Proc. Nat. Acad. Sci.
USA
Vol. 72, No. 9, pp. 3463-3467, September 1975
Biochemistry
Degradation of penicillin G to phenylacetylglycine by D-alanine
carboxypeptidase from Bacillus stearothermophilus
(gas-liquid chromatography/mass spectrometry/infrared spectroscopy)
SVEN HAMMARSTROM* AND JACK L. STROMINGER
The Biological Laboratories, Harvard University, C ambridge, Massachusetts 021.38
Contributed by Jack L. Strominger, July 9, 1975
ABSTRACT D-Alanine carboxypeptidase from Bacillus
stearothermophilus is a membrane-bound enzyme which is
inhibited by covalent interaction with penicillin G. The peni-
cilloyl enzyme spontaneously reactivates and simultaneously
releases a penicillin G degradation product; 0.2 itmol of the
latter was isolated after incubation of 4.2 gmol of [8.14Clpen-
icillin G with 10 g of membrane protein. It was identified as
phenylacetylglycine by chromatographic techniques, in-
frared spectroscopy, and mass spectrometry. A mechanism
for the degradation is proposed in which the remaining part
of penicillin G would be released as 5,5-dimethyl-A2-thiazo-
line-4-carboxylic acid. The implications of this finding are
discussed.
Penicillins kill bacteria by inhibiting the crosslinking of pep-
tidoglycan strands in the biosynthesis of bacterial cell walls.
It has been proposed that these antibiotics are structural ana-
logs of the acyl-D-alanyl-D-alanine which is cleaved by a
transpeptidase during the crosslinking and that penicillins
inhibit the reaction by acylating the substrate site of this en-
zyme (1). Multiple penicillin binding components have re-
cently been isolated from several bacterial strains (reviewed
in ref. 2). The major binding component in Bacillus subtilis
and B. stearothermophilus is D-alanine carboxypeptidase (3,
4), an enzyme which catalyzes a reaction analogous to the
transpeptidation. In this reaction water rather than the
amino-group of a pentaglycine chain serves as acceptor for
the acyl-D-alanyl residue. D-Alanine carboxypeptidase is
also inhibited by penicillins which bind covalently to the en-
zyme (4, 5). This binding may be reversed by hydroxyl-
amine or ethanethiol (6) or spontaneously (7, 8). Native pen-
icilloyl-carboxypeptidase is required for the release reac-
tions, suggesting that these reactions are enzymatically cata-
lyzed (8). The penicillin derivatives released in the presence
of hydroxylamine or ethanethiol have been identified (6),
whereas the spontaneously released product was not identi-
fied (7, 8). This report describes the identification of the
spontaneously released [8-14C]penicillin G degradation
product as phenylacetylglycine based on thin-layer and gas-
liquid chromatography, infrared spectroscopy, and mass
spectrometry.
MATERIALS AND METHODS
Chemicals. Penicillin G was generously supplied by E. R.
Squibb & Sons and cephalothin from Eli Lilly. 6-Aminopen-
icillanic acid was obtained from Sigma Chemical Co. [8-
14C]Penicillin G, K+-salt, (specific activity: 38 mCi/mmol)
Abbreviations: m.p., melting point; m/e, mass to charge ratio.
* Present address: Department of Chemistry, Karolinska Institutet,
S-10401 Stockholm 60, Sweden.
3463
was obtained from Amersham-Searle. Chloroform, ethyl ac-
etate, acetone, and hexane of nanograde quality were pur-
chased from Mallinckrodt.
Phenylacetylglycine Methyl Ester. Methyl phenylacetate
(prepared from phenylacetic acid by HCI-methanol), was
refluxed for 72 hr in anhydrous methanol containing gly-
cine, Na+-salt (9). The crude crystalline product [melting
point (m.p.) 140-141°; reported m.p. 139-141' (9)] was con-
verted to the methyl ester (HCI-methanol) and crystallized
from ethanol [m.p. 87°; reported m.p. 86.50 (10)]. The gas-
liquid chromatogram (compare Fig. 3, mass detector record-
ing) showed a single peak with an equivalent chain length of
C-14.4 (SE-30 column). The mass spectrum (Fig. 5, lower
panel) showed intense ions at mass/charge (m/e) 207 (M+),
176 (M - 31, -OCH3), 148 (M - 59, -COOCH3), 118
(qCH=C=O)+, 116 (O=C-NH-CH2COOCH3)+, 92
(C7H8)+, 91 (C7H7)+, 88 (H2N=-CH-COOCH3)+, 65
(C5H5)+, 63, 56, 44, 39, and 30.
Preparation of the [8"4C]Penicillin G Degradation
Product. B. stearothermophilus, strain ATCC 15952, was
grown as previously described (4). Membranes were pre-
pared by grinding the cells with glass beads and differential
centrifugation (3). The membranes were resuspended in
0.05 M Tris-HCl buffer, pH 7.5, to a protein concentration
of 20 mg/ml. Five hundred milliliters of this suspension and
1 ml cephalothin (1 mg/ml) were incubated at 370 for 10
min. After this, 1 ml of [8-14C]penicillin G (1.5 mg = 4.2
,gmol; specific activity: 3.8 mCi/mmol) was added and incu-
bation was continued for another 10 min. The mixture was
then centrifuged at 100,000 X g for 1 hr, the pellet was
washed once with 0.05 M Tris, pH 7.5, resuspended in 200
ml of 0.01 M sodium cacodylate buffer, pH 6.5, and incu-
bated at 550 for 50 min. After addition of 200 ml of water,
the suspension was centrifuged at 100,000 X g for 1 hr. The
pellet was discarded and the supernatant solution was Iyoph-
ilized. The residue thus obtained was dissolved in 0.02 N
HCI and extracted twice with ethyl acetate. The combined
extracts were concentrated to dryness in vacuo and the resi-
due was stored at -20° (yield: 0.67 gCi-0. 18 ,imol). Part of
the release product was treated with diazomethane and pu-
rified by preparative thin-layer chromatography (see
below).
Stability of Benzylpenicilloic Acid at pH 6.5 and 55°.
[8-14C]Benzylpenicilloic acid was prepared from [8-
'4C]penicillin G (3.8 mCi/mmol) by incubation with peni-
cillinase (Calbiochem). The product (1.6 ,Ci) was dissolved
in 20 ml 0.01 M sodium cacodylate buffer, pH 6.5, and incu-
bated at 550 for 50 min. The residue after Iyophilization was
dissolved in 0.02 N HCI and extracted with ethyl acetate.
The extracts were concentrated in vacuo and aliquots were
3464 Biochemistry: Hammarstrom and Strominger
analyzed by thin-layer chromatography before and after
methylation. This showed that there was no conversion of
benzylpenicilloic acid to phenylacetylglycine under the con-
ditions used in the isolation of the degradation product.
Analytical Methods. Melting points were determined
with a Thomas-Hoover capillary melting point apparatus
and are uncorrected. Thin-layer chromatography was per-
formed on precoated Silica Gel 60 F-254 plates (thickness
0.25 mm) from E. Merck. The two solvent systems used
were: chloroform-acetone-hexane, 9:1:1 (v/v/v) for methyl
ester derivatives and acetone-acetic acid, 19:1 (v/v) fQr car-
boxylic acids and their salts. Reference compounds were de-
tected by exposure to iodine vapors; radioactivity was de-
tected by autoradiography using blue-sensitive x-ray film
(Kodak, SB54). For preparative thin-layer chromatography,
the plates were developed twice with ethyl acetate before
use. After chromatography, the appropriate zones were
scraped off using a razor blade and the compounds were
eluted from the silica gel with ethyl acetate. An F & M
model 400 gas chromatograph with a stream splitter, a hy-
drogen flame ionization detector, and a Packard model 894
combustion oven/gas flow counter was used for gas-liquid
radiochromatography and a Perkin-Elmer 990 gas chroma-
tograph-Hitachi RMU 6L mass spectrometer operated on
line with an IBM 1800 computer for data acquisition and
processing (11) was used for gas-liquid chromatography-
mass spectrometry and direct inlet-mass spectrometry. The
gas chromatograph or direct inlet effluents were continuous-
ly scanned (m/e 30 to m/e 460) at 4 sec intervals. Mass spec-
tra and mass chromatograms were generated after each run
by the computer (11). Glass columns (inside diameter 3 mm,
length 90 cm) containing 0.75% SE-30 t on 100/140 mesh
Gas Chrom P were used in both types of gas chromato-
graphs. The mass spectrometer was operated with an elec-
tron energy of 70 eV, a trap current of 80 MA and an accel-
erating voltage of 2.2 kV. Infrared spectra were recorded
with a Perkin-Elmer model 567 instrument equipped with a
beam condensor unit. A Beckman model LS 230 liquid scin-
tillation spectrometer was used for radioactivity determina-
tions. Protein was determined by the method of Lowry et al.
(12) with 1% sodium dodecyl sulfate added to reagent A (cf.
4).
RESULTS
The radioactive product released from [8-'4C]benzylpenicil-
loyl-D-alanine carboxypeptidase had an RF value of 0.54 on
silica gel thin-layer chromatography (Fig. 1, left) when ace-
tone-acetic acid, 19:1 (v/v), was used as solvent system. The
RF value was 0 when chloroform-acetone-hexane, 9:1:1
(v/v/v) was used instead. It changed to 0.34 after treatment
of the degradation product with diazomethane, suggesting
the presence of a carboxyl group. The penicillin G release
product contained substantial amounts of contaminating lip-
ids, which could be quantitatively removed by preparative
thin-layer chromatography (70-80% recovery of radioactivi-
ty). After this step the methylated release product was pure
as judged by thin-layer (Fig. 1, right) and gas-liquid chro-
matography (compare Fig. 3 below). The RF value was
identical to that of the methylated release product before
purification.
To ascertain that our conditions for gas-liquid chromatog-
raphy would permit analysis of penicillins and related com-
t SE-30 is a methyl silicone stationary phase for gas-liquid chroma-
tography.
Proc. Nat. Acad. Sci. USA 72 (1975)
Rf
'henylacetylglycine Penicillin G,
-0.5 methyl ester
'henylacetylglycine; Penicilloic acid G,
methyl ester -- dimethyl ester
0-J -O
FIG. 1. Thin-layer chromatograms (Silica Gel G) of penicillin
G degradation product (1.5 nCi) plus 50 ;ig of synthetic phenyl-
acetylglycine [solvent system: acetone-acetic acid, 19:1 (v/v)J, left,
and methylated penicillin G degradation product (2.6 nCi) plus
100 jg of synthetic phenylacetylglycine methyl ester [solvent sys-
tem: chloroform-acetone-hexane, 9:1:1 (v/v/v)], right. The 14C
degradation product was detected by autoradiography and the
synthetic compounds (broken white lines) by exposure to iodine
vapors.
pounds, methyl ester derivatives of benzylpenicillin, benzyl-
penicilloic acid, S-methyl benzylpenicillenic acid, N-methyl
and N,N-dimethyl 6-aminopenicillanic acid were analyzed
by gas-liquid chromatography-mass spectrometry using a
90 cm 0.75% SE-30 column at temperatures between 130
and 200°. These compounds were all eluted intact from the
gas chromatograph as shown by mass spectrometry. A gas-
liquid radiochromatogram of the methyl ester of the [8-
14C]penicillin G, used to prepare the release product, is
shown in Fig. 2. The mass spectrum of the eluted material
was identical to that obtained by direct probe analysis (13).
Fig. 3 shows a gas-liquid radiochromatogram of methylated
[8-'4C]penicillin G degradation product. The equivalent
chain length was between C-14 and C-15. This is 8-9 carbon
units less than the equivalent chain length of penicillin G
methyl ester (C-23.1), suggesting that a part of the latter
molecule had been cleaved off in the release reaction.
An infrared spectrum of 66 nmol of the methylated deg-
radation product pressed into a micro KBr disk (Fig. 4, solid
curve) showed two absorption bands (3255 and 3080 cm-')
5~~~~~~~4
~
0 5
Mass
10
0
0
0 5 10
Time (min)
FIG. 2. Gas-liquid radiochromatogram of [8-14C]penicillin G
methyl ester (2.7 nCi, 25 Mg). The same preparation of ['4C]penicil-
lin G, K+-salt, was used for the incubations. Conditions for gas-
liquid chromatography: column (length, 90 cm; inside diameter, 3
mm) with 0.75% SE-30 on 100/140 mesh Gas Chrom P; column
temperature, 180°; flash heater temperature, 2000; carrier gas, N2
at 60 ml/min.
 Biochemistry: Hammarstrdm and Strominger Proc. Nat. Acad. Sci. USA 72 (1975) 3465

&
-1-1
:3.
lb Rolb
V0 q)
3.3

,;:T
q

2
Q.

Time (min)
FIG. 3. Gas-liquid radiochromatogram of methylated penicil-
lin G degradation product (5.2 nCi) plus 20 gg of synthetic phenyl-
acetylglycine methyl ester. The conditions for gas-liquid chroma-
tography were the same as those described in the legend to Fig. 2
except that the column and flash heater temperatures were 1200
and 160°, respectively.
in the N-H stretch region, a band at 1753 cm-l in the ester
carbonyl region, another band at 1641 cm-l, and a band at
1562 cm-1, (amide I and II regions, respectively), aromatic
C C absorption at 1498 cm-l, and C-O ester absorption
at 1212 cm-'. There was no absorption in the f3-lactam car-
bonyl region (about 1780 cm-'). The infrared spectrum thus
suggested that the compound was a secondary amide with
an ester group and an aromatic ring. No evidence for the
presence of other functional groups was obtained from the
infrared spectrum.
Mass spectra of the methylated degradation product ob-
tained by gas-liquid chromatography-mass spectrometry
and by direct inlet mass spectrometry were very similar, in-
dicating that the methylated release product was eluted in-
tact from the gas chromatograph. There were no ions at m/e
174, 142, 128, or 114 (Fig. 5, upper panel). These ions are
abundant in the mass spectra of the methyl ester derivatives
of penicillin G, benzylpenicilloic acid, benzylpenilloic acid,
and N-rmethyl-6-aminopenicillanic acid f, and have been at-
+ S. hammarstrom and J. L. Strominger, unpublished results.
Wavelength (mim)
50 100 150 200 250
m/e
FIG. 5. Mass spectra of methylated [8-_4C]penicillin G degra-
dation product (upper panel) and synthetic phenylacetylglycine
methyl ester (lower panel). The spectra were recorded during di-
rect probe inlet analyses with an electron energy of 70 eV.
tributed by high resolution mass spectrometry of penicillin
G methyl ester to fragment ions originating in the thiazoli-
dine ring (13). Their absence in the mass spectrum of the re-
lease product indicated that the thiazolidine ring had proba-
bly been eliminated, which would be compatible with the
retention behavior of this compound during gas-liquid chro-
matography. Ions in the mass spectrum at m/e 91 (C7H7+,
tropylium ion) and m/e 118 (,CH=C==O) showed that the
phenacetyl side chain of penicillin G was unchanged in the
degradation product. Other ions at m/e 88 [H2N=CH-
COOCH3]+, 116 [O=C= NH-CH2-COOCH3]+, 148
[0-CH2-CO-NH-CH2]+, 176 [X-CH2-CO-NH-
CH2CO]+ and 207 [W-CH2-CO-NH-CH2-
COOCH3]+ indicated the structure phenylacetylglycine
methyl ester.

NO
I-0
(0)
_-

E
C
(0
T

Wavenumber (cm-')
FIG. 4. Infrared spectrum of methylated [8-'4C]penicillin G degradation product, prepared as described under Materials and Methods
(0.25 gCi, 66 nmol), pressed into a micro KBr disk (solid line) and synthetic phenylacetylglycine, methyl ester (broken line). Fifty-seven na-
nomoles of the radioactive compound were recovered after the analysis by dissolving the KBr in water and extracting with ethyl acetate.
3466 Biochemistry: Hammarstrom and Strominger
I f i~X COOH
Ce HoCH2<N (2H,
N/Q ,' H
0 H
2I COOH
SO
C6 HS CH2
°0
+ N(CCH3
,' H
COOH
3 4
kH20
C6H5CH2CONHCH2
COOH
5
FIG. 6. Possible mechanisms for the formation of
Phenylacetylglycine and its methyl ester were synthesized
for direct comparison with the degradation product. The ra-
dioactive penicillin degradation product cochromato-
graphed with synthetic phenylacetylglycine on thin-layer
chromatography as the free acid and as the methyl ester
(Fig. 1) and on gas-liquid chromatography as the methyl
ester (Fig. 3). The infrared spectrum of the methyl ester
(Fig. 4, broken curve) was similar to that of the methylated
degradation product and the differences could be attributed
to nonvolatile impurities eluted from the silica gel during
thin-layer chromatography. Furthermore, the mass spec-
trum of the synthetic methyl ester (Fig. 5, lower panel) was
identical to that of the methylated release product. Based on
these results, the penicillin G degradation product released
by B. stearothermophilus D-alanine carboxypeptidase was
identified as phenylacetylglycine. In a control experiment,
incubation of [8-'4C]benzylpenicilloic acid under the same
conditions that were used to obtain the [8-'4C]penicillin G
release product, there was no formation of phenylacetylgly-
cine. Instead, most of the penicilloic acid was recovered in-
tact.
DISCUSSION
D-Alanine carboxypeptidases from B. subtilis (14) and B.
stearothermophilus (4) are membrane-bound enzymes
which have been purified to homogeneity using convention-
al techniques or covalent affinity chromatography. Penicil-
lin G inhibits both enzymes by covalent interaction (5, 4).
The binding of penicillin G may involve acylation of an
amino-acid residue at the substrate site of the enzyme by the
f3-lactam carbonyl group of penicillin [penicilloylation (1)].
Evidence for the binding of an intact benzylpenicilloyl
group has come from the identification of benzylpenicil-
loylhydroxamate and a-ethanethiobenzylpenicilloate as re-
lease products after treatment of penicilloyl-carboxypepti-
dase with hydroxylamine or ethanethiol (6).
Preincubation of B. stearothermophilus membranes with
cephalothin permits selective binding of [8-'4C]penicillin G
to the membrane bound D-alanine carboxypeptidase (4).
The resulting penicilloyl-carboxypeptidase is readily sepa-
rated from free penicillin G by two centrifugations. Subse-
Proc. Nat. Acad. Sci. USA 72 (1975)
CeHsCH2CON N 2(
Enzyme\
:nzyme-XH 1 \
3 C6H5CH2CONHCH2 S CH3
3 eCXx + r [CH3
N
o / H
6E1Enzyme 4
COOH
H20
FEnzyme-XH
ra w
3
C6H6CH2CONH CH2
COOH
5
phenylacetyiglycine (5) from benzylpenicilloyl-D-alanine carboxypeptidase (1).
quent incubation of the penicilloyl-enzyme at 550 for 50
min leads to release of about 97% of the radioactivity as a
penicillin G degradation product and causes reactivation of
the same percentage of the enzyme (8). Similar results have
been obtained using purified D-alanine carboxypeptidase
(8).
The identification of this release product as phenylacetyl-
glycine shows that the spontaneous reactivation of penicil-
loyl-carboxypeptidase is not a hydrolytic removal of the
penicilloyl group from the enzyme. The latter reaction
would give benzylpenicilloic acid, a compound which was
stable under the conditions used for the release reaction and
the isolation of release product. Thus, unlike a D-alanine car-
boxypeptidase from Escherichia coli (15), the corresponding
enzyme from B. stearothermophilus does not catalyze a
lactamase reaction. The conversion of penicillin G to phen-
ylacetylglycine does not occur after denaturation of benzyl-
penicilloyl-D-alanine carboxypeptidase by several tech-
niques, which suggests that it is enzymatically catalyzed.
The chemical degradation of penicillin G methyl ester to
phenylacetylglycine has been reported (16). It occurs in tri-
fluoroacetic acid at room temperature, probably via the in-
termediate proposed for the penillic acid rearrangement
[(17); see Fig. 6, compound 2]. Protonation of N-4 in 1 fol-
lowed by fragmentation between C-5 and C-6 gave 2-ben-
zyl-2-oxazolin-5-one and methyl D-5,5-dimethyl-A2-thiazo-
line-4-carboxylate (Fig. 6, 3 and the methyl ester of 4,
respectively). 3 is unstable and undergoes rapid hydrolysis to
5, phenylacetylglycine, in aqueous solutions (18). The degra-
dation of penicillin G to phenylacetylglycine by D-alanine
carboxypeptidase may follow a similar mechanism. Two
possibilities are outlined in Fig. 6. In pathway I, the forma-
tion of 2 from benzylpenicilloyl carboxypeptidase, 1, is anal-
ogous to the formation of 2 from penicillin G (17). Although
formation of 2 leads to scission of the penicilloyl-enzyme
bond, 2 may remain tightly bound to the enzyme by nonco-
valent forces until fragmentation into 3 and 4 has occurred.
Alternatively (pathway II), the enzyme may activate the
penicilloyl residue for cleavage without the necessity for for-
mation of 2. In this mechanism phenylacetylglycyl enzyme,
6, would be an intermediate.
 Biochemistry: Hammarstrbm and Strominger
An alternative degradation was also considered, namely
the formation of benzylpenamaldic acid by ring opening be-
tween the sulfur and C-5 followed by scission of this Schiff
base to benzylpenaldic acid (a-formylphenylacetyl glycine)
and D-penicillamine (17). Benzylpenaldic acid as well as its
reduction and oxidation products, N-phenylacetylserine and
phenylacetylaminomalonic acid, were synthesized and dis-
tinguished from the degradation product. All three com-
pounds were stable under the conditions used for examina-
tion of phenylacetylglycine (gas chromatography and mass
spectrometry), thus excluding the possibility that phenyl-
acetylglycine was formed from one of these compounds dur-
ing the analyses.
The unusual degradation of penicillin G described here
may provide information regarding the mechanism of ac-
tion of D-alanine carboxypeptidase. This enzyme catalyzes
the cleavage of a number of acyl-D-alanyl-D-alanine com-
pounds. Kinetic evidence supports the proposal that an acyl-
enzyme intermediate is formed in the cleavage §. At this
stage, the terminal D-alanine residue would be enzyme
bound and presumably the next step of the reaction would
be the release of D-alanine from the enzyme surface. If peni-
cillin G is an analog of the substrate, opening of the f3-lactam
ring with formation of penicilloyl carboxypeptidase would
be analogous to the cleavage of the D-alanyl-D-alanine pep-
tide bond (1). The subsequent cleavage of the penicillin mol-
ecule between C-5 and C-6 may result from an enzyme-cat-
alyzed reaction which normally functions to release D-ala-
nine. For example, protonation of the amino group of D-ala-
nine might facilitate its release while protonation of N-4 in 1
could facilitate cleavage of the C-5-C-6 bond according to
either of the pathways outlined in Fig. 6. This cleavage and
the release of D-5,5-dimethyl-A2-thiazoline-4-carboxylic
acid, 4, (or of D-alanine) may also be essential for deacyla-
tion of the enzyme. Thus, the cleavage may provide clues to
the late events in the D-alanine carboxypeptidase reaction,
namely the steps in which D-alanine is released and the en-
zyme deacylated.
The work was supported by research grants from the National
Institutes of Health (Al-09152) and National Science Foundation
§T. Nishino and J. L. Strominger, submitted for publication.
Proc. Nat. Acad. Sci. USA 72 (1975) 3467
(GB-79747). S.H. was the recipient of an exchange visitor grant
from the Swedish Medical Research Council. Use of the National
Institutes of Health mass spectrometry facility at Massachusetts In-
stitute of Technology, Grant RR0317, to K. Biemann, is gratefully
acknowledged. The sample of crude penicillin degradation product
was prepared by Ms. Eileen Willoughby and Dr. Peter Blumberg
gave much helpful advice.
1. Tipper, D. J. & Strominger, J. L. (1965) Proc. Nat. Acad. Sci.
USA 54, 1133-1141.
2. Blumberg, P. M. & Strominger, J. L. (1974) Bacteriol. Rev. 38,
291-335.
3. Blumberg, P. M. & Strominger, J. L. (1972) J. Biol. Chem.
247,8107-8113.
4. Yocum, R. R., Blumberg, P. M. & Strominger, J. L. (1974) J.
Biol. Chem. 249,4863-4871.
5. Umbreit, J. N. & Strominger, J. L. (1973) J. Biol. Chem. 248,
6767-6771.
6. Lawrence, P. J. & Strominger, J. L. (1970) J. Biol. Chem. 245,
3653-3659.
7. Strominger, J. L., Willoughby, E., Kamiryo, T., Blumberg,
P. M. & Yocum, R. R. (1974) Ann. N.Y. Acad. Scf. 235, 210-
224.
8. Blumberg, P. M., Yocum, R. R., Willoughby, E. 4 Strominger,
J. L. (1974) J. Biol. Chem. 249, 6828-6835.
9. Ford, J. H. (1949) J. Am. Chem. Soc. 71, 3842.
10. (1965) in Dictionary of Organic Compounds, eds. Pollock, J.
R. A. & Stevens, R. (Oxford Univ. Press, New York), Vol. 4,
4th ed., p. 2649.
11. Hites, R. A. & Biemann, K. (1968) Anal. Chem. 40, 1217-
1221.
12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193, 265-275.
13. Richter, W. & Biemann, K. (1964) Monatsh. Chem. 95, 766-
778.
14. Umbreit, J. N. & Strominger, J. L. (1973) J. Biol. Chem. 248,
6759-6766.
15. Tamura, T., Imae, Y. & Strominger, J. L. (1975), J. Biol.
Chem., in press.
16. Bell, M. R., Carlson, J. A. & Oesterlin, R. (1972) J. Org. Chem.
37,2733-2735.
17. Johnson, J. R., Woodward, R. B. & Robinson, R. (1949) in The
Chemistry of Penicillin, eds. Clarke, H. T., Johnson, J. R. &
Robinson, R. (Princeton Univ. Press, Princeton, N.J.), pp.
440-454.
18. Jansen, A. B. A. & Robinson, R. (1967) Monatsh. Chem. 98,
1017-1026.