- Email: [email protected]
Synthesis of a 125I-radiolabeled penicillin for penicillin-binding proteins studies
ANALYTICAL
BIOCHEMISTRY
128, 164- 168 ( 1983)
Synthesis of a ‘251-Radiolabeled Penicillin for Penicillin-Binding Proteins Studies J. M. MASSON CNRS-CERCOA
AND R. LABIA’
B.P. 28 94320 Thiais, France
Received June 16, 1982 Radioiodination of penicillin X (phydroxybenzylpenicillin) with ‘25INa, using the chloramine-T method, is simple and almost quantitative. The product thus obtained can be used without further purification for the penicillin-binding proteins (PBPs) assay. The chemical structure of ‘251-penicillin X is very close to that of penicillin G, and the affinity of the two compounds for the PBPs are very similar. ‘251-penicillin X can then advantageously replace [‘4C]penicillin G in these experiments, as its specific radioactivity is 2OQO Ci/mol, in our preparations, instead of about 50 Ci/mol; thus, the experimental procedure is simplified and the autoradiography exposure time is reduced to 12-48 h.
The binding of /3-lactam antibiotics on penicillin-binding proteins (PBPs)* presently is an important aspect of their mode of action. This is usually performed with penicillin G, 14C-labeled on the first carbon atom of the side chain. Presently, only this radiolabeled penicillin is commercially available. However, due to the small number of PBPs molecules per cell, the specific radioactivity of [‘4C]penicillin G (40-60 Ci/mol) appears too low and the method requires very long exposure times for the autoradiography step, 20-60 days or more, even if various improvements such as fluorography (1) and preflash of the film (2) are used. To shorten the exposure time, some authors have used penicillins with a higher specific activity, such as tritiated penicillin (3), iodinated furazlocillin (4), or more recently a iodinated derivative of ampicillin (5). Unfortunately, in addition to particular problems for each of these molecules, any compound of this kind with a high specific radioactivity is subjected to extensive radiolysis, ’ To whom correspondence should be addressed. * Abbreviations used: PBP, penicillin-binding protein; TLC, thin-layer chromatography. 0003-2697/83/010164-05$03.00/O Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form resewed.
due to the fragility of the P-la&am ring. This aspect seriously complicates the use of such molecules. We therefore aimed at the preparation of a highly radioactive ‘251-penicillin fulfilling the following conditions: (i) a rapid and simple way of radiolabeling, (ii) a readily usable reaction product without any purification step, and (iii) a compound structurally as closely related to penicillin G as possible. Finally, we opted for the radioiodination of penicillin X (p-hydroxybenzylpenicillin) (Fig. I), which seemed to us a relevant approach to the problem. METHODS
Synthesis of penicillin X. Ethyl chloroformate (630 ~1) is added slowly to a solution of 1 g of p-hydroxyphenylacetic acid and 830 ~1 of N,N-dimethylaniline in 14 ml of dry acetone maintained at ‘-5 “C under vigorous agitation. The temperature is slowly shifted from -5 to +5”C (30 min) in order to complete the formation of the mixed anhydrid. Then a cold extemporaneous solution of 1.43 g of 6-aminopenicillanic acid (6-APA) and .6 g Na2C03 in 14 ml of water is added quickly 164
SYNTHESIS
OF ‘ZSI-PENICILLIN
FIG. I. Chemical structure of penicillin X.
and the reaction mixture is stirred for 30 min at f4”C and for an additional hour without refrigeration. Acetone is extracted with 2 X 30 ml of methylene chloride and the aqueous phase is then covered with 14 ml of methyl isobutyl ketone and acidified to pH 2 with a 50% solution of phosphoric acid. During this step, the aqueous phase becomes turbid, because penicillin X tends to precipitate, but this precipitate disappears as penicillin X shifts to the organic phase. The aqueous phase is quickly washed with 2 X 5 ml of methyl isobutyl ketone, and the methyl isobutyl ketone fractions are pooled, washed once with 20 ml of water, and then extracted with a sodium bicarbonate solution until pH 6 is reached. After this solution is lyophylized, we finally obtain 2.1 g (85%) of penicillin X sodium salt. Thin-layer chromatography. TLC was performed on silicate plates buffered at pH 6 with phosphate buffer 0.1 M; the solvent used for elution was the superior phase of n-butanol:acetic acid:water (4: 1:5). The chromatograms were revealed in uv light or with an iodometric assay of penicillins (6). Radioactive material was revealed by autoradiography on Fuji RX medical film, exposure time being usually about half an hour. Radioiodination of penicillin X. ‘251Na (Amersham France) is provided as a sodium hydroxyde solution (100 mCi/ml). All other compounds are solubilized in potassium phosphate buffer 0.1 M, pH 7.5. To 23 ~1 of phosphate buffer are successively added 10 ~1 of the penicillin X solution (2 mg/ml), 2 ~1 of the ‘251Na solution, and 10 ~1 of a chloramine-T solution (2 mg/ml). After 1 min of incubation at room temperature, 5 ~1 of a INa solution (2 mgfml) is added and the mixture is incubated for 40 min at room temperature. The reaction is
FOR PBPs STUDIES
165
then stopped with 10 ~1 of a solution of sodium metabisulfite (2 mg/ml). The final volume is then adjusted with phosphate buffer according to the concentration of penicillin X needed for the various experiments. Preparation of membrane extracts. Escherichia coli K12 cells are grown to mid log phase (70-80 NTU) in 20 ml of trypticase soja broth. The cultures are sonicated with a Branson B- 15 sonicator equipped with a microtip (30%, 5 min) while refrigerating on ice. The extracts are centrifuged for 10 min at 3000g. The supernatants are kept and centrifuged for 1 h at 48,OOOg. The pellets are washed once with phosphate buffer 0.1 M, pH 7.5, and resuspended in the same buffer to a final volume of 50 yl. Extracts are stored at -80°C until use. Labeling of the PBPs and competition experiments. The PBP assays are performed according to Spratt and Pardee (7), but with 5 ~1 of membrane extracts in a final reaction volume of 50 ~1. Competition experiments with other &lactam antibiotics are performed according to Curtis et al. (8) in a final volume of 50 ~1. Acrylamide concentrations were 4% in the stacking gel and 8% in the running gel, whereas N,N’-methylenebisacrylamide was at 0.066% in the stacking gel and 0.13% in the running gel. After electrophoresis, the gels were stained with Coomassie blue and dried on a Bio-Rad Model 224 slab gel drier. Autoradiography was done at -80°C with a preflashed Fuji RX medical film, using only one Cronex Hi plus intensifying screen. Usual exposure times ranged from 12 to 48 h. RESULTS
Penicillin X. Penicillin X is obtained with a 85% yield, and its purity is usually about 80 to 90% depending on the synthesis. On TLC plates, we can see that the main impurity is penicilloi’c acid (Fig. 2). With the chromatographic system we used, penicillin X and penicillin G migrate very similarly.
166
MASSON
%P 0
00
0 0
0
III1 ABC0
II EF
c I G
FIG. 2. Thin-layer chromatography. Lane A, penicillin Cl; lane B, penicilldic acid (obtained by the action of 0.1 N NaOH); lane C, penicillin X; lane D, corresponding penicillolc acid (also obtained by alkaline hydrolysis); lane E, p-hydroxyphenylacetic acid; lane F, NaI; lane G, products of radioiodination of penicillin X as revealed by autoradiography.
Iodination of penicillin X. The iodination of penicillin X by the chloramine-T process is very fast and almost quantitative. At the end of the reaction, there remains less than 1% of ‘251Na (Fig. 2). Under these experimental conditions the stability of the p-lactam ring is very good for the time of the reaction. The penicillin we start with contains about 5% of penicilldic acids. Penicilldic acids are known to reduce iodine, as do ICl and chloramine-T involved in the radiolabeling process. This only leads to a slight overcon-
AND LABIA
sumption of chloramine-T, because during this step the possible reaction of ICl regenerates an iodide ion which is then recycled. However, we cannot exclude the formation of p-hydroxyphenylacetamido-2-malonic acid, a degradation product of penicillin X that could be also iodinated, although we were unable to detect any. Anyway, such a molecule can be regarded as inert compared with the penicillins for biological activity. The specific activity of the ‘251-penicillin X that we obtained is 2000 Ci/mol. The antimicrobial activities of penicillin X and of its iodinated derivative are quite identical to that of penicillin G. The three compounds have a MIC of 32 pg/ml for E. coli K12. Labeling of the PBPs. We can see from Fig. 3 that ‘251-penicillin X labels all the PBPs of E. coli in a manner very similar to that described for [i4C]penicillin G (9). Competition experiments with 1251-penicillin X (Fig. 4) show that mecillinam binds exclusively PBP 2, the first target of cefotaxime is PBP 3, cefoxitin does not bind PBP 2, and cefsulodin shows affinity for PBP IA, and IB only. These results and the Iso data (Table 1) are in good agreement with the ones obet al. (10) using tained by Curtis [ “C]penicillin G.
IA IB-
234-
516a
bcdefghi
FIG. 3. Penicillin-binding proteins of E. coli K 12. Labeling was performed as described under Methods. Final concentration of ‘251-penicillin X ranges from (a) 40 pg/ml to (i) 0.15 pg/rnl in twofold dilutions.
SYNTHESIS
a
OF ‘251-PENICILLIN
b
c
d
167
FOR PBPs STUDIES
e
f
g
h
FIG. 4. Competition experiments with unlabeled @Iactam antibiotics (a) Control; (b) cefotaxime, 0.05 &ml: (c) cefotaxime, 0.5 rg/ml; (d) cefsulodin, 0.5 &ml; (e) cefsulodin, 5 pg/ml: (f~ cefoxitin, 0.5 pg/ ml; (g) cefoxitin, 5 rg/ml; and (h) mecillinam, 1 pg/ml.
Among all the radioactive molecules described until now, only penicillin G provided a good saturation of all the PBPs, and it was therefore the only one convenient to use in competition studies. This could be done with the commercially available penicillin G, with the drawbacks we have seen, or with tritiated penicillin G (3) but this noncommercial product cannot be synthesized routinely in a biochemical laboratory (12). In addition, the iodinated derivative of furazlocillin (4) whose synthesis was not described, shows a mode of binding to the PBPs different from that of penicillin G. This is the case too for the iodinated derivative of ampicillin described by Schwarz et al. (5). The iodinated derivative of penicillin X that we described
DISCUSSION
Penicillin X is a natural /3-lactam which presents no specific therapeutic properties when compared with penicillin G or penicillin V. Thus, penicillin X is not commercially available, and we therefore had to synthesize it. Iodinated derivatives of penicillin X have been described very early (1 I), but by an unreliable way of synthesis, and these iodinated compounds were not radioactive. Penicillin X and its iodinated derivative have chemical structure very close to that of penicillin G, their microbial properties are very close, and their modes of binding to PBPs are identical, which is an interesting and fundamental property in our field. TABLE HALF-SATURATION
CONCENTRATIONS(~&
I
OBTAINED
IN COMPETITION
EXPERIMENTS
PBP No. P-Lactam antibiotics “SI-Penicillin Mecillinam Cefotaxime Cefsulodin Cefoxitin
X
1A
1B
2
3
4
5
6
0.7 -0 0.1 0.41 0.12
1.7 1 3 2.8
3.5 0.05 5 -
0.9 0.04 1.5
0.3 and 2.5 -
2.5 0.32
3 0.37
’ No competition detected.
19
168
MASSON
here seems therefore especially advisable for use in PBPs studies. Due to its high specific radioactivity (2000 Cifmol), ‘251-penicillin X allows very short exposure times (12-48 h) in autoradiography. Thus, it was of no use to increase this specific activity. The experiment is also less expensive and less time consuming because 23 - diphenyloxazole/dimethylsulfoxide or Enhance (Amersham) is no longer needed. The high specific activity of i2’I-penicillin X leads to extensive radiolysis, and it is advisable to use it immediately after its synthesis because it is more than half destroyed within 48 h, thus giving a lower labeling of the PBPs but no artifact labeling. This drawback is compensated for by the fact that iodination of penicillin X is simple and takes less than an hour. No further purification being needed, the iodination step can be performed just prior to the PBP assay and the product used immediately, which considerably simplifies the problems of radioactive contamination. In conclusion, the main advantage of 12% penicillin X is that radioactive iodine is introduced in a mild way on a penicillin selected to provide a chemical reactivity especially suited for this experiment. The flexi-
AND LABIA
bility of the method was confirmed extensive use in the laboratory.
by an
REFERENCES 1. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 2. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56, 335-341. 3. Williamson, R., and Tomasz, A. (1980) J. Bacterial. 144, 105-l 13. 4. Schmidt, L. S., Botta, G., and Park, J. T. (1981) J. Bacterial.
145, 632-637.
5. Schwarz, U., Seeger, K., Wengenmayer, F., and Strecker, H. (1981) FEMS Microbial. Lett. 10, 107-109. 6. Comelis, G., Wauters, G., and Vanderhaeghe, H. (1973) Ann. Microbial. (Inst. Pasteur) 124 B, 139-152. 7. Spratt, B. G., and Pardee, A. B. (1975) Nature (London) 254, 5 16-5 17. 8. Curtis, N. A. C., Brown, C., Boxall, M., and Boulton, M. G. (1979) Antimicrob. Agents Chemother. 15, 332-336.
9. Spratt, B. G. (1977) Eur. J. Biochem. 72, 341-352. 10. Curtis, N. A. C., Orr, D., Ross, G. W., and Boulton, M. G. (1979) Antimicrob. Agents Chemother. 16, 533-539.
11. Clarke, H. T., Johnson, J. R., and Robinson, R. (1949) in The Chemistry of Penicillin, pp. 680687, Princeton Univ. Press, Princeton, N. J. 12. Rosegay, A. (1981) J. Labelled Compd. Radiopharm. 18, 1337-l 340.
BIOCHEMISTRY
128, 164- 168 ( 1983)
Synthesis of a ‘251-Radiolabeled Penicillin for Penicillin-Binding Proteins Studies J. M. MASSON CNRS-CERCOA
AND R. LABIA’
B.P. 28 94320 Thiais, France
Received June 16, 1982 Radioiodination of penicillin X (phydroxybenzylpenicillin) with ‘25INa, using the chloramine-T method, is simple and almost quantitative. The product thus obtained can be used without further purification for the penicillin-binding proteins (PBPs) assay. The chemical structure of ‘251-penicillin X is very close to that of penicillin G, and the affinity of the two compounds for the PBPs are very similar. ‘251-penicillin X can then advantageously replace [‘4C]penicillin G in these experiments, as its specific radioactivity is 2OQO Ci/mol, in our preparations, instead of about 50 Ci/mol; thus, the experimental procedure is simplified and the autoradiography exposure time is reduced to 12-48 h.
The binding of /3-lactam antibiotics on penicillin-binding proteins (PBPs)* presently is an important aspect of their mode of action. This is usually performed with penicillin G, 14C-labeled on the first carbon atom of the side chain. Presently, only this radiolabeled penicillin is commercially available. However, due to the small number of PBPs molecules per cell, the specific radioactivity of [‘4C]penicillin G (40-60 Ci/mol) appears too low and the method requires very long exposure times for the autoradiography step, 20-60 days or more, even if various improvements such as fluorography (1) and preflash of the film (2) are used. To shorten the exposure time, some authors have used penicillins with a higher specific activity, such as tritiated penicillin (3), iodinated furazlocillin (4), or more recently a iodinated derivative of ampicillin (5). Unfortunately, in addition to particular problems for each of these molecules, any compound of this kind with a high specific radioactivity is subjected to extensive radiolysis, ’ To whom correspondence should be addressed. * Abbreviations used: PBP, penicillin-binding protein; TLC, thin-layer chromatography. 0003-2697/83/010164-05$03.00/O Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form resewed.
due to the fragility of the P-la&am ring. This aspect seriously complicates the use of such molecules. We therefore aimed at the preparation of a highly radioactive ‘251-penicillin fulfilling the following conditions: (i) a rapid and simple way of radiolabeling, (ii) a readily usable reaction product without any purification step, and (iii) a compound structurally as closely related to penicillin G as possible. Finally, we opted for the radioiodination of penicillin X (p-hydroxybenzylpenicillin) (Fig. I), which seemed to us a relevant approach to the problem. METHODS
Synthesis of penicillin X. Ethyl chloroformate (630 ~1) is added slowly to a solution of 1 g of p-hydroxyphenylacetic acid and 830 ~1 of N,N-dimethylaniline in 14 ml of dry acetone maintained at ‘-5 “C under vigorous agitation. The temperature is slowly shifted from -5 to +5”C (30 min) in order to complete the formation of the mixed anhydrid. Then a cold extemporaneous solution of 1.43 g of 6-aminopenicillanic acid (6-APA) and .6 g Na2C03 in 14 ml of water is added quickly 164
SYNTHESIS
OF ‘ZSI-PENICILLIN
FIG. I. Chemical structure of penicillin X.
and the reaction mixture is stirred for 30 min at f4”C and for an additional hour without refrigeration. Acetone is extracted with 2 X 30 ml of methylene chloride and the aqueous phase is then covered with 14 ml of methyl isobutyl ketone and acidified to pH 2 with a 50% solution of phosphoric acid. During this step, the aqueous phase becomes turbid, because penicillin X tends to precipitate, but this precipitate disappears as penicillin X shifts to the organic phase. The aqueous phase is quickly washed with 2 X 5 ml of methyl isobutyl ketone, and the methyl isobutyl ketone fractions are pooled, washed once with 20 ml of water, and then extracted with a sodium bicarbonate solution until pH 6 is reached. After this solution is lyophylized, we finally obtain 2.1 g (85%) of penicillin X sodium salt. Thin-layer chromatography. TLC was performed on silicate plates buffered at pH 6 with phosphate buffer 0.1 M; the solvent used for elution was the superior phase of n-butanol:acetic acid:water (4: 1:5). The chromatograms were revealed in uv light or with an iodometric assay of penicillins (6). Radioactive material was revealed by autoradiography on Fuji RX medical film, exposure time being usually about half an hour. Radioiodination of penicillin X. ‘251Na (Amersham France) is provided as a sodium hydroxyde solution (100 mCi/ml). All other compounds are solubilized in potassium phosphate buffer 0.1 M, pH 7.5. To 23 ~1 of phosphate buffer are successively added 10 ~1 of the penicillin X solution (2 mg/ml), 2 ~1 of the ‘251Na solution, and 10 ~1 of a chloramine-T solution (2 mg/ml). After 1 min of incubation at room temperature, 5 ~1 of a INa solution (2 mgfml) is added and the mixture is incubated for 40 min at room temperature. The reaction is
FOR PBPs STUDIES
165
then stopped with 10 ~1 of a solution of sodium metabisulfite (2 mg/ml). The final volume is then adjusted with phosphate buffer according to the concentration of penicillin X needed for the various experiments. Preparation of membrane extracts. Escherichia coli K12 cells are grown to mid log phase (70-80 NTU) in 20 ml of trypticase soja broth. The cultures are sonicated with a Branson B- 15 sonicator equipped with a microtip (30%, 5 min) while refrigerating on ice. The extracts are centrifuged for 10 min at 3000g. The supernatants are kept and centrifuged for 1 h at 48,OOOg. The pellets are washed once with phosphate buffer 0.1 M, pH 7.5, and resuspended in the same buffer to a final volume of 50 yl. Extracts are stored at -80°C until use. Labeling of the PBPs and competition experiments. The PBP assays are performed according to Spratt and Pardee (7), but with 5 ~1 of membrane extracts in a final reaction volume of 50 ~1. Competition experiments with other &lactam antibiotics are performed according to Curtis et al. (8) in a final volume of 50 ~1. Acrylamide concentrations were 4% in the stacking gel and 8% in the running gel, whereas N,N’-methylenebisacrylamide was at 0.066% in the stacking gel and 0.13% in the running gel. After electrophoresis, the gels were stained with Coomassie blue and dried on a Bio-Rad Model 224 slab gel drier. Autoradiography was done at -80°C with a preflashed Fuji RX medical film, using only one Cronex Hi plus intensifying screen. Usual exposure times ranged from 12 to 48 h. RESULTS
Penicillin X. Penicillin X is obtained with a 85% yield, and its purity is usually about 80 to 90% depending on the synthesis. On TLC plates, we can see that the main impurity is penicilloi’c acid (Fig. 2). With the chromatographic system we used, penicillin X and penicillin G migrate very similarly.
166
MASSON
%P 0
00
0 0
0
III1 ABC0
II EF
c I G
FIG. 2. Thin-layer chromatography. Lane A, penicillin Cl; lane B, penicilldic acid (obtained by the action of 0.1 N NaOH); lane C, penicillin X; lane D, corresponding penicillolc acid (also obtained by alkaline hydrolysis); lane E, p-hydroxyphenylacetic acid; lane F, NaI; lane G, products of radioiodination of penicillin X as revealed by autoradiography.
Iodination of penicillin X. The iodination of penicillin X by the chloramine-T process is very fast and almost quantitative. At the end of the reaction, there remains less than 1% of ‘251Na (Fig. 2). Under these experimental conditions the stability of the p-lactam ring is very good for the time of the reaction. The penicillin we start with contains about 5% of penicilldic acids. Penicilldic acids are known to reduce iodine, as do ICl and chloramine-T involved in the radiolabeling process. This only leads to a slight overcon-
AND LABIA
sumption of chloramine-T, because during this step the possible reaction of ICl regenerates an iodide ion which is then recycled. However, we cannot exclude the formation of p-hydroxyphenylacetamido-2-malonic acid, a degradation product of penicillin X that could be also iodinated, although we were unable to detect any. Anyway, such a molecule can be regarded as inert compared with the penicillins for biological activity. The specific activity of the ‘251-penicillin X that we obtained is 2000 Ci/mol. The antimicrobial activities of penicillin X and of its iodinated derivative are quite identical to that of penicillin G. The three compounds have a MIC of 32 pg/ml for E. coli K12. Labeling of the PBPs. We can see from Fig. 3 that ‘251-penicillin X labels all the PBPs of E. coli in a manner very similar to that described for [i4C]penicillin G (9). Competition experiments with 1251-penicillin X (Fig. 4) show that mecillinam binds exclusively PBP 2, the first target of cefotaxime is PBP 3, cefoxitin does not bind PBP 2, and cefsulodin shows affinity for PBP IA, and IB only. These results and the Iso data (Table 1) are in good agreement with the ones obet al. (10) using tained by Curtis [ “C]penicillin G.
IA IB-
234-
516a
bcdefghi
FIG. 3. Penicillin-binding proteins of E. coli K 12. Labeling was performed as described under Methods. Final concentration of ‘251-penicillin X ranges from (a) 40 pg/ml to (i) 0.15 pg/rnl in twofold dilutions.
SYNTHESIS
a
OF ‘251-PENICILLIN
b
c
d
167
FOR PBPs STUDIES
e
f
g
h
FIG. 4. Competition experiments with unlabeled @Iactam antibiotics (a) Control; (b) cefotaxime, 0.05 &ml: (c) cefotaxime, 0.5 rg/ml; (d) cefsulodin, 0.5 &ml; (e) cefsulodin, 5 pg/ml: (f~ cefoxitin, 0.5 pg/ ml; (g) cefoxitin, 5 rg/ml; and (h) mecillinam, 1 pg/ml.
Among all the radioactive molecules described until now, only penicillin G provided a good saturation of all the PBPs, and it was therefore the only one convenient to use in competition studies. This could be done with the commercially available penicillin G, with the drawbacks we have seen, or with tritiated penicillin G (3) but this noncommercial product cannot be synthesized routinely in a biochemical laboratory (12). In addition, the iodinated derivative of furazlocillin (4) whose synthesis was not described, shows a mode of binding to the PBPs different from that of penicillin G. This is the case too for the iodinated derivative of ampicillin described by Schwarz et al. (5). The iodinated derivative of penicillin X that we described
DISCUSSION
Penicillin X is a natural /3-lactam which presents no specific therapeutic properties when compared with penicillin G or penicillin V. Thus, penicillin X is not commercially available, and we therefore had to synthesize it. Iodinated derivatives of penicillin X have been described very early (1 I), but by an unreliable way of synthesis, and these iodinated compounds were not radioactive. Penicillin X and its iodinated derivative have chemical structure very close to that of penicillin G, their microbial properties are very close, and their modes of binding to PBPs are identical, which is an interesting and fundamental property in our field. TABLE HALF-SATURATION
CONCENTRATIONS(~&
I
OBTAINED
IN COMPETITION
EXPERIMENTS
PBP No. P-Lactam antibiotics “SI-Penicillin Mecillinam Cefotaxime Cefsulodin Cefoxitin
X
1A
1B
2
3
4
5
6
0.7 -0 0.1 0.41 0.12
1.7 1 3 2.8
3.5 0.05 5 -
0.9 0.04 1.5
0.3 and 2.5 -
2.5 0.32
3 0.37
’ No competition detected.
19
168
MASSON
here seems therefore especially advisable for use in PBPs studies. Due to its high specific radioactivity (2000 Cifmol), ‘251-penicillin X allows very short exposure times (12-48 h) in autoradiography. Thus, it was of no use to increase this specific activity. The experiment is also less expensive and less time consuming because 23 - diphenyloxazole/dimethylsulfoxide or Enhance (Amersham) is no longer needed. The high specific activity of i2’I-penicillin X leads to extensive radiolysis, and it is advisable to use it immediately after its synthesis because it is more than half destroyed within 48 h, thus giving a lower labeling of the PBPs but no artifact labeling. This drawback is compensated for by the fact that iodination of penicillin X is simple and takes less than an hour. No further purification being needed, the iodination step can be performed just prior to the PBP assay and the product used immediately, which considerably simplifies the problems of radioactive contamination. In conclusion, the main advantage of 12% penicillin X is that radioactive iodine is introduced in a mild way on a penicillin selected to provide a chemical reactivity especially suited for this experiment. The flexi-
AND LABIA
bility of the method was confirmed extensive use in the laboratory.
by an
REFERENCES 1. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 2. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56, 335-341. 3. Williamson, R., and Tomasz, A. (1980) J. Bacterial. 144, 105-l 13. 4. Schmidt, L. S., Botta, G., and Park, J. T. (1981) J. Bacterial.
145, 632-637.
5. Schwarz, U., Seeger, K., Wengenmayer, F., and Strecker, H. (1981) FEMS Microbial. Lett. 10, 107-109. 6. Comelis, G., Wauters, G., and Vanderhaeghe, H. (1973) Ann. Microbial. (Inst. Pasteur) 124 B, 139-152. 7. Spratt, B. G., and Pardee, A. B. (1975) Nature (London) 254, 5 16-5 17. 8. Curtis, N. A. C., Brown, C., Boxall, M., and Boulton, M. G. (1979) Antimicrob. Agents Chemother. 15, 332-336.
9. Spratt, B. G. (1977) Eur. J. Biochem. 72, 341-352. 10. Curtis, N. A. C., Orr, D., Ross, G. W., and Boulton, M. G. (1979) Antimicrob. Agents Chemother. 16, 533-539.
11. Clarke, H. T., Johnson, J. R., and Robinson, R. (1949) in The Chemistry of Penicillin, pp. 680687, Princeton Univ. Press, Princeton, N. J. 12. Rosegay, A. (1981) J. Labelled Compd. Radiopharm. 18, 1337-l 340.