184,100-103
ANALYTICALBIOCHEMISTRY
Purification Charles
(1990)
of Gramicidin A
J. Stankovic,l
Jose M. Delfino,*
and Stuart L. Schreiber’,’ Department of Chemistry and *Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511
Received
June
26,1989
A simple chromatographic purification of the naturally occurring ion channel-forming pentadecapeptide gramicidin A (gA) is presented. This procedure allows gA to be isolated in gram quantities from the commercially available mixture of isomers after chromatography on silica gel. The gramicidin A obtained in this manner is greater than 95% pure as determined by ‘HNMR, HPLC, and amino acid analysis. o 1990 Academic Press,
Inc.
Gramicidin A (gA)3 is an ion channel-forming antibiotic produced by Bacillus breuis (ATCC 8185). As a result of the relatively simple structure and commercial availability (see later) of this peptide, gA represents the most widely studied ion channel molecule. We have been interested in preparing and examining the properties of covalently linked dimers of gramicidin A using rationally designed asymmetric dicarboxylic acid linkers (1). In the course of these studies it became apparent that a convenient method for obtaining gA free from the natural congeners present in the commercial sources was required. The commercial sample of gramicidin (gA’)4 has the structure OHCHN-L-Val-Gly-L-Ala-DLeu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-TrpD-Leu-L-Trp-D-Leu-L-Trp-CONHCHzCHzOH and consists of a mixture of residues at amino acid 11 [approx 72% Trp (gA), 9% Phe (gB), and 19% Tyr (gC)]
’ Present address: Department of Chemistry, Harvard University, Cambridge, MA 02138. x To whom correspondence should be addressed. 3 Abbreviations used: gA, gramicidin A, gB, gramicidin B; gC, gramicidin C; gA’, gramicidin A’; DMSO, dimethyl sulfoxide; DMAB, paradimethylaminobenzaldehyde; C:M:W:AA, chloroform:methanol:water:acetic acid. 4 The nomenclature employed is that proposed by Urry et al. (2) and refers to the natural mixture of gramicidins.
100
(3).5 Each of these is a further mixture that consists of 515% isoleucine (in place of valine) at position 1 (4). Currently there are two possible routes to pure gA6: automated solid-phase peptide synthesis (6), and preparative HPLC purification (7) from natural gA’. Both of these methods can produce (Val’) gA in very high purity, but at a relatively high cost. The latter technique requires the use of equipment not generally accessible to all researchers7 and is impractical to perform on a preparative scale. In this paper we describe a fast and simple purification of gA using the now common organic chemistry technique of flash chromatography (8). This procedure allows for the purification of 2-4 g of the gA’ mixture at a time to yield, after a single cycle, 1-2 g of >95% pure gA. MATERIALS
AND METHODS
Chemicals. Gramicidin A’ (gA’) was used as purchased from Sigma Chemical Company. Solvents for TLC and flash chromatography were reagent grade and used as received from J. T. Baker Chemical Company. Thin-layer chromatography. Chromatography fractions were assayed by TLC using HPTLC plates, Silica Gel-60, from EM Reagents. Plates were eluted with chloroform:methanol:water:acetic acid (C:M:W:AA) 250:30: 4:1, visualized with uv quenching (254 nm), and stained with p-dimethylaminobenzaldehyde (DMAB) followed by brief heating. Flash chromatography. A 5 X 18-cm column was slurry packed with silica gel 60, 0.040-0.063 mm (230400 mesh ASTM), from EM Reagents using C:M:W:AA ’ This resuit is also confirmed by our own analysis. ’ gA can also be purified by countercurrent distribution, but this method is cumbersome and no longer generally used. In addition, the equipment needed to achieve the 2000 or so cycles necessary for separation is no longer available. See Ref. (5). r This method requires the use of preparative HPLC and a 3-m reverse-phase column, neither of which is standard lab equipment.
0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
PURIFICATION
OF
WWwW gC-(pink)
gB-(orange)
FIG. 1. Schematic diagram of the HPTLC of gA’ as received from Sigma (A) and of purified gA (B) eluted with CHCls:MeOH:H20: AcOH (100:30:4:1). Colors in parentheses indicate the colors of the spots on visualization withpare-dimethylaminobenzaldehyde stain.
400:30:4:1. In this solvent system gA has an Rf of approx 0.1. Approximately 1.2 g of gA’ was dissolved in a minimal amount of the same solvent system; excess chloroform was supplied as needed to dissolve the sample.8 The column was then eluted with the same solvent system. Twenty 60-ml fractions eluting at 40-60 ml/min were collected. The solvent mixture was then changed to 300: 30:4:1 (in which gA has an Rf of approx 0.2) and 10 more fractions were collected. Finally, CHC4:MeOH (1:l) was used to elute the last 10 fractions. Fractions 17-28 (containing most of the gA) were pooled and concentrated on a rotary evaporator with minimal heating to avoid degradation of the tryptophans in gA. After most of the chloroform was evaporated, the solution was passed through a 2.5 X 20-cm column of AG-501 (BioRad) ion-exchange resin to remove the remaining acetic acid, salts, and silica gel. The column was eluted with methanol until gA was no longer observed by TLC. This sample was concentrated and rechromatographed according to the above process to yield ca. 700 mg of gA (>95% pure by ‘H-NMR, HPLC and amino acid analysis) . HPLC analysis. The purity of gA was monitored by HPLC using an IBM Instruments Model LC/9533 chromatograph. Samples were analyzed with a 4.5 X 250-mm C-18 column that was eluted with MeOH:H20:CH&N (68:12:20) following the conditions of Prasad et al. (typically between 10 and 20 pg of gA was injected at 1 ml/ min (9)). HPLC grade methanol and acetonitrile from J. T. Baker Chemical Company and water (deionized and distilled) were degassed and used without further purification. ‘H-NMR. NMR spectra were measured on a Bruker WM-500 operating at 500 MHz. Samples were dissolved in DMSO-c-2, and chemical shifts are reported relative to DMSO-dF, (H) at 2.49 ppm. is to be avoided because little or no separation.
101
A
Amino acid analysis. Approximately 0.5 mg of gramicidin was dissolved in 50 ~1 of acetic acid (J. T. Baker) and 200 ~1 of 6 N HCl containing 2% phenol (J. T. Baker). The sample was then degassed, sealed, and heated at 105110°C for 70 h (10). The sample was cooled, opened, and concentrated in vucuo. Samples were analyzed on a 121M Beckman amino acid analyzer at the Yale Protein and Nucleic Acid Chemistry Facility.
-I I T
s The use of excess methanol sample to elute too quickly with
GRAMICIDIN
it causes
the
RESULTS
AND
DISCUSSION
Flash chromatography has been widely used in organic synthesis since its introduction in 1978, but it has not spread widely to other areas of chemistry (i.e., biochemistry or organometallic chemistry). Nevertheless, we have found it to be an excellent way to purify gA’ and other related compounds. The absence of previous reports on the preparative purification of gA’ ’ on silica gel is undoubtedly due to its propensity to separate into conformational isomers by TLC in various solvent systems.” However, other investigators have observed that in solvent systems similar to those reported herein, gA is a single spot by TLC, and even that gA and gB separate into two single spots by TLC (4,13). Based on the observation that gA’ separated into three distinct spots on HPTLC plates with C:M:W:AA (100:30:4:1) (see Fig. lA), and that each of these spots stained a different color with DMAB stain, (suggesting a difference in constitu’ gC can be separated from gA and gB by preparative num oxide plates (11). lo Even synthetic gA shows multiple spots by TLC.
TLC
on alumi-
See Refs.
(3,12).
FIG. 2. HPLC traces of gA’ as received from Sigma (A) and of gA after purification (B). Typically, between 10 and 20 pg of gA was injected onto a 4.5 X 250-mm C-18 column and eluted with MeOH:H20: CH,CN (68:12:20) at 1 ml/min. Each peak is doubled as a result of the presence of valine and isoleucine isomers at residue 1.
102
STANKOVIC.
DELFINO,
tion and not just conformation), these conditions were then applied to the preparative purification of gA’. Purification of gA’ by flash chromatography on silica gel and pooling of the fractions containing mostly gA (as detected by TLC) provide a sample enriched in gA. Recycling this material once more through the same procedure and combining the fractions that contain only gA by TLC provide a sample of gA > 95% pure as judged by ‘H-NMR, HPLC, and amino acid analysis. Figures l-3 show the TLC, HPLC, and ‘H-NMR of gramicidin A’ as received from Sigma and of gramicidin A purified by the above method. Each analysis clearly shows the presence of substantial quantities of gB and gC in the commercial batch of gA’ and the absence of either of these components in the purified sample. This is confirmed by the amino acid analysis which shows less than 2% of either gB or gC (see Table 1). It should be noted that the Rf values indicated are somewhat variable and depend a great deal on the exact composition of the solvent system used. Since separa-
AND
SCHREIBER TABLE
1
Amino Acid Analysis of Gramicidin” Natural Amino acid GUY Ala Val’ Ile Leu T yr Phe TWd
Observed 1.01 2.02 3.70 0.17 3.96 0.18 0.04 3.56
gA (Sigma)
Purified
Expected*
Observed
1 2 3.75-3.85 0.15-0.25 4 0.20 0.05-0.10 3.70-3.80
1.02 2.03 3.64 0.16 3.93 0.01 0.01 3.91
gA Expected 1 2 3.75-3.85 0.15-0.25 4 0 0 4
’ Expressed as moles of amino acid per mole of peptide. Moles of peptide calculated as the average of (moles of Ala)/2 and (moles of Leu)/4. * The range of values given for some residues arises from variability in the biosynthesis and/or the commercial purification. ’ Lower values for valine can be accounted for by the poor hydrolysis of the Val-Val-Val sequence. d Values for Trp are uncorrected for losses during hydrolysis.
tion is presumably modulated by the deactivation of the silica gel by water, this variability is most likely due to the variable water content of the silica gel.” Thus, the solvent compositions listed here should be used only as a guideline. The Rf of gA in the solvent system to be used for elution of the column should be checked prior to use, and adjusted as necessary by addition of chloroform or methanol to provide the specified Rf values. The advantages of this method include the use of a simple, readily available technique to provide gram quantities of gramicidin A at low cost and in a relatively short period. Although the gA so produced still contains a small amount (515%) of the analog with isoleucine at position 1, this preparation is quite appropriate for subsequent reactions or conductance studies. Of course, the presence of this mixture is irrelevant to studies that replace the first amino acid with a new natural or nonnatural amino acid. In the case of our recently reported synthesis of linked dimers the isoleucine-containing hybrids are removed by further purification (HPLC) of the product dimers. In summary this technique provides a simple and efficient route to synthetically useful amounts of gA suitable for the preparation of semisynthetic analogs.
PPM FIG. 3. ‘H-NMR at 500 MHz of the aromatic region of gA as received from Sigma (A) and of purified gA (B). Samples were dissolved in DMSO-ds and chemical shifts are reported relative to DMSO-d,(H) at 2.49 ppm. The protons indicated for gC are the o&o protons of tyrosine, and the protons indicated for gB are the phenyl protons of phenylalanine; the remaining signals are from the tryptophan protons.
ACKNOWLEDGMENTS S.L.S. is pleased to acknowledge and Pfizer for a graduate fellowship
ii This is supported pounds is not possible are used.
the NIGMS (awarded
for generous support, to C.J.S.). J.M.D. ac-
by the fact that the separation if mixtures of only chloroform
of these comand methanol
PURIFICATION knowledges support and the CONICET,
from the John E. Fogarty Republica Argentina.
International
OF Center
REFERENCES 1. Stankovic, C. J., Heinemann, S. H., Delfino, J. M., S&worth, F. J., and Schreiber, S. L. (1989) Science 11,813-817. 2. Glickson, J. D., Mayers, D. F., Settine, J. M., and Urry, D. W. (1972) Biochemistry 11, 477-486; Urry, D. W., Glickson, J. D., Mayers, D. F., and Haider, J. (1972) Biochemistry 11,487-493. 3. Sarges, 2019. 4. Sarges, Sarges,
R., and Witkop,
B. (1965)
J. Amer.
Chem.
Sot. 87,2011,
R., and Witkop, R., and Witkop,
B. (1965) J. Amer. Chem. Sot. 87.2027; B. (1965) Biochemistry 4,249l.
5. Gregory, J. D., and Craig, L. C. (1948) J. Biol. Chem. Ramacbandran, L. K. (1963) Biochemistry 2,1138.
172, 839;
GRAMICIDIN
A
103
6. Prasad, K. U., Trapane, T. L., Busath, D., Szabo, G., and Urry, D. W. (1982) J. Protein Chem. 1, 191-202; Urry, D. W., Walker, J. T., and Trapane, T. L. (1982) J. Membr. Biol. 69,225-231. 7. Koeppe, R. E., and Weiss, L. B. (1981) J. Chromatogr. 208,414418. 8. Still, W. C., Kahn, M., and Mitra, A. (1978) J. Org. Chem. 49,576. 9. Prasad, K. U., Alonso-Romanowski, S., Venkatachalam, C. M., Trapane, T. L., Urry, D. W. (1985) Biochemistry 25,456-463. 10. Weinstein, S., Durkin, J. T., Veatch, W. R., Blout, E. R. (1985) Biochemistry 24,4374-4382. 11. Pepinsky, R. B., and Feigenson, G. W. (1978) Anal. Biochem. 86, 512-518. 12. Veatch, W. R., Fossel, E. T., and Blout, E. R. (1974) Biochemistry
13,5249-5256. 13. Prasad, K. U., Trapane, T. L., Busath, D., Szabo, G., and Urry, D. W. (1982) Znt. J. Peptide Protein Res. 19,162-171.
ANALYTICALBIOCHEMISTRY
Purification Charles
(1990)
of Gramicidin A
J. Stankovic,l
Jose M. Delfino,*
and Stuart L. Schreiber’,’ Department of Chemistry and *Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511
Received
June
26,1989
A simple chromatographic purification of the naturally occurring ion channel-forming pentadecapeptide gramicidin A (gA) is presented. This procedure allows gA to be isolated in gram quantities from the commercially available mixture of isomers after chromatography on silica gel. The gramicidin A obtained in this manner is greater than 95% pure as determined by ‘HNMR, HPLC, and amino acid analysis. o 1990 Academic Press,
Inc.
Gramicidin A (gA)3 is an ion channel-forming antibiotic produced by Bacillus breuis (ATCC 8185). As a result of the relatively simple structure and commercial availability (see later) of this peptide, gA represents the most widely studied ion channel molecule. We have been interested in preparing and examining the properties of covalently linked dimers of gramicidin A using rationally designed asymmetric dicarboxylic acid linkers (1). In the course of these studies it became apparent that a convenient method for obtaining gA free from the natural congeners present in the commercial sources was required. The commercial sample of gramicidin (gA’)4 has the structure OHCHN-L-Val-Gly-L-Ala-DLeu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-TrpD-Leu-L-Trp-D-Leu-L-Trp-CONHCHzCHzOH and consists of a mixture of residues at amino acid 11 [approx 72% Trp (gA), 9% Phe (gB), and 19% Tyr (gC)]
’ Present address: Department of Chemistry, Harvard University, Cambridge, MA 02138. x To whom correspondence should be addressed. 3 Abbreviations used: gA, gramicidin A, gB, gramicidin B; gC, gramicidin C; gA’, gramicidin A’; DMSO, dimethyl sulfoxide; DMAB, paradimethylaminobenzaldehyde; C:M:W:AA, chloroform:methanol:water:acetic acid. 4 The nomenclature employed is that proposed by Urry et al. (2) and refers to the natural mixture of gramicidins.
100
(3).5 Each of these is a further mixture that consists of 515% isoleucine (in place of valine) at position 1 (4). Currently there are two possible routes to pure gA6: automated solid-phase peptide synthesis (6), and preparative HPLC purification (7) from natural gA’. Both of these methods can produce (Val’) gA in very high purity, but at a relatively high cost. The latter technique requires the use of equipment not generally accessible to all researchers7 and is impractical to perform on a preparative scale. In this paper we describe a fast and simple purification of gA using the now common organic chemistry technique of flash chromatography (8). This procedure allows for the purification of 2-4 g of the gA’ mixture at a time to yield, after a single cycle, 1-2 g of >95% pure gA. MATERIALS
AND METHODS
Chemicals. Gramicidin A’ (gA’) was used as purchased from Sigma Chemical Company. Solvents for TLC and flash chromatography were reagent grade and used as received from J. T. Baker Chemical Company. Thin-layer chromatography. Chromatography fractions were assayed by TLC using HPTLC plates, Silica Gel-60, from EM Reagents. Plates were eluted with chloroform:methanol:water:acetic acid (C:M:W:AA) 250:30: 4:1, visualized with uv quenching (254 nm), and stained with p-dimethylaminobenzaldehyde (DMAB) followed by brief heating. Flash chromatography. A 5 X 18-cm column was slurry packed with silica gel 60, 0.040-0.063 mm (230400 mesh ASTM), from EM Reagents using C:M:W:AA ’ This resuit is also confirmed by our own analysis. ’ gA can also be purified by countercurrent distribution, but this method is cumbersome and no longer generally used. In addition, the equipment needed to achieve the 2000 or so cycles necessary for separation is no longer available. See Ref. (5). r This method requires the use of preparative HPLC and a 3-m reverse-phase column, neither of which is standard lab equipment.
0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
PURIFICATION
OF
WWwW gC-(pink)
gB-(orange)
FIG. 1. Schematic diagram of the HPTLC of gA’ as received from Sigma (A) and of purified gA (B) eluted with CHCls:MeOH:H20: AcOH (100:30:4:1). Colors in parentheses indicate the colors of the spots on visualization withpare-dimethylaminobenzaldehyde stain.
400:30:4:1. In this solvent system gA has an Rf of approx 0.1. Approximately 1.2 g of gA’ was dissolved in a minimal amount of the same solvent system; excess chloroform was supplied as needed to dissolve the sample.8 The column was then eluted with the same solvent system. Twenty 60-ml fractions eluting at 40-60 ml/min were collected. The solvent mixture was then changed to 300: 30:4:1 (in which gA has an Rf of approx 0.2) and 10 more fractions were collected. Finally, CHC4:MeOH (1:l) was used to elute the last 10 fractions. Fractions 17-28 (containing most of the gA) were pooled and concentrated on a rotary evaporator with minimal heating to avoid degradation of the tryptophans in gA. After most of the chloroform was evaporated, the solution was passed through a 2.5 X 20-cm column of AG-501 (BioRad) ion-exchange resin to remove the remaining acetic acid, salts, and silica gel. The column was eluted with methanol until gA was no longer observed by TLC. This sample was concentrated and rechromatographed according to the above process to yield ca. 700 mg of gA (>95% pure by ‘H-NMR, HPLC and amino acid analysis) . HPLC analysis. The purity of gA was monitored by HPLC using an IBM Instruments Model LC/9533 chromatograph. Samples were analyzed with a 4.5 X 250-mm C-18 column that was eluted with MeOH:H20:CH&N (68:12:20) following the conditions of Prasad et al. (typically between 10 and 20 pg of gA was injected at 1 ml/ min (9)). HPLC grade methanol and acetonitrile from J. T. Baker Chemical Company and water (deionized and distilled) were degassed and used without further purification. ‘H-NMR. NMR spectra were measured on a Bruker WM-500 operating at 500 MHz. Samples were dissolved in DMSO-c-2, and chemical shifts are reported relative to DMSO-dF, (H) at 2.49 ppm. is to be avoided because little or no separation.
101
A
Amino acid analysis. Approximately 0.5 mg of gramicidin was dissolved in 50 ~1 of acetic acid (J. T. Baker) and 200 ~1 of 6 N HCl containing 2% phenol (J. T. Baker). The sample was then degassed, sealed, and heated at 105110°C for 70 h (10). The sample was cooled, opened, and concentrated in vucuo. Samples were analyzed on a 121M Beckman amino acid analyzer at the Yale Protein and Nucleic Acid Chemistry Facility.
-I I T
s The use of excess methanol sample to elute too quickly with
GRAMICIDIN
it causes
the
RESULTS
AND
DISCUSSION
Flash chromatography has been widely used in organic synthesis since its introduction in 1978, but it has not spread widely to other areas of chemistry (i.e., biochemistry or organometallic chemistry). Nevertheless, we have found it to be an excellent way to purify gA’ and other related compounds. The absence of previous reports on the preparative purification of gA’ ’ on silica gel is undoubtedly due to its propensity to separate into conformational isomers by TLC in various solvent systems.” However, other investigators have observed that in solvent systems similar to those reported herein, gA is a single spot by TLC, and even that gA and gB separate into two single spots by TLC (4,13). Based on the observation that gA’ separated into three distinct spots on HPTLC plates with C:M:W:AA (100:30:4:1) (see Fig. lA), and that each of these spots stained a different color with DMAB stain, (suggesting a difference in constitu’ gC can be separated from gA and gB by preparative num oxide plates (11). lo Even synthetic gA shows multiple spots by TLC.
TLC
on alumi-
See Refs.
(3,12).
FIG. 2. HPLC traces of gA’ as received from Sigma (A) and of gA after purification (B). Typically, between 10 and 20 pg of gA was injected onto a 4.5 X 250-mm C-18 column and eluted with MeOH:H20: CH,CN (68:12:20) at 1 ml/min. Each peak is doubled as a result of the presence of valine and isoleucine isomers at residue 1.
102
STANKOVIC.
DELFINO,
tion and not just conformation), these conditions were then applied to the preparative purification of gA’. Purification of gA’ by flash chromatography on silica gel and pooling of the fractions containing mostly gA (as detected by TLC) provide a sample enriched in gA. Recycling this material once more through the same procedure and combining the fractions that contain only gA by TLC provide a sample of gA > 95% pure as judged by ‘H-NMR, HPLC, and amino acid analysis. Figures l-3 show the TLC, HPLC, and ‘H-NMR of gramicidin A’ as received from Sigma and of gramicidin A purified by the above method. Each analysis clearly shows the presence of substantial quantities of gB and gC in the commercial batch of gA’ and the absence of either of these components in the purified sample. This is confirmed by the amino acid analysis which shows less than 2% of either gB or gC (see Table 1). It should be noted that the Rf values indicated are somewhat variable and depend a great deal on the exact composition of the solvent system used. Since separa-
AND
SCHREIBER TABLE
1
Amino Acid Analysis of Gramicidin” Natural Amino acid GUY Ala Val’ Ile Leu T yr Phe TWd
Observed 1.01 2.02 3.70 0.17 3.96 0.18 0.04 3.56
gA (Sigma)
Purified
Expected*
Observed
1 2 3.75-3.85 0.15-0.25 4 0.20 0.05-0.10 3.70-3.80
1.02 2.03 3.64 0.16 3.93 0.01 0.01 3.91
gA Expected 1 2 3.75-3.85 0.15-0.25 4 0 0 4
’ Expressed as moles of amino acid per mole of peptide. Moles of peptide calculated as the average of (moles of Ala)/2 and (moles of Leu)/4. * The range of values given for some residues arises from variability in the biosynthesis and/or the commercial purification. ’ Lower values for valine can be accounted for by the poor hydrolysis of the Val-Val-Val sequence. d Values for Trp are uncorrected for losses during hydrolysis.
tion is presumably modulated by the deactivation of the silica gel by water, this variability is most likely due to the variable water content of the silica gel.” Thus, the solvent compositions listed here should be used only as a guideline. The Rf of gA in the solvent system to be used for elution of the column should be checked prior to use, and adjusted as necessary by addition of chloroform or methanol to provide the specified Rf values. The advantages of this method include the use of a simple, readily available technique to provide gram quantities of gramicidin A at low cost and in a relatively short period. Although the gA so produced still contains a small amount (515%) of the analog with isoleucine at position 1, this preparation is quite appropriate for subsequent reactions or conductance studies. Of course, the presence of this mixture is irrelevant to studies that replace the first amino acid with a new natural or nonnatural amino acid. In the case of our recently reported synthesis of linked dimers the isoleucine-containing hybrids are removed by further purification (HPLC) of the product dimers. In summary this technique provides a simple and efficient route to synthetically useful amounts of gA suitable for the preparation of semisynthetic analogs.
PPM FIG. 3. ‘H-NMR at 500 MHz of the aromatic region of gA as received from Sigma (A) and of purified gA (B). Samples were dissolved in DMSO-ds and chemical shifts are reported relative to DMSO-d,(H) at 2.49 ppm. The protons indicated for gC are the o&o protons of tyrosine, and the protons indicated for gB are the phenyl protons of phenylalanine; the remaining signals are from the tryptophan protons.
ACKNOWLEDGMENTS S.L.S. is pleased to acknowledge and Pfizer for a graduate fellowship
ii This is supported pounds is not possible are used.
the NIGMS (awarded
for generous support, to C.J.S.). J.M.D. ac-
by the fact that the separation if mixtures of only chloroform
of these comand methanol
PURIFICATION knowledges support and the CONICET,
from the John E. Fogarty Republica Argentina.
International
OF Center
REFERENCES 1. Stankovic, C. J., Heinemann, S. H., Delfino, J. M., S&worth, F. J., and Schreiber, S. L. (1989) Science 11,813-817. 2. Glickson, J. D., Mayers, D. F., Settine, J. M., and Urry, D. W. (1972) Biochemistry 11, 477-486; Urry, D. W., Glickson, J. D., Mayers, D. F., and Haider, J. (1972) Biochemistry 11,487-493. 3. Sarges, 2019. 4. Sarges, Sarges,
R., and Witkop,
B. (1965)
J. Amer.
Chem.
Sot. 87,2011,
R., and Witkop, R., and Witkop,
B. (1965) J. Amer. Chem. Sot. 87.2027; B. (1965) Biochemistry 4,249l.
5. Gregory, J. D., and Craig, L. C. (1948) J. Biol. Chem. Ramacbandran, L. K. (1963) Biochemistry 2,1138.
172, 839;
GRAMICIDIN
A
103
6. Prasad, K. U., Trapane, T. L., Busath, D., Szabo, G., and Urry, D. W. (1982) J. Protein Chem. 1, 191-202; Urry, D. W., Walker, J. T., and Trapane, T. L. (1982) J. Membr. Biol. 69,225-231. 7. Koeppe, R. E., and Weiss, L. B. (1981) J. Chromatogr. 208,414418. 8. Still, W. C., Kahn, M., and Mitra, A. (1978) J. Org. Chem. 49,576. 9. Prasad, K. U., Alonso-Romanowski, S., Venkatachalam, C. M., Trapane, T. L., Urry, D. W. (1985) Biochemistry 25,456-463. 10. Weinstein, S., Durkin, J. T., Veatch, W. R., Blout, E. R. (1985) Biochemistry 24,4374-4382. 11. Pepinsky, R. B., and Feigenson, G. W. (1978) Anal. Biochem. 86, 512-518. 12. Veatch, W. R., Fossel, E. T., and Blout, E. R. (1974) Biochemistry
13,5249-5256. 13. Prasad, K. U., Trapane, T. L., Busath, D., Szabo, G., and Urry, D. W. (1982) Znt. J. Peptide Protein Res. 19,162-171.
