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Reductive methods for isotopic labeling of antibiotics
ANALYTICAL
BIOCHEMISTRY
Reductive
18
1,90-95
Methods
(1989)
for Isotopic
Labeling of Antibiotics
W. Scott Champney Department
of
Biochemistry,
College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614
Received February 6,1989 MATERIALS Methods for the reductive methylation of the amino groups of eight different antibiotics using ‘HCOH or H14COH are presented. The reductive labeling of an additional seven antibiotics by NaB3H4 is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented. o loss Academic Press,
Inc.
Detailed studies on the mechanisms of antibiotic function require the use of isotopically labeled drugs for quantitative measurements of uptake and binding characteristics. Of the numerous antibiotics affecting the ribosome, a very limited number are commercially available as labeled compounds. Methods for the synthesis of radioactive antibiotics are difficult and often expensive and are generally not easily performed in most laboratories. A set of precedures based on reductive methods for protein labeling (l-4) has been devised for the isotopic labeling of 13 aminoglycoside and macrolide antibiotics. In addition, two different methods for the quantitation of these compounds have also been established on the basis of the reactivity of their amino groups with fluorescamine (5) and of aldehyde and ketone groups with 2,4-dinitrophenylhydrazine (6). A phosphocellulose paper binding assay for measuring the specific activity of the methyl-labeled antibiotics has also been developed. The ease of preparation of these drugs should help to overcome the limited availability of such molecules for cellular uptake studies, as recently emphasized by Taber et al. (7). The potential usefulness of these compounds for the study of uptake kinetics and ribosome binding is demonstrated.
AND
METHODS
Muterzizls. Both 3HCOH (36 mCi/mmol) and H14COH (53 mCi/mmol) came from New England Nuclear; NaB3H4 (1.4 Ci/mmol) was from ICN Radiochemicals. NaCNBH3, fluorescamine, and all antibiotics except neamine were from Sigma Chemical Co. Neamine was a gift from Dr. Richard Keene (Upjohn Co.). NaBH4 was from Alfa Chemicals, 2,4-dinitrophenylhydrazine was from Eastman Chemicals, and P81 phosphocellulose paper was from Whatman. Bio-Gel P2 (100-200 mesh) was a product of Bio-Rad. Reductive methyl&ion. The standard reaction mixture contained 1 mg of antibiotic, 20 pg NaCNBH3, 10 &i 3HCOH (280 nmol), and 10 mM NaP04 buffer (pH 7.3) in a volume of 0.2 ml. The mixture was incubated at 37°C for 15 min, an additional 10 pg of NaCNBH3 was added, and the incubation was continued for an additional 15 min. Samples of 1-5 ~1 were removed for the phosphocellulose paper assay (see following) and 1~1 of 37% HCOH was added to the reaction mixture which was stored frozen. Labeling with Hr4COH was also performed at 10 &i (200 nmol) per reaction. Borohydride reduction. The standard reaction mixture contained 1 mg of antibiotic, 25 &i NaB3H4 (0.01 N NaOH), and 0.1 M Na borate (pH 9.0) in a final volume of 0.2 ml. The mixture was incubated at 25°C for 10 min and at 37°C for an additional IO min. NaBH4 was added to give a final concentration of 0.05 M, and the mixture was brought to pH 6.5 with 5 ~1 glacial acetic acid and stored frozen. Gel filtration chromatography. The labeled antibiotics were separated from unincoporated 3HCOH or NaB3H4 by chromatography on a Bio-Gel P2 column (0.9 X 25 cm) in 0.1 M NH4 acetate. Fractions of 0.5 ml were collected and sampled for radioactivity by spotting 25 or 50 ~1 on filter paper disks which were air-dried. 3H radioactivity was measured in a liquid scintillation counter, and fractions containing labeled antibiotic were pooled and dried by lyphilization. Antibiotics were dissolved in 0.1 ml of water and 1 ~1 was removed and counted for 3H radioactivity on GF/A glass fiber filters
90 All
Copyright 0 1989 rights of reproduction
0003-2697/89 $3.00 by Academic Press, Inc. in any form reserved.
ANTIBIOTIC
LABELING
BY
60% compared to equivalent samples counted on GF/A filters. Fluwescamine assay. Assay mixtures contained variable amounts of antibiotics in 0.9 ml of 0.1 M Na borate (pH 9.0). A volume of 0.1 ml of fluorescamine (0.25% w/v in dimethyl sulfoxide) was added with vigorous mixing. The fluorescence of the product was measured in a Perkin-Elmer 650-40 fluorescence spectrophotometer at 485 nm (slit 20 nm) with excitation at 390 nm (slit 10 nm).
2000
2,4-Dinitrophenylhydrazinc assay. Assay mixtures contained variable amounts of antibiotics in 0.5 ml of 0.1 M Na borate (pH 9.0). A volume of 0.5 ml of 2,4-dinitrophenylhydrazine (0.4% w/v in 2 N HCl) was added with mixing. After standing for 20-30 min, the 2,4-dinitrophenylhydrazone product was read in a spectrophotometer at 443 nm. Stock solutions of 12.5 mM formaldehyde and 13.5 mM acetone were used as standards in the assay.
500
0
10
3-o
20
Fraction
0 ,
Functional assays. The inhibitory activity of labeled antibiotics was tested with a zone of inhibition assay. Volumes of l-5 ~1 of labeled antibiotics were spotted on filter paper disks (Whatman No. 3) which were placed on soft agar lawns of sensitive Escherichia coli cells [strain SK901 (S)] on LB agar plates (9) along with samples of unlabeled drugs. The diameter of the zone of inhibition was measured after incubation at 37°C and compared with the zone size of unlabeled samples. Antibiotic uptake assays were conducted as described previously (10-12). Briefly, E. coli cells at a density of 2 X 108/ml were suspended in 1 ml of 10 mM Hepes buffer containing 0.5% glucose at 37°C. Labeled antibiotic was added and 0.2-ml samples were removed at intervals, filtered on Millipore filters (HAWP, 0.45 p), washed three times with 5 ml of Hepes buffer containing 0.3% NaCl, dried, and counted for bound radioactivity.
No.
FIG. 1. Gel filtration chromatography of [3H]hygromycin B on a Bio-Gel P2 column. The absorbance at 250 nm (0) and the “H cpm (0) in 50 ~1 of each 0.5-ml column fraction were measured.
at an efficiency of 60%. The counting fluid was 0.25% (w/v) 2,5-diphenyloxazole in toluene. Phosphocellulose paper assay. The specific activity of methyl-labeled antibiotics was determined by spotting samples of three different volumes from the reaction mixture on Z.&cm phosphocellulose paper disks. The disks were washed with 10 ml of water to remove unincorporated 3HCOH and the radioactivity remaining was measured by liquid scintillation counting. Radioactivity measured on phosphocellulose paper was quenched by
TABLE
Reductive Methylation
Antibiotic Neomycin B Tobramycin Kanamycin Neamine Gentamycin Hygromycin B Spectinomycin Streptomycin
91
REDUCTION
1
and Quantitative
Specific activity (mCi/mmol)
PC disk assay (w-drd
5.1-27 3.2-45 3.6-34 1.9-34 3.4-47 2.3-19 2.0-7.8 1.2-5.8
20,000 48,000 36,000 45,500 32,000 20,000 12,500 5300
Assay of Antibiotic
Amino-methyl groups 12 10 8 8 7 5 2 1
Amino Groups
Relative
fluorescence 650 400 450 900 225 197 0 0
Primary amino groups 6 5 4 4 3 3 0 0
Note. Specific activities are based on radioactivity present in column-purified antibiotic samples. The range represents labeling at different ratios of ‘HCOH to antibiotic. The phosphocellulose (PC disk) binding data are from the slopes of Fig. 2. The expected number of labeled amino-methy groups is from the structure of the compound (16). The relative fluorescence at 485 nm per pg of antibiotic is from the slopes of Fig. 3. The number of fluorescamine reactive primary amino groups is from the structure of the compound (16).
92
W.
x
0 Tobra.
60: 50 -
=
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3
30-
E
zo-
tf
lo-
E
30000 5.D
,I:
ti 0. E B
o-
OKana. 0 uiea. Genta. 0 HYQ~O.
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SCOTT
2
4
R-CH3
6
6
CHAMPNEY
hyde by gel filtration chromatography (Fig. 1). In this instance the absorbance of hygromycin B was used to monitor the elution and recovery of the antibiotic from the column. Seven different aminoglycoside antibiotics and spectinomycin were labeled by this procedure. The range of specific activities found for different labeling conditions is listed in Table 1. Generally, 3HCOH was reacted at a molar ratio of 0.1-0.2 with each antibiotic, to give derivatives with the lower specific activities listed. By reducing the antibiotic concentration in the reaction mixture to give a 5- to lo-fold molar excess of 3HCOH, derivatives with higher specific activities were generated with maximal labeling of all reactive amino groups. The final specific activity of the compound can be varied depending on its intended use and the amount of isotope available. Comparable specific activities were obtained when H14COH was used. For any reaction condition, a measure of the specific activity of the antibiotic could be determined by a simple
I
0 Neo.
101214
groups
20000
L c-+-J
1ooot
2000 0 k 0
1
Antibiotic
2
3
-
4
(pgldisk)
FIG. 2.
Phosphocellulose paper disk assay. Three different volumes of the indicated antibiotics were removed from the reaction mixture and filtered through phosphocellulose paper disks as described under Materials and Methods. The dried disks were measured for 3H cpm by scintillation counting. (0) neamine; (D) hygromycin B; (A) streptomycin. Inset: The micromolar specific activity of eight antibiotics reacted with excess ‘HCOH is correlated with the expected number of labeled amino methyl groups in each compound.
R-NH2 groups ‘0 3
lOOO-
E
Antibiotic binding to 70 S ribosomes was based on a filter binding method (13-14). High-salt-washed ribosomes were prepared as described (8) and were reacted with variable amounts of labeled antibiotics at 37°C in R buffer (10 mM Tris-HCl, pH 7.6,lO mM Mg acetate, 50 mM NH,Cl, 3 mM mercaptoethanol). Each sample was filtered on a Millipore filter, washed three times with 5 ml of R buffer, and counted for bound radioactivity.
P) 5 .z z
a
600 -
600 -
400 -
200 -
o!
RESULTS
AND
DISCUSSION
The aminoglycoside antibiotics and spectinomycin contain primary amino groups which can be reduced to dimethyl amino derivatives after reactions with aldehydes via a Schiff base intermediate (2-4). Using 3HCOH or H14COH and the reducing agent sodium cyanoborohydride, labeled derivatives of these antibiotics can be readily formed. The labeled compounds can be readily separated from the unincorporated formalde-
0
I
I
1
2
.
Antibiotic FIG. 3.
I
I
3
4
.
I
5
. 0
(pglml)
Fluorescamine assay standard curves. The relative fluorescence of three fluorescamine-labeled antibiotics is shown-function of concentration. (0) neamine; (0) tobramycin; (Cl) gentamycin. Inset: The nanomolar fluorescence of six aminoglycoside antibiotics is eorrelated with the number of primary amines in their structure.
ANTIBIOTIC TABLE
2
Reductive Labeling and Quantitation of Antibiotic and Ketone Groups Specific activity (mCi/mmol)
Antibiotic Virginiamycin Tylosin Spiramycin Streptomycin Spectinomycin Oleandomycin Erythromycin
M,
89 69 50 36 24 24 11
LABELING
2,4-DNP assay (A443/100 0.92 0.32 0.22 0.15 0.13 0.04 0.03
rg)
Aldehyde
Aldehyde and ketone groups 4 3 2 1 1 2 2
Note. Specific activities are based on radioactivity present in column-purified antibiotic samples. The 2,4-dinitrophenylhydrazone absorbance at 443 nm is from the slopes of Fig. 4. The number of reactive aldehyde and ketone groups is from the structure of the compound (16).
filter paper binding test. The antibiotics listed in Table 1 each bound quantitatively to disks of phosphocellulose paper after washing to remove unincorporated formaldehyde. Figure 2 shows the slopes found for the binding of three different compounds (neamine, hygromycin B, and streptomycin) to the ion-exchange paper. The specific activities in counts per minute per microgram for each drug are listed in Table 1 along with the expected number of methyl amino groups. The inset to Fig. 2 shows the micromolar specific activity for the seven antibiotics tested as a function of the number of methyl amino groups. Except for neomycin, there was a good correlation between the observed specific activity and the reactive substituents. By sampling the reaction mixture, a measure of the specific activity can be obtained before purification of the antibiotic. An additional amount of isotope or antibiotic can be added to give any desired final specific activity. An assay based on this principle has been described by LeGoffic et al. (15) to measure the specific activity of labeled tobramycin. Fluorescamine reacts with primary amines to give a strongly fluorescent covalent derivative (5). This reactivity forms the basis of a sensitive quantitative assay for six of the aminoglycoside antibiotics. As Fig. 3 indicates, the relative fluorescence of the fluorescamine derivative was proportional to the antibiotic concentration. Each of the six drugs could be easily detected at the microgram level (Table 1). The inset to Fig. 3 reveals that with the exception of tobramycin, there was a good correlation between the nanomolar fluorescence and the number of reactive primary amino groups. The fluorescamine and formaldehyde reactivity of these antibiotics is not quantitatively the same since fluorescamine will react only with primary amines, whereas formaldehyde can form an additional labeled dimethyl group with the monomethyl amines existing in the structure of com-
BY
93
REDUCTION
pounds like hygromycin, gentamycin, spectinomycin, and streptomycin (16). For this reason, streptomycin and spectinomycin can be labeled by reductive methylation but are not reactive with fluorescamine (Table 1). Reductive procedures can also be used to label compounds without amino groups, including several different macrolide antibiotics as well as the compounds virginiamycin MI, streptomycin, and spectinomycin. Each of these contain one or more aldehyde or ketone groups which can be quantitatively reduced with NaB3H4 at pH 9.0 (2,16). Table 2 lists the seven compounds which have been labeled by this method. Each was also purified by gel filtration chromatography like the methyl-labeled antibiotics. The specific activities found for each compound are listed in Table 2. Five of the seven showed a good correlation between the observed specific activity and the number of reactive aldehyde and ketone groups. Oleandomycin and erythromycin did not react with sodium borohydride to the extent expected. The specific activity of the [3H]erythromycin is comparable to that of
0
100
200
Antibiotic
300
400
500
(pglml)
FIG. 4. 2,4-Dinitrophenylhydrazine assay standard curves. The absorbance at 443 nm of the 2,4-dinitrophenylhydrazone derivatives of five antibiotics is shown as a function of concentration. 0, virginiamycin; Cl, tylosin; (m) spiramycin; (A) streptomycin; (A) spectinomycin. Inset: The micromolar absorbance of the 2,4-dinitrophenylhydrazone derivatives of seven antibiotics is correlated with the number of reactive aldehyde and ketone groups in each molecule. The arrow indicates the micromolar absorbance of acetone and formaldehyde used as standards.
94
W.
50
Time
SCOTT
100
(min.)
15000
E E ” 3 = z .g 0 E = c a i m
12000
9000
6000
3000
5’0
Time FIG. 5. Antibiotic uptake assays. methyl drugs: (0) [3H]tobramycin hygromycin (2 pg/ml, 0.01 &i/ml); rg/ml, 0.3 &i/ml). (B) Kinetics of drugs: (0) [sH]spiramycin (10 spectinomycin (15 pg/ml, 1.1 &i/ml); rg/ml, 0.9 &i/ml).
loo
CHAMPNEY
and erythromycin did not react proportionally. These macrolides are known to be poorly soluble in water (19) and this may account for their lowered reactivity in both cases. Three different tests were performed to examine the functional activity of the labeled antibiotics. Each compound tested was found to be inhibitory to bacterial cell growth using a simple zone of inhibition test. The diameters of the zones were proportional to the amount of antibiotic spotted on the disk. As a second functional test the kinetics of uptake of labeled drug into resting bacterial cells were also determined. Figure 5A shows the uptake observed for three of the methyl-labeled compounds (tobramycin, hygromycin B, and spectinomycin) and Fig. 5B shows similar kinetics for three of the reduced compounds (spiramycin, spectinomycin, and oleandomycin). Cellular uptake was observed for all eight of the methyl-labeled compounds prepared and tested. Of the seven antibiotics prepared by borohydride reduction, five demonstrated uptake kinetics like those shown in Fig. 5B. Only erythromycin showed no appreciable entry into cells under the conditions tested. [3HJTylosin was not tested in this assay. These kinetics are similar to those observed by others for the uptake of labeled streptomycin (10,12) and gentamycin (11). The final functional assay tested the ribosomal binding activity of the labeled compounds. Figure 6 shows the saturation curves for the binding of three borohydridereduced antibiotics to 70 S ribosomes. The binding of streptomycin and erythromycin was saturated at a 5 to 1 molar ratio to ribosomes while spectinomycin binding
(min.) (A) Kinetics of uptake of three 3H (3 pg/ml, 0.02 /.L!i/ml); (A) [3H]and (0) [sH]spectinomycin (50 uptake of three 3H-labeled reduced pg/ml, 0.6 &i/ml); (Cl) [3H]and (m) [3H]oleandomycin (25
cn
[14C]erythromycin made by a synthetic procedure (17). Others have described the reductive labeling of both streptomycin (13) and virginiamycin S (18) by similiar methods. Of the 13 antibiotics tested, only streptomycin and spectinomycin were capable of being labeled by both procedures, as their structures would predict (16). A quantitative assay for these antibiotics was developed on the basis of the reactivity of their aldehyde and ketone groups with 2,4-dinitrophenylhydrazine to give a phenylhydrazone derivative. Figure 4 shows standard curves for five of these antibiotics reacted with 2,4-dinitrophenylhydrazine and Table 2 lists the relative absorbance for each. The micromolar absorbance for five of the seven compounds was in good agreement with the total number of reactive aldehyde and ketone groups (inset to Fig. 4), but as in the labeling assay, oleandomycin
g 2 5 0 p
0.6
0.4
0
500
Antibiotic
1000
input,
15
pmol
FIG. 6. Ribosome binding assay. 70 S ribosomes (4 Az6,, units; 96 pmol) were incubated with different amounts of three sH-labeled reduced antibiotics and the binding was measured by a filtration assay. The specific activities of the antibiotics used are listed in Table 2. (0) streptomycin; (A) erythromycin; (0) spectinomycin.
ANTIBIOTIC
LABELING
was incomplete under the conditions examined. The binding of labeled streptomycin (13,20) and erythromytin (14,17) to E. coli ribosomes has been previously documented. These methods provide an easy, rapid, and inexpensive set of procedures for the isolation, purification, and quantitation of 13 different radiolabeled antibiotics. These labeled compounds will facilitate an examination of the inhibitory effects of these drugs and should permit quantitative studies on their uptake, cellular distribution, and ribosomal binding features. Since many other antibiotics have structures containing amino, aldehyde, or ketone groups, these techniques should have broad application for antibiotic labeling.
BY
95
REDUCTION
4. Jentoft, N., and Dearborn, D. G. (1979) J. Biol. Chem. 254,43594365. 5. Undenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Science 178,871-872. 6. Touchstone, J. C., and Dobbins, M. F. (1983) Practice of Thin Layer
Chromatography, 2 ed., p. 174, Wiley, New York. H. W., Mueller, J. P., Miller, P. F., and Arrow, A. S. (1987) Microbial. Rev. 5 1,439-457. 8. Champney, W. S. (1980) Biochim. Biophys. Acta 609,464-474. 9. Miller, J. H. (1972) Experiments in Molecular Genetics, p. 433, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 10. Bryan, L. E., and VanDen Elzen, H. M. (1976) Antimicrob. Agents Chemother. 9,928-938. 11. Ahmad, M. H., Rechenmacher, A., and Bock, A. (1980) Antimicrab. Agents Chemother. 18,798-806. 12. Campbell, B. D., and Kadner, R. J. (1980) Biochim. Biophys. Acta
7. Taber,
593,1-10. ACKNOWLEDGMENTS I am very grateful for the excellent technical Kiersten Hutchinson on this work. This research NIH Grant GM 38492.
assistance of Miss was supported by
REFERENCES 1. Rice, R. H., and Means,
G. E. (1971)
J. Biol.
Chem.
2. Means, G. E. (1977) in Methods in Enzymology and Timasheff, S. N., Eds.), Vol. 47, 469-478, New York. 3. Dottavio-Martin,
562-565.
D., and Ravel,
J. M. (1978)
246,831-832. (Hirs, C. H. W., Academic Press,
Anal.
Biochem.
87,
13. Chang, F. N., and Flaks, J. G. (1972) Antimicrob. Agents Chemother. 2,294-307. 14. Pestka, S. (1974) Antimicrob. Agents Chemother. 6,474-478. 15. LeGoffic, F., Capmau, M.-L., Tangy, F., and Baillarge, M. (1979) Eur. J. Biochem. 102,73-81. 16. Vazquez, D. (1979) Inhibitors of Protein Biosynthesis, SpringerVerlag, Berlin/New York. 17. Mao, J. C.-H., and Putterman, M. (1969) J. Mol. Biol. 44, 347361. 18. deBethune, M.-P., and Nierhaus, N. H. (1978) Eur. J. Biochem.
86,187-191. 19. Omura, S. (1984) Macrolide Antibiotics (Omura, 125, Academic Press, New York. 20. Bock, A., Petzet, A., and Pipersberg, W. (1979) 317-321.
S., Ed.), FEBS
Lett.
pp. 85-
104,
BIOCHEMISTRY
Reductive
18
1,90-95
Methods
(1989)
for Isotopic
Labeling of Antibiotics
W. Scott Champney Department
of
Biochemistry,
College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614
Received February 6,1989 MATERIALS Methods for the reductive methylation of the amino groups of eight different antibiotics using ‘HCOH or H14COH are presented. The reductive labeling of an additional seven antibiotics by NaB3H4 is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented. o loss Academic Press,
Inc.
Detailed studies on the mechanisms of antibiotic function require the use of isotopically labeled drugs for quantitative measurements of uptake and binding characteristics. Of the numerous antibiotics affecting the ribosome, a very limited number are commercially available as labeled compounds. Methods for the synthesis of radioactive antibiotics are difficult and often expensive and are generally not easily performed in most laboratories. A set of precedures based on reductive methods for protein labeling (l-4) has been devised for the isotopic labeling of 13 aminoglycoside and macrolide antibiotics. In addition, two different methods for the quantitation of these compounds have also been established on the basis of the reactivity of their amino groups with fluorescamine (5) and of aldehyde and ketone groups with 2,4-dinitrophenylhydrazine (6). A phosphocellulose paper binding assay for measuring the specific activity of the methyl-labeled antibiotics has also been developed. The ease of preparation of these drugs should help to overcome the limited availability of such molecules for cellular uptake studies, as recently emphasized by Taber et al. (7). The potential usefulness of these compounds for the study of uptake kinetics and ribosome binding is demonstrated.
AND
METHODS
Muterzizls. Both 3HCOH (36 mCi/mmol) and H14COH (53 mCi/mmol) came from New England Nuclear; NaB3H4 (1.4 Ci/mmol) was from ICN Radiochemicals. NaCNBH3, fluorescamine, and all antibiotics except neamine were from Sigma Chemical Co. Neamine was a gift from Dr. Richard Keene (Upjohn Co.). NaBH4 was from Alfa Chemicals, 2,4-dinitrophenylhydrazine was from Eastman Chemicals, and P81 phosphocellulose paper was from Whatman. Bio-Gel P2 (100-200 mesh) was a product of Bio-Rad. Reductive methyl&ion. The standard reaction mixture contained 1 mg of antibiotic, 20 pg NaCNBH3, 10 &i 3HCOH (280 nmol), and 10 mM NaP04 buffer (pH 7.3) in a volume of 0.2 ml. The mixture was incubated at 37°C for 15 min, an additional 10 pg of NaCNBH3 was added, and the incubation was continued for an additional 15 min. Samples of 1-5 ~1 were removed for the phosphocellulose paper assay (see following) and 1~1 of 37% HCOH was added to the reaction mixture which was stored frozen. Labeling with Hr4COH was also performed at 10 &i (200 nmol) per reaction. Borohydride reduction. The standard reaction mixture contained 1 mg of antibiotic, 25 &i NaB3H4 (0.01 N NaOH), and 0.1 M Na borate (pH 9.0) in a final volume of 0.2 ml. The mixture was incubated at 25°C for 10 min and at 37°C for an additional IO min. NaBH4 was added to give a final concentration of 0.05 M, and the mixture was brought to pH 6.5 with 5 ~1 glacial acetic acid and stored frozen. Gel filtration chromatography. The labeled antibiotics were separated from unincoporated 3HCOH or NaB3H4 by chromatography on a Bio-Gel P2 column (0.9 X 25 cm) in 0.1 M NH4 acetate. Fractions of 0.5 ml were collected and sampled for radioactivity by spotting 25 or 50 ~1 on filter paper disks which were air-dried. 3H radioactivity was measured in a liquid scintillation counter, and fractions containing labeled antibiotic were pooled and dried by lyphilization. Antibiotics were dissolved in 0.1 ml of water and 1 ~1 was removed and counted for 3H radioactivity on GF/A glass fiber filters
90 All
Copyright 0 1989 rights of reproduction
0003-2697/89 $3.00 by Academic Press, Inc. in any form reserved.
ANTIBIOTIC
LABELING
BY
60% compared to equivalent samples counted on GF/A filters. Fluwescamine assay. Assay mixtures contained variable amounts of antibiotics in 0.9 ml of 0.1 M Na borate (pH 9.0). A volume of 0.1 ml of fluorescamine (0.25% w/v in dimethyl sulfoxide) was added with vigorous mixing. The fluorescence of the product was measured in a Perkin-Elmer 650-40 fluorescence spectrophotometer at 485 nm (slit 20 nm) with excitation at 390 nm (slit 10 nm).
2000
2,4-Dinitrophenylhydrazinc assay. Assay mixtures contained variable amounts of antibiotics in 0.5 ml of 0.1 M Na borate (pH 9.0). A volume of 0.5 ml of 2,4-dinitrophenylhydrazine (0.4% w/v in 2 N HCl) was added with mixing. After standing for 20-30 min, the 2,4-dinitrophenylhydrazone product was read in a spectrophotometer at 443 nm. Stock solutions of 12.5 mM formaldehyde and 13.5 mM acetone were used as standards in the assay.
500
0
10
3-o
20
Fraction
0 ,
Functional assays. The inhibitory activity of labeled antibiotics was tested with a zone of inhibition assay. Volumes of l-5 ~1 of labeled antibiotics were spotted on filter paper disks (Whatman No. 3) which were placed on soft agar lawns of sensitive Escherichia coli cells [strain SK901 (S)] on LB agar plates (9) along with samples of unlabeled drugs. The diameter of the zone of inhibition was measured after incubation at 37°C and compared with the zone size of unlabeled samples. Antibiotic uptake assays were conducted as described previously (10-12). Briefly, E. coli cells at a density of 2 X 108/ml were suspended in 1 ml of 10 mM Hepes buffer containing 0.5% glucose at 37°C. Labeled antibiotic was added and 0.2-ml samples were removed at intervals, filtered on Millipore filters (HAWP, 0.45 p), washed three times with 5 ml of Hepes buffer containing 0.3% NaCl, dried, and counted for bound radioactivity.
No.
FIG. 1. Gel filtration chromatography of [3H]hygromycin B on a Bio-Gel P2 column. The absorbance at 250 nm (0) and the “H cpm (0) in 50 ~1 of each 0.5-ml column fraction were measured.
at an efficiency of 60%. The counting fluid was 0.25% (w/v) 2,5-diphenyloxazole in toluene. Phosphocellulose paper assay. The specific activity of methyl-labeled antibiotics was determined by spotting samples of three different volumes from the reaction mixture on Z.&cm phosphocellulose paper disks. The disks were washed with 10 ml of water to remove unincorporated 3HCOH and the radioactivity remaining was measured by liquid scintillation counting. Radioactivity measured on phosphocellulose paper was quenched by
TABLE
Reductive Methylation
Antibiotic Neomycin B Tobramycin Kanamycin Neamine Gentamycin Hygromycin B Spectinomycin Streptomycin
91
REDUCTION
1
and Quantitative
Specific activity (mCi/mmol)
PC disk assay (w-drd
5.1-27 3.2-45 3.6-34 1.9-34 3.4-47 2.3-19 2.0-7.8 1.2-5.8
20,000 48,000 36,000 45,500 32,000 20,000 12,500 5300
Assay of Antibiotic
Amino-methyl groups 12 10 8 8 7 5 2 1
Amino Groups
Relative
fluorescence 650 400 450 900 225 197 0 0
Primary amino groups 6 5 4 4 3 3 0 0
Note. Specific activities are based on radioactivity present in column-purified antibiotic samples. The range represents labeling at different ratios of ‘HCOH to antibiotic. The phosphocellulose (PC disk) binding data are from the slopes of Fig. 2. The expected number of labeled amino-methy groups is from the structure of the compound (16). The relative fluorescence at 485 nm per pg of antibiotic is from the slopes of Fig. 3. The number of fluorescamine reactive primary amino groups is from the structure of the compound (16).
92
W.
x
0 Tobra.
60: 50 -
=
40-
3
30-
E
zo-
tf
lo-
E
30000 5.D
,I:
ti 0. E B
o-
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SCOTT
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4
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6
6
CHAMPNEY
hyde by gel filtration chromatography (Fig. 1). In this instance the absorbance of hygromycin B was used to monitor the elution and recovery of the antibiotic from the column. Seven different aminoglycoside antibiotics and spectinomycin were labeled by this procedure. The range of specific activities found for different labeling conditions is listed in Table 1. Generally, 3HCOH was reacted at a molar ratio of 0.1-0.2 with each antibiotic, to give derivatives with the lower specific activities listed. By reducing the antibiotic concentration in the reaction mixture to give a 5- to lo-fold molar excess of 3HCOH, derivatives with higher specific activities were generated with maximal labeling of all reactive amino groups. The final specific activity of the compound can be varied depending on its intended use and the amount of isotope available. Comparable specific activities were obtained when H14COH was used. For any reaction condition, a measure of the specific activity of the antibiotic could be determined by a simple
I
0 Neo.
101214
groups
20000
L c-+-J
1ooot
2000 0 k 0
1
Antibiotic
2
3
-
4
(pgldisk)
FIG. 2.
Phosphocellulose paper disk assay. Three different volumes of the indicated antibiotics were removed from the reaction mixture and filtered through phosphocellulose paper disks as described under Materials and Methods. The dried disks were measured for 3H cpm by scintillation counting. (0) neamine; (D) hygromycin B; (A) streptomycin. Inset: The micromolar specific activity of eight antibiotics reacted with excess ‘HCOH is correlated with the expected number of labeled amino methyl groups in each compound.
R-NH2 groups ‘0 3
lOOO-
E
Antibiotic binding to 70 S ribosomes was based on a filter binding method (13-14). High-salt-washed ribosomes were prepared as described (8) and were reacted with variable amounts of labeled antibiotics at 37°C in R buffer (10 mM Tris-HCl, pH 7.6,lO mM Mg acetate, 50 mM NH,Cl, 3 mM mercaptoethanol). Each sample was filtered on a Millipore filter, washed three times with 5 ml of R buffer, and counted for bound radioactivity.
P) 5 .z z
a
600 -
600 -
400 -
200 -
o!
RESULTS
AND
DISCUSSION
The aminoglycoside antibiotics and spectinomycin contain primary amino groups which can be reduced to dimethyl amino derivatives after reactions with aldehydes via a Schiff base intermediate (2-4). Using 3HCOH or H14COH and the reducing agent sodium cyanoborohydride, labeled derivatives of these antibiotics can be readily formed. The labeled compounds can be readily separated from the unincorporated formalde-
0
I
I
1
2
.
Antibiotic FIG. 3.
I
I
3
4
.
I
5
. 0
(pglml)
Fluorescamine assay standard curves. The relative fluorescence of three fluorescamine-labeled antibiotics is shown-function of concentration. (0) neamine; (0) tobramycin; (Cl) gentamycin. Inset: The nanomolar fluorescence of six aminoglycoside antibiotics is eorrelated with the number of primary amines in their structure.
ANTIBIOTIC TABLE
2
Reductive Labeling and Quantitation of Antibiotic and Ketone Groups Specific activity (mCi/mmol)
Antibiotic Virginiamycin Tylosin Spiramycin Streptomycin Spectinomycin Oleandomycin Erythromycin
M,
89 69 50 36 24 24 11
LABELING
2,4-DNP assay (A443/100 0.92 0.32 0.22 0.15 0.13 0.04 0.03
rg)
Aldehyde
Aldehyde and ketone groups 4 3 2 1 1 2 2
Note. Specific activities are based on radioactivity present in column-purified antibiotic samples. The 2,4-dinitrophenylhydrazone absorbance at 443 nm is from the slopes of Fig. 4. The number of reactive aldehyde and ketone groups is from the structure of the compound (16).
filter paper binding test. The antibiotics listed in Table 1 each bound quantitatively to disks of phosphocellulose paper after washing to remove unincorporated formaldehyde. Figure 2 shows the slopes found for the binding of three different compounds (neamine, hygromycin B, and streptomycin) to the ion-exchange paper. The specific activities in counts per minute per microgram for each drug are listed in Table 1 along with the expected number of methyl amino groups. The inset to Fig. 2 shows the micromolar specific activity for the seven antibiotics tested as a function of the number of methyl amino groups. Except for neomycin, there was a good correlation between the observed specific activity and the reactive substituents. By sampling the reaction mixture, a measure of the specific activity can be obtained before purification of the antibiotic. An additional amount of isotope or antibiotic can be added to give any desired final specific activity. An assay based on this principle has been described by LeGoffic et al. (15) to measure the specific activity of labeled tobramycin. Fluorescamine reacts with primary amines to give a strongly fluorescent covalent derivative (5). This reactivity forms the basis of a sensitive quantitative assay for six of the aminoglycoside antibiotics. As Fig. 3 indicates, the relative fluorescence of the fluorescamine derivative was proportional to the antibiotic concentration. Each of the six drugs could be easily detected at the microgram level (Table 1). The inset to Fig. 3 reveals that with the exception of tobramycin, there was a good correlation between the nanomolar fluorescence and the number of reactive primary amino groups. The fluorescamine and formaldehyde reactivity of these antibiotics is not quantitatively the same since fluorescamine will react only with primary amines, whereas formaldehyde can form an additional labeled dimethyl group with the monomethyl amines existing in the structure of com-
BY
93
REDUCTION
pounds like hygromycin, gentamycin, spectinomycin, and streptomycin (16). For this reason, streptomycin and spectinomycin can be labeled by reductive methylation but are not reactive with fluorescamine (Table 1). Reductive procedures can also be used to label compounds without amino groups, including several different macrolide antibiotics as well as the compounds virginiamycin MI, streptomycin, and spectinomycin. Each of these contain one or more aldehyde or ketone groups which can be quantitatively reduced with NaB3H4 at pH 9.0 (2,16). Table 2 lists the seven compounds which have been labeled by this method. Each was also purified by gel filtration chromatography like the methyl-labeled antibiotics. The specific activities found for each compound are listed in Table 2. Five of the seven showed a good correlation between the observed specific activity and the number of reactive aldehyde and ketone groups. Oleandomycin and erythromycin did not react with sodium borohydride to the extent expected. The specific activity of the [3H]erythromycin is comparable to that of
0
100
200
Antibiotic
300
400
500
(pglml)
FIG. 4. 2,4-Dinitrophenylhydrazine assay standard curves. The absorbance at 443 nm of the 2,4-dinitrophenylhydrazone derivatives of five antibiotics is shown as a function of concentration. 0, virginiamycin; Cl, tylosin; (m) spiramycin; (A) streptomycin; (A) spectinomycin. Inset: The micromolar absorbance of the 2,4-dinitrophenylhydrazone derivatives of seven antibiotics is correlated with the number of reactive aldehyde and ketone groups in each molecule. The arrow indicates the micromolar absorbance of acetone and formaldehyde used as standards.
94
W.
50
Time
SCOTT
100
(min.)
15000
E E ” 3 = z .g 0 E = c a i m
12000
9000
6000
3000
5’0
Time FIG. 5. Antibiotic uptake assays. methyl drugs: (0) [3H]tobramycin hygromycin (2 pg/ml, 0.01 &i/ml); rg/ml, 0.3 &i/ml). (B) Kinetics of drugs: (0) [sH]spiramycin (10 spectinomycin (15 pg/ml, 1.1 &i/ml); rg/ml, 0.9 &i/ml).
loo
CHAMPNEY
and erythromycin did not react proportionally. These macrolides are known to be poorly soluble in water (19) and this may account for their lowered reactivity in both cases. Three different tests were performed to examine the functional activity of the labeled antibiotics. Each compound tested was found to be inhibitory to bacterial cell growth using a simple zone of inhibition test. The diameters of the zones were proportional to the amount of antibiotic spotted on the disk. As a second functional test the kinetics of uptake of labeled drug into resting bacterial cells were also determined. Figure 5A shows the uptake observed for three of the methyl-labeled compounds (tobramycin, hygromycin B, and spectinomycin) and Fig. 5B shows similar kinetics for three of the reduced compounds (spiramycin, spectinomycin, and oleandomycin). Cellular uptake was observed for all eight of the methyl-labeled compounds prepared and tested. Of the seven antibiotics prepared by borohydride reduction, five demonstrated uptake kinetics like those shown in Fig. 5B. Only erythromycin showed no appreciable entry into cells under the conditions tested. [3HJTylosin was not tested in this assay. These kinetics are similar to those observed by others for the uptake of labeled streptomycin (10,12) and gentamycin (11). The final functional assay tested the ribosomal binding activity of the labeled compounds. Figure 6 shows the saturation curves for the binding of three borohydridereduced antibiotics to 70 S ribosomes. The binding of streptomycin and erythromycin was saturated at a 5 to 1 molar ratio to ribosomes while spectinomycin binding
(min.) (A) Kinetics of uptake of three 3H (3 pg/ml, 0.02 /.L!i/ml); (A) [3H]and (0) [sH]spectinomycin (50 uptake of three 3H-labeled reduced pg/ml, 0.6 &i/ml); (Cl) [3H]and (m) [3H]oleandomycin (25
cn
[14C]erythromycin made by a synthetic procedure (17). Others have described the reductive labeling of both streptomycin (13) and virginiamycin S (18) by similiar methods. Of the 13 antibiotics tested, only streptomycin and spectinomycin were capable of being labeled by both procedures, as their structures would predict (16). A quantitative assay for these antibiotics was developed on the basis of the reactivity of their aldehyde and ketone groups with 2,4-dinitrophenylhydrazine to give a phenylhydrazone derivative. Figure 4 shows standard curves for five of these antibiotics reacted with 2,4-dinitrophenylhydrazine and Table 2 lists the relative absorbance for each. The micromolar absorbance for five of the seven compounds was in good agreement with the total number of reactive aldehyde and ketone groups (inset to Fig. 4), but as in the labeling assay, oleandomycin
g 2 5 0 p
0.6
0.4
0
500
Antibiotic
1000
input,
15
pmol
FIG. 6. Ribosome binding assay. 70 S ribosomes (4 Az6,, units; 96 pmol) were incubated with different amounts of three sH-labeled reduced antibiotics and the binding was measured by a filtration assay. The specific activities of the antibiotics used are listed in Table 2. (0) streptomycin; (A) erythromycin; (0) spectinomycin.
ANTIBIOTIC
LABELING
was incomplete under the conditions examined. The binding of labeled streptomycin (13,20) and erythromytin (14,17) to E. coli ribosomes has been previously documented. These methods provide an easy, rapid, and inexpensive set of procedures for the isolation, purification, and quantitation of 13 different radiolabeled antibiotics. These labeled compounds will facilitate an examination of the inhibitory effects of these drugs and should permit quantitative studies on their uptake, cellular distribution, and ribosomal binding features. Since many other antibiotics have structures containing amino, aldehyde, or ketone groups, these techniques should have broad application for antibiotic labeling.
BY
95
REDUCTION
4. Jentoft, N., and Dearborn, D. G. (1979) J. Biol. Chem. 254,43594365. 5. Undenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. (1972) Science 178,871-872. 6. Touchstone, J. C., and Dobbins, M. F. (1983) Practice of Thin Layer
Chromatography, 2 ed., p. 174, Wiley, New York. H. W., Mueller, J. P., Miller, P. F., and Arrow, A. S. (1987) Microbial. Rev. 5 1,439-457. 8. Champney, W. S. (1980) Biochim. Biophys. Acta 609,464-474. 9. Miller, J. H. (1972) Experiments in Molecular Genetics, p. 433, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 10. Bryan, L. E., and VanDen Elzen, H. M. (1976) Antimicrob. Agents Chemother. 9,928-938. 11. Ahmad, M. H., Rechenmacher, A., and Bock, A. (1980) Antimicrab. Agents Chemother. 18,798-806. 12. Campbell, B. D., and Kadner, R. J. (1980) Biochim. Biophys. Acta
7. Taber,
593,1-10. ACKNOWLEDGMENTS I am very grateful for the excellent technical Kiersten Hutchinson on this work. This research NIH Grant GM 38492.
assistance of Miss was supported by
REFERENCES 1. Rice, R. H., and Means,
G. E. (1971)
J. Biol.
Chem.
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104,