Vol.
169,
No.
June
29,
1990
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
RESEARCH
Pages
CHICKEN
HYPOTENSIVE PEPTIDE: CHARACTERIZATION
PURIFICATION
916-920
AND
G. F. Barbato1y2 and R. F. Widemanl
Intercollege
Received
April
16,
1 Department of Poultry Science and Graduate Programs inlPhysiology and2Genetics The Pennsylvania State University University Park, PA 16802
1990
SUMMARY: We have purified and isolated a novel, hypotensive peptide from avian ventricular tissue. Ventricular homogenateshave been shown to exhibit potent hypotensive activity in the avian and mammalian species,with little natriuresis or diuresis. Using avian mean arterial pressure (MAP) asa bioassay,we were able to purify a peptide which decreasedMAP 30% in adult, female chickens. Amino acid analysisindicated that it contained 20 amino acids (including two cysteine residues), and was not similar to the amino acid composition of mammalian atria1 natriuretic factors, or other known hypotensive peptides. 01990 Academic Press, Inc.
Over the past decade, the discovery of a class of biologically active peptides in mammalian atria has led to an explosion in cardiovascular research. These atria1 natriuretic factors (ANF,
1) produce potent natriuretic, diuretic and vasodilatory effects when administered intravenously (2-4). Efforts to observe ANF-like compounds in other species have met with mixed success. Membrane-bound secretory granules have been observed in cardiocytes of a wide variety of non-mammalian vertebrates, but cardiac tissue homogenates from these species never produce a diuresis or natriuresis equivalent to rat or human ANF (5). Indeed, extracts of chicken hearts produce a mild diuresis and natriuresis in rats, but little, or none, in chickens (5,6). Noted, however, was a marked kaliuresis and hypotension (7). It has not been determined whether the physiological effects of the avian heart homogenate are due to a unique, chemical substance,or to multiple factors contained in the mixture. In the work reported here, we used solid phase extraction techniques and reversed-phase HPLC to isolate factors involved in the hypotensive action of chicken ventricular homogenate. 0006-291XMI Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
916
Vol.
169,
No.
MATERIALS
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Blood uressure bioassay. Mean arterial pressure (MAP) was recorded as reported previously (6,7). Briefly, the carotid artery of an adult, female chicken is cannulated with PE-50 tubing , filled with heparinized saline, and attached to a blood pressure transducer. The transducer is connected to a Grass Model 79D physiograph. The left, brachial vein is also cannulated and used to inject all substances to be tested for pressor activity. Solid phase extraction. Avian ventricular tissue was homogenized, using a Tekmar ‘Tissumizer, in 10 w/v (i.e., 1 g tissue in 10 ml buffer) of an acid extraction medium (containing 0.1 N HCl; 1% TFA, 1% formic acid; 1% NaCl; and 15% CH,CN). The resulting homogenate was centrifuged for 30 min. at 32,000 x g, and the supernatant filtered through a 0.45 micron filter. The filtrate was divided into aliquots and frozen at -20“ C. Samples were thawed, dried in a Savant Speed Vat (Savant Instruments, Inc.; Farmingdale, NY) and resuspended in 50 mM ammonium acetate with pH = 5.5. [NOTE: SO mA4 ammonium acetate (pH=7.0) was also used in a concurrent experiment, but the same results were observed.] Five anion and cation exchange Sep-Paks (Waters QMA and CMA packing, respectively) were linked together, activated with 10 column volumes of 75% CH,CN, and equilibrated with 20 column volumes of 50 mM ammonium acetate. The tissue homogenate was then passed over the columns with a 50 ml glass syringe. The unretained eluate (the neutral pool) was collected in a 50 ml sample tube. The QMA and CMA columns were then disconnected and retaining proteins eluted with 10 column volumes of 50 mM ammonium acetate with 1 M NaCl added. The QMA-retained proteins were designated the basic pool and the CMA-retained proteins were the acidic pool. All three fractions were dried in the Speed Vat, and the salts removed via elution on three C,, SepPak (Waters), using 0.1% TFA in 80% CH,CN. Aliquots of each fraction were then dried in the Speed Vat and tested for pressor activity. A large amount of fat was observed in the neutral pool, so the dried samples were treated with 20 ml CHCl,:CH,OH (2:l). The fat was aspirated and the remaining liquid dried under a stream of dry N,. At each step during the purification, aliquots were assayed for both MAP and total protein content (8). Reversed-phase HPLC. HPLC equipment consisted of a Rainin Instruments gradient HPLC system, with Gilson HPLC System Controller software (v. 1.2), running on an IBM PS/2 Model 30. All separations were performed with a Phenomenex (Ranch0 Palos Verdes , CA), Spherex, 5 micron, C,, analytical (0.46 x 25 cm) or semi-preparative (1 x 25 cm) column. Detection of column effluent was provided by a Gilson 116 programmable UV detector (at 256 and 280 nm). Fractions were collected at 1 min. intervals with a Gilson 203 fraction collector. A simple TFA:CH,CN gradient was employed, with mobile phase ‘A’ consisting of 0.1% TFA in H,O and mobile phase ‘B’ consisting of 75% CH,CN in H,O. Semi-preparative separations were performed at a flow rate of 4.72 ml/min., while analytical separations were performed at 1.0 ml/min. Repurification of the bioactive fraction was performed under similar conditions using a shallow gradient. The bioactive fraction from this sample was re-purified on HPLC using 0.13% HFBA (heptafluorobutyric acid) as the aqueous phase (9). Amino Acid Analvsis. Purified chicken hypotensive peptide (CHP), from the final HPLC run, was dried in the Speed Vat, and hydrolyzed under the gas-phase conditions described by S. Stein (e.g., 10). Essentially, triplicate dried samples were placed in a vacuum dessicator with 10 ml of 6 N HCI (Sequanal Grade, Pearce Chemical Co.) containing 4% thioglycolic acid. The dessicator was evacuated and placed in a drying oven at 110°C for 24 hr. The samples were then resuspended in 0.1 M HCl and assayed for amino acid content 917
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
using pre-column OPA derivatization (11). Amino acid analysis was confirmed by the Protein and Peptide Laboratory in the Biotechnology Institute at Penn State, using standard hydrolysis techniques and PTC amino acid analysis.
RESULTS AND DISCUSSION Table 1 presents the results of the purification process. Solid phase extraction of the tissuehomogenate resulted in a discrete separation of the hypotensive activity into the unretained, neutral pool. Indeed, m activity was observed in the acidic or basic pools. Figure 1 presents analytical chromatograms resulting from the HPLC purification of the neutral pool. Hypotensive activity was only observed in 2 -3 adjacent fractions from the separation, which were pooled and re-purified. Each purification step resulted in fewer UV absorbing peaks, and ultimately, in only one peak which consistently eluted at the sametime under the sameconditions. Section C of Figure 1 represents the chromatogram of a repurified fraction from Section B, using HFBA in the aqueous phase. Elution time of the UV absorbing peak shifted slightly, but consistently, 1 minute later. HPLC separation resulted in a 250,000-fold purification of the original homogenate. High yields were obtained throughout the entire purification procedure, ranging from 100 to 28 percent. While at first glance this seemsunusual, it would appear to be an artefact of the bioassay. Since decreasein MAP is the measure of bioactivity, there is a clear limit to how much blood pressure can be reduced. Since the homogenate causesa 30% decreasein blood pressure, there is little room for increased activity due to purification. Since small amounts of the peptide effected major changesin MAP, we were clearly not assayingthe purification on the linear portion of the dose-responsecurve for CHP. Amino acid analysis of the purified peptide resulted in the discovery of a 20-mer peptide, containing two (2) cysteine residues(Table 2). This would suggestthat the peptide contains a sulfide bridge and is, at least, partially circular. The peptide also contains tyrosine, which is consistent with the chromatograms, which indicate significant absorption at 280 nm. These characteristics are typical of mammalian variants of atria1 natriuretic
Table
1. Purification of chicken hypotensive peptide (CHP) from avian ventricular tissue
Stage
Protein Specific Activity3 (mg) (DMAP/mg protein) Homogenate 127,510 0.0006 0.0059 32,000 x g supernatant 13,400 Neutral pool 1,247 0.055 CHCI3:CH3OH supematant 426 0.1 0.8 HPLC (semi-prep) 33’ HPlC (analytical) 0.52 44.8 ‘protein determined by abs. @ 280 nm 2protein determined by amino acid analysis 3~MAP: Decrease In mean arterial pressure (mm Hg) from baseline
918
Yield (%) 100 98 85 53 33 28
Purification (-fold) 1 IO 100 300 4000 250,000
Vol.
169,
No.
BIOCHEMICAL
3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Time (min.) Figure 1. Anatytical chromatographs of HPLC purification of CHP -- shaded areas correspond to fractions of column eluate which lowered MAP. A) neutral pool. B) active fraction from A. C) active fraction from B. [Solid line = 256 nm; Dotted line = 280 nm]. The x-axis represents absorbance and % of mobile phase
8’ (see text).
factor (ANF). Table 2 compares the amino acid composition of CHP to ANF and other hypoactive peptides isolated from mammalian species. It is clear that CHP does not conform to any of the amino acid profiles of these peptides, and is a novel hypotensive peptide.
Table
2. Amino acid composition abbreviations
AMINO ACID 1 CHF
hANF(99-126)
D/N
E/Q
1
0
202130100112132
2
5050511 505051100202202 405031100201202 41 41
rANF(99-126)
2
rANF(103-126) pVlP pPHl hPHM
2
1 1 1
5 2 2
121 2 2
Nomenclature
follows
S
of CHP compared to other hypotensive peptides (amino acid conform to standard one-letter symbols)
N.E.J.Med.
H
G
T
R
A
Y
M
V
F
Q
L
I
K
C
TOTAL 201 28
10202102
28 24
02222121 2 1 1
2
210211 316:1278(1987).
919
1
0
1 2 1205030
03130 0 5
1
2
0
28 27 27
Vol.
169,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENT This work was supported
by a Biomedical Research Support Grant awarded to the
authors.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
DeBold, A. J. (1979) Proc. Sot. Exp. Biol. Med. 170,133-138. Palluk, R., Gaida, W., and Hoefke, W. (1985) Life Sci. 36,1415-1420. Samson, W. K. (1985) Neuroendocrinology 40,277- 279. Cameron, V. A., Espiner, E. A., Nicholls, M. G., and Skidmore, D. S. (1988) Endocrinology 122,407-414. DeBold, A. J., and Salerno, T. A. (1983) Can. J. Physiol. Pharmacol. 61,127-130. Reilley, T., Gregg, C. M., Wideman, R. F., Jr., and Jarett-Zaczek, D. (1987) Proc. Sot. Exp. Biol. Med. 186,288-293. Gregg, C. F., and Wideman, R. F., Jr. (1986) Amer. J. Physiol. 251, R543-R551. Markwell, M. A. K., Haas, S. M., Tolbert, N. E., and Bieber, L. L. (1981) Methods in Enzymology 72,296-303. Bennett, J. C., Browne, C., and Solomon, S. (1981) Biochemistry 20,4530-4538. Lahm, H.-W., and S. Stein (1985) J. Chromatography 326,357-361. Sista, H. S. (1986) J. Chromatog. 359,231-240.
920
169,
No.
June
29,
1990
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
COMMUNICATIONS
RESEARCH
Pages
CHICKEN
HYPOTENSIVE PEPTIDE: CHARACTERIZATION
PURIFICATION
916-920
AND
G. F. Barbato1y2 and R. F. Widemanl
Intercollege
Received
April
16,
1 Department of Poultry Science and Graduate Programs inlPhysiology and2Genetics The Pennsylvania State University University Park, PA 16802
1990
SUMMARY: We have purified and isolated a novel, hypotensive peptide from avian ventricular tissue. Ventricular homogenateshave been shown to exhibit potent hypotensive activity in the avian and mammalian species,with little natriuresis or diuresis. Using avian mean arterial pressure (MAP) asa bioassay,we were able to purify a peptide which decreasedMAP 30% in adult, female chickens. Amino acid analysisindicated that it contained 20 amino acids (including two cysteine residues), and was not similar to the amino acid composition of mammalian atria1 natriuretic factors, or other known hypotensive peptides. 01990 Academic Press, Inc.
Over the past decade, the discovery of a class of biologically active peptides in mammalian atria has led to an explosion in cardiovascular research. These atria1 natriuretic factors (ANF,
1) produce potent natriuretic, diuretic and vasodilatory effects when administered intravenously (2-4). Efforts to observe ANF-like compounds in other species have met with mixed success. Membrane-bound secretory granules have been observed in cardiocytes of a wide variety of non-mammalian vertebrates, but cardiac tissue homogenates from these species never produce a diuresis or natriuresis equivalent to rat or human ANF (5). Indeed, extracts of chicken hearts produce a mild diuresis and natriuresis in rats, but little, or none, in chickens (5,6). Noted, however, was a marked kaliuresis and hypotension (7). It has not been determined whether the physiological effects of the avian heart homogenate are due to a unique, chemical substance,or to multiple factors contained in the mixture. In the work reported here, we used solid phase extraction techniques and reversed-phase HPLC to isolate factors involved in the hypotensive action of chicken ventricular homogenate. 0006-291XMI Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
916
Vol.
169,
No.
MATERIALS
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Blood uressure bioassay. Mean arterial pressure (MAP) was recorded as reported previously (6,7). Briefly, the carotid artery of an adult, female chicken is cannulated with PE-50 tubing , filled with heparinized saline, and attached to a blood pressure transducer. The transducer is connected to a Grass Model 79D physiograph. The left, brachial vein is also cannulated and used to inject all substances to be tested for pressor activity. Solid phase extraction. Avian ventricular tissue was homogenized, using a Tekmar ‘Tissumizer, in 10 w/v (i.e., 1 g tissue in 10 ml buffer) of an acid extraction medium (containing 0.1 N HCl; 1% TFA, 1% formic acid; 1% NaCl; and 15% CH,CN). The resulting homogenate was centrifuged for 30 min. at 32,000 x g, and the supernatant filtered through a 0.45 micron filter. The filtrate was divided into aliquots and frozen at -20“ C. Samples were thawed, dried in a Savant Speed Vat (Savant Instruments, Inc.; Farmingdale, NY) and resuspended in 50 mM ammonium acetate with pH = 5.5. [NOTE: SO mA4 ammonium acetate (pH=7.0) was also used in a concurrent experiment, but the same results were observed.] Five anion and cation exchange Sep-Paks (Waters QMA and CMA packing, respectively) were linked together, activated with 10 column volumes of 75% CH,CN, and equilibrated with 20 column volumes of 50 mM ammonium acetate. The tissue homogenate was then passed over the columns with a 50 ml glass syringe. The unretained eluate (the neutral pool) was collected in a 50 ml sample tube. The QMA and CMA columns were then disconnected and retaining proteins eluted with 10 column volumes of 50 mM ammonium acetate with 1 M NaCl added. The QMA-retained proteins were designated the basic pool and the CMA-retained proteins were the acidic pool. All three fractions were dried in the Speed Vat, and the salts removed via elution on three C,, SepPak (Waters), using 0.1% TFA in 80% CH,CN. Aliquots of each fraction were then dried in the Speed Vat and tested for pressor activity. A large amount of fat was observed in the neutral pool, so the dried samples were treated with 20 ml CHCl,:CH,OH (2:l). The fat was aspirated and the remaining liquid dried under a stream of dry N,. At each step during the purification, aliquots were assayed for both MAP and total protein content (8). Reversed-phase HPLC. HPLC equipment consisted of a Rainin Instruments gradient HPLC system, with Gilson HPLC System Controller software (v. 1.2), running on an IBM PS/2 Model 30. All separations were performed with a Phenomenex (Ranch0 Palos Verdes , CA), Spherex, 5 micron, C,, analytical (0.46 x 25 cm) or semi-preparative (1 x 25 cm) column. Detection of column effluent was provided by a Gilson 116 programmable UV detector (at 256 and 280 nm). Fractions were collected at 1 min. intervals with a Gilson 203 fraction collector. A simple TFA:CH,CN gradient was employed, with mobile phase ‘A’ consisting of 0.1% TFA in H,O and mobile phase ‘B’ consisting of 75% CH,CN in H,O. Semi-preparative separations were performed at a flow rate of 4.72 ml/min., while analytical separations were performed at 1.0 ml/min. Repurification of the bioactive fraction was performed under similar conditions using a shallow gradient. The bioactive fraction from this sample was re-purified on HPLC using 0.13% HFBA (heptafluorobutyric acid) as the aqueous phase (9). Amino Acid Analvsis. Purified chicken hypotensive peptide (CHP), from the final HPLC run, was dried in the Speed Vat, and hydrolyzed under the gas-phase conditions described by S. Stein (e.g., 10). Essentially, triplicate dried samples were placed in a vacuum dessicator with 10 ml of 6 N HCI (Sequanal Grade, Pearce Chemical Co.) containing 4% thioglycolic acid. The dessicator was evacuated and placed in a drying oven at 110°C for 24 hr. The samples were then resuspended in 0.1 M HCl and assayed for amino acid content 917
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
using pre-column OPA derivatization (11). Amino acid analysis was confirmed by the Protein and Peptide Laboratory in the Biotechnology Institute at Penn State, using standard hydrolysis techniques and PTC amino acid analysis.
RESULTS AND DISCUSSION Table 1 presents the results of the purification process. Solid phase extraction of the tissuehomogenate resulted in a discrete separation of the hypotensive activity into the unretained, neutral pool. Indeed, m activity was observed in the acidic or basic pools. Figure 1 presents analytical chromatograms resulting from the HPLC purification of the neutral pool. Hypotensive activity was only observed in 2 -3 adjacent fractions from the separation, which were pooled and re-purified. Each purification step resulted in fewer UV absorbing peaks, and ultimately, in only one peak which consistently eluted at the sametime under the sameconditions. Section C of Figure 1 represents the chromatogram of a repurified fraction from Section B, using HFBA in the aqueous phase. Elution time of the UV absorbing peak shifted slightly, but consistently, 1 minute later. HPLC separation resulted in a 250,000-fold purification of the original homogenate. High yields were obtained throughout the entire purification procedure, ranging from 100 to 28 percent. While at first glance this seemsunusual, it would appear to be an artefact of the bioassay. Since decreasein MAP is the measure of bioactivity, there is a clear limit to how much blood pressure can be reduced. Since the homogenate causesa 30% decreasein blood pressure, there is little room for increased activity due to purification. Since small amounts of the peptide effected major changesin MAP, we were clearly not assayingthe purification on the linear portion of the dose-responsecurve for CHP. Amino acid analysis of the purified peptide resulted in the discovery of a 20-mer peptide, containing two (2) cysteine residues(Table 2). This would suggestthat the peptide contains a sulfide bridge and is, at least, partially circular. The peptide also contains tyrosine, which is consistent with the chromatograms, which indicate significant absorption at 280 nm. These characteristics are typical of mammalian variants of atria1 natriuretic
Table
1. Purification of chicken hypotensive peptide (CHP) from avian ventricular tissue
Stage
Protein Specific Activity3 (mg) (DMAP/mg protein) Homogenate 127,510 0.0006 0.0059 32,000 x g supernatant 13,400 Neutral pool 1,247 0.055 CHCI3:CH3OH supematant 426 0.1 0.8 HPLC (semi-prep) 33’ HPlC (analytical) 0.52 44.8 ‘protein determined by abs. @ 280 nm 2protein determined by amino acid analysis 3~MAP: Decrease In mean arterial pressure (mm Hg) from baseline
918
Yield (%) 100 98 85 53 33 28
Purification (-fold) 1 IO 100 300 4000 250,000
Vol.
169,
No.
BIOCHEMICAL
3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Time (min.) Figure 1. Anatytical chromatographs of HPLC purification of CHP -- shaded areas correspond to fractions of column eluate which lowered MAP. A) neutral pool. B) active fraction from A. C) active fraction from B. [Solid line = 256 nm; Dotted line = 280 nm]. The x-axis represents absorbance and % of mobile phase
8’ (see text).
factor (ANF). Table 2 compares the amino acid composition of CHP to ANF and other hypoactive peptides isolated from mammalian species. It is clear that CHP does not conform to any of the amino acid profiles of these peptides, and is a novel hypotensive peptide.
Table
2. Amino acid composition abbreviations
AMINO ACID 1 CHF
hANF(99-126)
D/N
E/Q
1
0
202130100112132
2
5050511 505051100202202 405031100201202 41 41
rANF(99-126)
2
rANF(103-126) pVlP pPHl hPHM
2
1 1 1
5 2 2
121 2 2
Nomenclature
follows
S
of CHP compared to other hypotensive peptides (amino acid conform to standard one-letter symbols)
N.E.J.Med.
H
G
T
R
A
Y
M
V
F
Q
L
I
K
C
TOTAL 201 28
10202102
28 24
02222121 2 1 1
2
210211 316:1278(1987).
919
1
0
1 2 1205030
03130 0 5
1
2
0
28 27 27
Vol.
169,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENT This work was supported
by a Biomedical Research Support Grant awarded to the
authors.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
DeBold, A. J. (1979) Proc. Sot. Exp. Biol. Med. 170,133-138. Palluk, R., Gaida, W., and Hoefke, W. (1985) Life Sci. 36,1415-1420. Samson, W. K. (1985) Neuroendocrinology 40,277- 279. Cameron, V. A., Espiner, E. A., Nicholls, M. G., and Skidmore, D. S. (1988) Endocrinology 122,407-414. DeBold, A. J., and Salerno, T. A. (1983) Can. J. Physiol. Pharmacol. 61,127-130. Reilley, T., Gregg, C. M., Wideman, R. F., Jr., and Jarett-Zaczek, D. (1987) Proc. Sot. Exp. Biol. Med. 186,288-293. Gregg, C. F., and Wideman, R. F., Jr. (1986) Amer. J. Physiol. 251, R543-R551. Markwell, M. A. K., Haas, S. M., Tolbert, N. E., and Bieber, L. L. (1981) Methods in Enzymology 72,296-303. Bennett, J. C., Browne, C., and Solomon, S. (1981) Biochemistry 20,4530-4538. Lahm, H.-W., and S. Stein (1985) J. Chromatography 326,357-361. Sista, H. S. (1986) J. Chromatog. 359,231-240.
920
