G., Boucher, R., and Genes& J. (198 1) Hypertension 3,8 l-86. 5. Ikeda, M., and Arakawa, K. (1981) Jap. Circ. J. 45, 1083-1089. 6. Sluyterman, L. A. E., and Elgersma, 0. (1978) J. Chromatogr. 150, 17-30. 7. Sluyterman, L. A. E., and Wijdenes, J. (1978) J. Chromatogr.
150, 3 l-44.
8. Herbert, V., Lau, K. S., Gottlieb, C. W., and Bleicher, S. J. (1965) J. Clin. Endocrinol. 25, 1375-1384. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265 275.
10. O’Farrell, P. H. (1975) J. Biol. Chem. 250, 40074021. 11. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmh, U. K. (1977) J. Biol. Chem. 252, 1102-l 106. 12. Ouchterlony, 0. (1949) Acfa Pathol. Microbial. Stand. 26, 507-5 15. 13. Garcia, R., Schiffrin, E. L., Thibault, G., and Genest, J. (1981) Hypertension 3 (Suppl. I), 101106.
BIOCHEMISTRY
126, 295-300 (1982)
A Simple Method of Isolating Tonin Using Chromatofocusing’ ELVINS. Department
of Physiology,
P. CHENG ANDBRIAN
The University
of Sydney,
Sydney,
J. MORRIS’ New South
Wales 2006,
Australia
Received March I 1, 1982 A simple procedure for the purification of the serine protease tonin from rat submandibular glands was developed. The method involves steps of ammonium sulfate precipitation (50-80s) and gel filtration on Sephadex G-100 before complete purification by chromatofocusing on Polybuffer exchanger PBE 94 with a pH 7.4-5.0 gradient. Tonin eluted with high resolution at its isoelectric point of 6.2. After separation of Polybuffer 74 on a Bio-Gel column, the final product ran as a single IU, 3 1,500 band on sodium dodecyl sulfate gel electrophoresis. Digestion aureus VA protease yielded peptides of Mr 16,700, 10,000, and 9000. with Staphylococcus Pure tonin was used to produce affinity-purified antitonin having a binding capacity of 11
Tonin is a serine protease capable of hydrolyzing angiotensin II directly from angiotensinogen, 1- 14 tetradecapeptide renin substrate, and angiotensin I (1). By far the richest source of tonin is the submandibular3 gland of the adult rat, which has a tonin concentration of 4 mg/g (2). In fact, 8% of the protein in gland homogenates is tonin. Tonin was first purified by steps of differential centrifugation, ammonium sulfate precipitation, gel filtration on Sephadex G-l 50, and ionexchange chromatography on DEAE-cellulose, phosphocellulose, SP-Sephadex C25, and SP-Sephadex C50 (3). The purified tonin was shown to be homogeneous by analytical gel electrophoresis, and ultracentrifugation and was remarkably stable. A more rapid purification procedure was later developed (2). This involved differential centrifugation, followed by ion-exchange chromatography on DEAE-Bio-Gel A, phosphocellulose, DEAE-Sephacel, and finally preparative isoelectric focusing on Sephadex IEF.4 A tech-
nique involving affinity chromatography on Sephadex 4B coupled to antitonin, followed by chromatography on DEAE-cellulose and Sephadex G-100, has also been described (4). However, although this method involves few steps, it requires antitonin or pure tonin for raising antibodies for the affinity chromatography. To circumvent this, tonin has been isolated recently by chromatography on a CH-Sepharose 4B column to which soybean trypsin inhibitor had been coupled, followed by chromatography on DEAE-cellulose and Sephadex G-100 (5). The present paper describes a quick, simple, and reliable method of purifying tonin from the rat submandibular gland using the new technique of chromatofocusing, which is a column chromatographic technique in which proteins are separated according to their isoelectric point (6,7). METHODS
Isolation procedure. Submandibular glands were removed from male Wistar rats, greater than 400 g in weight, and frozen immediately in liquid N2. Tissue was stored at -20°C and thawed on the day of use. All subsequent procedures were performed at 0 to 4°C. Tissue was weighed, cut into small pieces, and homogenized in 0.0 1 M potassium phosphate
r This work was supported by grants from the National Heart Foundation of Australia, the National Health and Medical Research Council of Australia, and the Children’s Assistance Fund. ’ To whom correspondence should be addressed. 3 Often less correctly termed “submaxillary.” 4 Abbreviations used IEF, isoelectric focusing; BSA, bovine serum albumin: SDS, sodium dodecyl sulfate. 295
0003-2697/82/160295-06$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
296
CHENG
AND
buffer, pH 6.8, containing 0.25 M sucrose and 0.1% Tween 20 (5 ml/g tissue) using a motordriven Potter-Elvehjem homogenizer. The resulting suspension was centrifuged in a Sorvall RC-5B refrigerated centrifuge at 30,OOOg for 20 min. The supernatant was decanted and adjusted to pH 6.2, which is the isoelectric point of tonin (4). Finely crushed solid ammonium sulfate (special enzyme grade) was then added slowly with constant stirring to gradually increase the salt concentration to 50% of saturation. After further stirring for 20 min the sample was centrifuged at 10,OOOg for 10 min. The supernatant was decanted, made to 80% saturation, stirred, and centrifuged as before. The pellet was resuspended to 10 ml with distilled water, dialyzed against 2 changes of 2 liters distilled water overnight, centrifuged as before to remove debris, and either stored at -20°C or applied to a 2.5 X 92-cm column of Sephadex G-100 (Pharmacia) equilibrated in 0.025 M potassium phosphate buffer, pH 6.8, containing 0.1 M NaCl. Fractions of 13.8 ml were collected with a LKB 2070 Ultrorac at a flow rate of 32 ml/h, maintained by a Gilson peristaltic pump, and absorbance at 280 nm was monitored by a LISB Uvicord S spectrophotometer. Fractions with the highest tonin activity were pooled, dialyzed exhaustively for 24 h against 3 changes of distilled water, and lyophilized in a Dynavac FD-1 freeze dryer. Chromatofocusing was performed on a 1.5 X 30-cm column packed with Polybuffer exchanger PBE 94 (Pharmacia) and equilibrated with start buffer (0.025 M imidazoleHCl, pH 7.4) at a high constant flow rate (> 100 ml/h) until the gel bed had settled completely; this required about 10 to 15 bed volumes of start buffer. About 5 ml of eluant buffer (l/8 Polybuffer 74/HCl, pH 5.0) was run into the column before application of 5 to 10 ml of sample redissolved in start or eluant buffer. The column was then eluted with eluant buffer at a constant flow rate of 32 ml/h, and fractions of 13.8 ml were collected while monitoring absorbance at 280 nm as before. The pH was measured by an Anax digital pH meter. Fractions with the
MORRIS
highest tonin activity were pooled and dialyzed overnight against distilled water. To separate Polybuffer from tonin, protein was precipitated with ammonium sulfate at 85% of saturation, and the pellet obtained by centrifugation at 10,OOOg for 10 min was resuspended in a minimal volume (approx 0.4 ml) of 0.01 M potassium phosphate buffer, pH 6.8, before application to a 0.8 X 20-cm BioGel column which was eluted at 8 ml/h. One major peak of protein was obtained. These fractions were pooled, dialyzed exhaustively against distilled water for 24 h, and lyophilized. Tonin assay. Tonin activity in column fractions was detected by a quick and simple procedure based on the hydrolysis of ‘251-labeled angiotensin I under conditions in which proteolytic activities other than tonin were eliminated (1,3). Serine proteases other than tonin were inhibited by using phenylmethanesulfonyl fluoride at a concentration of 1 mM, which is insufficient to inhibit tonin; e.g., a molar ratio exceeding 10,000 results in only 40% inhibition (2). Samples of 10 and 100 ~1 were made up to 350 ~1 with incubation buffer (0.1 M potassium phosphate buffer, pH 6.8, containing 1 mM phenylmethanesulphonyl fluoride, 5 mM EDTA, 8.5 mM 2,2’-bipyridyl, 3.4 mM 8-quinolinol, and 0.1% bovine serum albumin). Two blanks were included in which buffer instead of sample was used. To each tube was added 50 ~1 12’1labeled angiotensin I (New England Nuclear, Boston, Mass., specific activity 900- 1100 &i/pg, approx 10,000 cpm) and the mixtures were incubated at 37°C for 15 min. Tubes were then placed in a boiling-water bath for 30 min to stop the reaction of tonin, which has a high thermal stabihty (3). After cooling on ice, 400 ~1 of angiotensin I antiserum was added to give a final titer of 1:20,000. Samples were incubated overnight at 4°C before addition of 400 ~10.1 M TrisHCl, pH 8, containing 0.5% Dextran T-10 and 2.5% charcoal (8). This rapidly absorbs free radioactivity, which was then separated from antibody-bound lz51-labeled angiotensin I by centrifugation at 1OOOg for I5 min.
ISOLATION
OF TONIN BY CHROMATOFOCUSING
After decanting the supernatant, radioactivity in the charcoal pellets was measured using a LKB 1282 Compugamma counter. The percentage of radioactivity absorbed by the charcoal (“free” radioactivity) for blanks was generally close to 45. Values >45, for samples, were most likely due to absorption of ‘251-labeled angiotensin II octapeptide (which does not bind to the angiotensin I antibody) since the single tyrosine residue in decapeptide angiotensin I is at position four and fragments smaller than angiotensin II are not formed under the reaction conditions employed (2). The tonin activity of the samples was then expressed on a 0 to 100 scale by using the formula Tonin activity (units) =
100
x
(t &&m,le
-
(d+blank) +
(1
-
(Y/~)blank),
where x = cpm lz51-labeled angiotensin I added and y = cpm in charcoal pellet. The results obtained, although semiquantitative, quickly pinpointed column fractions having the highest tonin activity. Protein measurement. Protein was quantified by the method of Lowry et al. (9) using bovine serum albumin (Commonwealth Serum Laboratories, Melbourne) as standard. SDS’ gel electrophoresis. Electrophoresis was performed on SDS-polyacrylamide gels in which the monomer concentration of the running gel was 12.5% (w/v) (10). Protein was visualized by staining with Coomassie blue. Peptide mapping. This was performed by the method of Cleveland et al. (11) which uses Staphylococcus aureus VA protease. The latter is useful because it is not denatured by SDS, so that the SDS (used to ensure that proteins migrate as single polypeptide chains during electrophoresis in a charge-independent fashion) does not have to be removed before digestion of the protein (11). In fact, no elution is needed, as the gel slice is applied directly on to the resolving gel and is then overlaid with S. aureus VA protease. More-
297
over, S. aureus VA protease usually generates relatively large peptide fragments rather than small ones. The site of cleavage is at the COOH-terminal side of aspartic and glutamic acid residues. Individual bands of 5 pg pure tonin were excised from the lanes of a stained SDS gel using a razor blade, trimmed to 2-4 mm wide, and then soaked for 30 min with occasional swirling in 1 ml of 0.125 M Tris-HCl, pH 6.8, containing 0.1% SDS and 1 mM EDTA. The gel slices were pushed to the bottom of the sample wells of a 15% acrylamide gel containing 0.1% SDS. Each slice was then overlaid with 0.01 ml of the buffer containing, in addition, 20% glycerol followed by 0.01 ml buffer/glycerol containing 0.04, 0.4, or 4 pg S. aureus VA protease. Electrophoresis was then performed normally, except that the current was turned off for 30 min when the bromphenol blue dye (which had been added to sample to track the electrophoretic front) neared the bottom of the stacking gel to allow digestion of tonin by VA protease. Immunological procedures. Antitonin antisera were raised in Castle Hill white rabbits by SCinjection of 1, 0.5, and 0.1 mg pure tonin in Freund’s complete adjuvant at 0, 3, and 7 weeks, respectively, with serum being collected on week 9. Antitonin was then isolated by affinity chromatography of a 3350% ammonium sulfate cut of serum on CHSepharose 4B to which tonin had been coupled according to the directions supplied by Pharmacia. The column was equilibrated in 20 mM sodium phosphate buffer, pH 7.2, containing 0.5% BSA and 0.03% NaN+ The rabbit antitonin immunoglobulin fraction was mixed with BSA to 0.5% and applied to the column, which was then washed with 10 ml 20 mM sodium phosphate buffer, pH 7.2, followed by extensive washing with the same buffer containing 1 M NaCl. When the absorbance of the eluate reached zero, antitonin was eluted with 20 mM citrate buffer, pH 3.0. After adjusting to neutral pH, antitonin was dialyzed against distilled water overnight, lyophilized, and then redissolved in 0.1 M sodium phosphate buffer, pH 7.4, containing
298
CHENG AND MORRIS TABLE
. loo-
-3
E 2
s 2 3 :
PURIFICATIONOFTONINFFCOM SUBMANDIBULARGLANDS
B
I =
1
j
3.8 g OFRAT
-2; 50-
,:
:
P 0 2
: ; ::: :
s I-" 0
1 100
.:
Protein (%I
190 155 78 4.9 4.2
100 82 34 2.6 2.2”
'1
: : i
Protein (a4
..' . . . ...' J 200
vohm
t , 300
of &ate
,.'..._.. .,,.... 400
.._ '.
...'
'..,
4 4
500
(ml)
FIG. I. Elution of tonin from Sephadex G- 100. Sample applied was a 50-80% (NH4)rS04 cut of an extract of rat submandibular glands. The nine fractions having highest tonin activity were pooled and prepared for the next column step. 0, Tonin activity; . * . + . , absorbance at 280 nm.
0.9% NaCl to a volume equal to the volume of the original sample of antiserum. Antisera and affinity-purified antitonin were tested by the Ouchterlony double-immunodiffusion method (12) and by titration with 1251-labeled tonin ( lo6 cpm/pg), prepared using a lactoperoxidase-glucose oxidase radioiodination kit (New England Nuclear). Portions of 2 ~1 (2 pg) tonin were mixed with various dilutions of affinity-purified antitonin and 10 ~1 ‘251-lab-eled tonin (20,000 cpm) in a final total volume of 0.3 ml with 0.025 M sodium phosphate buffer, pH 7.6, containing 0.5% BSA, 0.1 M NaCl, and 0.05% Nonidet-P-40. After an overnight incubation at 4”C, the immunoprecipitates were sedimented by centrifugation in an Eppendorf microfuge, washed in 0.025 M sodium phosphate buffer, pH 7.6,O. 1 M NaC1,
Volume of ekrate
Homogenate (NH4)$04 precipitate Sephadex G- 100 peak Chromatofocusing peak Bio-Gel peak a Electrophoretically
pure tonin.
and 0.25% NP-40, sured.
and radioactivity
mea-
RESULTS
The elution of tonin from Sephadex G- 100 is shown in Fig. 1. Chromatofocusing of this peak separated tonin from other proteins (Fig. 2). The peaks in tonin activity on these two columns occurred at positions on the columns equivalent to M, 3 1,000 and ~16.2 in each case, identical with values reported for tonin (3,4). The protein recovered from step to step is shown in Table 1. After sep aration of Polybuffer on Bio-Gel the tonin ran as a single band during SDS gel electrophoresis (Fig. 3). By comparison with the relative mobility of protein standards, the M, of this band was estimated as 3 1,500 (Fig. 4). Although the final preparation had high tonin activity, specific activity could not be
(ml)
FIG. 2. Elution of tonin from chromatofocusing column. The pooled Sephadex fractions were applied to the column of Polybuffer exchanger PBE 94 and eluted over the pH range 7.4-5.0. 0, Tonin activity; 0,pH; ..*‘., absorbance at 280 nm.
ISOLATION
299
OF TONIN BY CHROMATOFOCUSING
0.8 -
92.5 0.6 -
69
0.5 u!
‘;) 0 7 x r”
46
0.4 0.3 0.2 O.l-
1
I
25
I I Illll
2
1
3 4
Molecular
6
weight
810
I
x 10m4
FIG. 4. Calibration curve of protein standards on SDS gel shown in Fig. 3, showing estimation of M, of the pure tonin. Abbreviations of protein standards are apparent from the legend to Fig. 3.
13 FIG. 3. SDS gel electrophoresis of purified tonin (left lane) and crude extract of rat submandibular gland (right lane). Protein standards run simultaneously in an adjacent lane included, from top to bottom, phosphorylase b, bovine serum albumin, ovalbumin, cu-chymotrypsinogen, and ribonuclease A.
determined because the assay was only semiquantitative. Peptide mapping showed that S, aureus VA protease could produce three major polypeptide fragments of tonin (Fig. 5). Little digestion took place with 0.04 pg VA protease. With 0.4 pg a band appeared at M, 16,700 and with 4 pg additional bands appeared at M, 10,000 and 9000 accompanied by a decreased intensity of the tonin band. Tonin was highly antigenic in rabbits. The Ouchterlony immunodiffusion of crude antiserum or affinity-purified antitonin against pure tonin or total submandibular gland proteins revealed a single precepitin line, indicating monospecificity. Titration with rz51-labeled tonin demonstrated that 2 pg tonin was precipitated optimally (86%) by 180 ~1 of the affinity purified antitonin, with 50 ~1 required to precipitate 50%.
1
92.5, 69-
c;) 0 ‘;;
2
” ^,,^, :
3
, (~
46-
6 -a 25-
r”
1s‘ -b 13-;
_‘
FIG. 5. Peptide map of tonin obtained by digestion with 0.04 (lane l), 0.4 (lane 2), and 4 jig (lane 3) of S. aureu~ VA protease and electrophoresis. (a) Undigested tonin; (b, c, and d), the major peptides obtained after digestion.
300
CHENG AND MORRIS
DISCUSSION
The present method of using chromatofocusing for the purification of tonin would appear to be the easiest procedure yet described. The advantage of chromatofocusing is that it gives high resolution such as is obtained with separation techniques based on differences in p1 (isoelectric focusing) together with the high capacity of ion-exchange procedures (7). Moreover, the self-generating pH gradient eliminated the need for a gradient-making device. Furthermore, this simple chromatographic technique is relatively cheap, unlike preparative isoelectric focusing. The final yield in the method described was 28%, which is comparable to that obtained by methods involving affinity chromatography on CH-Sepharose 4B coupled to antitonin, 33% (4), or soybean trypsin inhibitor, 2 1% (5). Higher yields of 55% have been obtained by more cumbersome methods (2). Despite this, the rapidity and simplicity of the present procedure must outweigh considerations based on yield since rat submandibular glands are readily obtainable. Thus, the present method can quickly isolate 4 mg tonin from 4 g tissue and provides an avenue for the isolation of even larger amounts of tonin, which is receiving increasing attention as a putative pathogenic factor in hypertension (13).
ACKNOWLEDGMENT We thank David Tomlinson of Pharmacia and Dr. Joseph F. Y. Hoh for helpful advice.
REFERENCES 1. Boucher, R., Saidi, M., and Genest, J. (1972) in Hypertension 1972 (Genest, J., and Koiw, E., eds.), pp. 5 12-523, Springer-Verlag, Berlin. 2. Thibault, G., and Genest, J. (1981) Biochim. Biophys. Actu 660, 23-29.
3. Demassieux, S., Boucher, R., Grise, C., and Genest, J. (1976) Canad. J. Biochem. 54,788-795. 4. Ikeda, M., Gutkowska, J., Thibault, G., Boucher, R., and Genes& J. (198 1) Hypertension 3,8 l-86. 5. Ikeda, M., and Arakawa, K. (1981) Jap. Circ. J. 45, 1083-1089. 6. Sluyterman, L. A. E., and Elgersma, 0. (1978) J. Chromatogr. 150, 17-30. 7. Sluyterman, L. A. E., and Wijdenes, J. (1978) J. Chromatogr.
150, 3 l-44.
8. Herbert, V., Lau, K. S., Gottlieb, C. W., and Bleicher, S. J. (1965) J. Clin. Endocrinol. 25, 1375-1384. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265 275.
10. O’Farrell, P. H. (1975) J. Biol. Chem. 250, 40074021. 11. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmh, U. K. (1977) J. Biol. Chem. 252, 1102-l 106. 12. Ouchterlony, 0. (1949) Acfa Pathol. Microbial. Stand. 26, 507-5 15. 13. Garcia, R., Schiffrin, E. L., Thibault, G., and Genest, J. (1981) Hypertension 3 (Suppl. I), 101106.