ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
68, l-14
(1957)
A Spectrophotofluorometric
Study of Compounds of
Biological Interest
Daniel E. Duggan,’ Robert L. Bowman, Bernard B. Brodie and Sidney
Udenfriend
From the Laboratory
of Chemical Pharmacology and Laboratory of Technical Development, National Heart Institute, National Institutes of Health, Public Health
and Welfare,
Bethesda.,
Service,
U. S. Department of Hea.lth, Education,
Maryland
Received
October
8, 1956
INTRODUCTION
Since the observation of Desha (1) that the fluorescence exhibited by
solutions of quinine and aniline erythrosine when irradiated by ultraviolet light could be utilized for their quantitative determination, useful
fluorimetric methods have been devised for a number of biological constituents (2). The extreme sensitivity of fluorimetric assay offers a distinct advantage over other chemical and physical methods, but instrumental limitations have restricted its application to a relatively small
number of compounds. Among the most serious of these limitations have
been the unavailability
of activating light of high intensity over a wide
range of wavelengths, and the restriction of fluorescence detection to the
visible region of the spectrum. The adequate fluorimetric methods that
are available, notably those for riboflavine (4), thiamine (3), and adrenaline (5,6) are possible because each fluorophor absorbs light corresponding to the wavelength of one of the few available lines of the mercury
emission spectrum,2 and emits visible fluorescence. In addition, the fluorescence of thiamine and adrenaline must be induced by chemical
change, a requisite common to many fluorimetric procedures.
Recent papers from this laboratory (7-10) describe the design and
1 American Instrument
Co. postdoctoral
research fellow.
* Mercury-vapor
lamps ordinarily
used in fluorometers
emit utilizable
lines at
366, 405, and 436 ml. Riboflavine,
thiochrome,
adrenolutine,
and the quinoxaline derivative
of adrenaline
have activation
maxima at 440, 370, 405, and 435
mp, respectively.
2
DUGGAN, BOWMAN, BRODIE AND UDENFRIEND
analytical applications of an experimental spectrophotofluorometer capable of delivering high-intensity monochromatic activation at all wavelengths from approximately 220 to 800 rnp and of automatic spectral
analysis of resulting fluorescence throughout this same range. Two commercial instruments based upon this experimental spectrophotofluorometer have subsequently become available.3 All three instruments are
routinely employed in this laboratory for the determination in tissues of
serotonin (8, ll), tryptophan (9), and numerous other compounds of
biochemical and pharmacological interest.4
A survey of the potential applicability of spectrophotofluorometry to
sensitive qualitative and quantitative analyses revealed that a surprisingly large percentage of compounds absorbing light above 220 ml.r
emitted measurable fluorescence. The results of more detailed investigation of the fluorescence characteristics of naturally occurring biological
constituents indicate that spectrophotofluorometry provides the basis
for the assay of many compounds in tissues at the submicrogram level.
This paper presents a tabulation of the fluorescence characteristics of
a number of biological constituents and a discussion of some problems
encountered in the practical application of spectrophotofluorometry to
qualitative and quantitative analysis. Another paper (12) to be published will describe the fluorescence characteristics of a large number of
drugs together with extraction procedures suitable for their isolation
from tissues.
EXPERIMENTAL
Instrumentation
The instruments consist of the same essential components. A high-pressure
xenon arc lamp6 emitting a continuum from approximately 220 to 800 rnp is used
as the source of activating light. Two monochromators are employed: one to isolate light at the wavelength of activation; and a second, at right angles to the
first, to analyze the emitted fluorescence spectrum. In each instrument, provision
is made to control the resolving power of either monochromator by variable or
replaceable slits. An appropriate photomultiplier
tube’ is used to measure the
8 The Aminco-Bowman Spectrophotofluorometer
and the Farrand Spectrofluorometer.
4 Tyrosine, tocopherol, 5-hydroxyindoleacetic
acid, lysergic acid diethylamide,
barbital, chlorpromazine, and reserpine.
6 Hanovia 150-w. xenon lamp.
0 TheRCA lP21 “ultraviolet-sensitive”
and lP23 “visible-sensitive”
phototube
are interchangeable throughout the major portion of the spectrum. The lP21
tube is preferred for the measurement of fluorescence below 330 nn~ (see Fig. 5).
SPECTROPEOTOFLUOROMETRIC
70-
I
I
STUDY
I
3
I
605040?
2,30&202
c- IO2
-x 0
I
200
400
Mh
600
FIG. 1. E&radio1 (0.1 a./ml.)
in absolute ethanol. Activation
at 285 w) ; fluorescence spectrum (peak at 330 mp).
spectrum (peak
intensity of fluorescence. The photomultiplier
signal may be read directly on a
galvanometer or supplied to the vertical input of a cathode ray oscilloscope or
recorder. The horizontal signal in the latter cases is supplied by an appropriate
device coupled to the wavelength scale of the fluorescence monochromator. The
resulting plot.of intensity versus wavelength is the jluorescence spectrum. Actiuation spectra are obtained in a similar manner by taking the X signal from the
activating monochromator while the fluorescence monochromator is fixed at the
wavelength of peak fluorescence. Activation and fluorescence spectra are typified
hv those of estradiol as illustrated in Fig. 1.
Determination
of Fluorescence Characteristics
Samples for analysis were prepared by the dilution of an aqueous or alcoholic
solution (1 mg./ml.) of the material to be tested to a concentration of 1.0 pg./ml.
in each of the following media 7: 0.1 N sulfuric acid, 0.1 M phosphate buffer pH
7.0, 1 N ammonium hydroxide (pH ll), 1.0 N sodium hydroxide (pH 14). For each
The lP28 is slightly more sensitive at the red end of the spectrum for the detection of fluorescence in the red region, but the use of an S-l response-type photomultiplier may be required.
7 The variation of media was restricted to these four aqueous solutions because
they constitute a sufficient over-all range and gradation of pH values w to preclude the omission of any possible ionized form of the compounds studied. It is
entirely probable that in many cases maximal fluorescence intensity might be
achieved at some intermediate pH value.
4
DUGGAN,
BOWMAN,
BRODIE
TABLE
Compound
Adenine
Adenosine
Adenylic acid
Adrenaline
p-Aminobenaoic acid
Anthranilic acid
ATP
DPNH
3,4-Dihydroxyphenethylamine
3,4-Dihydroxyphenylacetic
acid
3,4-Dihydroxyphenylalanine
3,4-Dihydroxyphenylserine
Equilenin
Equilin
Estradiol
Estrone
Folic acid
Folinic acid
Guanine
Homogentisic acid
Homovanillic acid
3-Hydroxyanthranilic
acid
5-Hydroxyanthranilic
acid
p-Hydroxycinnamic
acid
3-Hydroxykynurenine
5-Hydroxykynurenine
p-Hydroxymandelic
acid
p-Hydroxyphenylacetic
acid
p-Hydroxyphenylpyruvic
acid
p-Hydroxyphenylserine
5-Hydroxyindole
5-Hydroxyindoleacetic
acid
Kynurenine
Indole
Kynurenic acid
Kynurenic acid
Noradrenaline
Pteroic acid
Pyridoxal
Pyridoxamine
Pyridoxine
Riboflavine
Activation
AND
UDENFRIEND
I
max.
Fluorescence
max.
PH
Practical
sells.
w*
w*
280
285
285
285
295
300
285
340
285
375
395
395
325
345
405
395
435
325
1
1
1
1
11
7
1
7
1
0.1
0.05
0.07
0.006
0.002
0.001
0.09
0.02
0.01
280
.330
7
0.04
285
280
250, 290, 340
290
285
285
365
370
285
290
270
320
340
350
365
375
300
280
290
290
290
300
370
280
325
325
285
365
330
335
340
270, 370, 445
325
320
370
345, 420
330
325
450
460
365
340
315
415
430
440
460
460
380
310
345
320
355
355
490
355
405
440
325
450
385
400
400
520
7
1
(I
0
a
0
7
7
1
7
7
7
7
7
11
11
7
7
7
1
1
7
11
7
7
11
1
7
7
7
7
7
0.01
0.03
0.001
0.1
0.01
0.08
0.01
0.15
0.1
0.02
0.2
0.001
0.001
0.02
0.002
0.005
0.006
0.03
0.1
0.05
0.003
0.002
0.03
0.002
0.003
0.005
0.006
0.005
0.002
0.001
0.0015
0.0002
pg./ml.
SPECTROPHOTOFLUOROMETRIC
TABLE
Compound
I-continued
Activation
Serotonin
Serotonin
Tocopherol
Thiochrome
Tryptophan
Tryptamine
Tyrosine
Tyramine
Uric acid
Vitamin X
Vitamin B~z
Xanthine
Xanthurenic acid
5
STUDY
d
295
295
295
370
285
290
275
275
325
325
275
315
350
max.
Fluorescence
max.
PR
mb
340
540
330
445
365
360
310
310
370
470
305
435
460
Practical
sells.
P&T./ml.
2
0
a
10
11
7
7
1
a
7
1
11
0.003
0.005
0.01
0.01
0.003
0.002
0.005
0.02
0.7
0.01
0.003
0.08
0.005
a Sample in 99% ethyl alcohol.
b Maxima determined to f5 mp.
sample the fluorescent spectrum was rapidly scanned on the oscilloscope screen
while changing the activating wavelength IO-20 nqu per scan until the entire activation range of 200-500 rnF had been covered. Upon the appearance of a fluorescence band, the scan was stopped at the peak and the wavelength of maximum
activation was accurately determined by manipulation of the activating monochromator. Precise fluorescent spectra were then obtained in each of the media
with the activating monochromator set to the optimum wavelength as determined
above. That pH at which fluorescence intensity was found to be a maximum was
chosen for further study, and the ultimate sensitivity under these conditions determined by serial dilution.
RESULTS
For each of the compounds tested and found to exhibit fluorescence,
the wavelength of maximum activation and fluorescence, the pH at
which fluorescence intensity is maximal, and the practical sensitivity
are given in Table I.
The practical sensitivity is defined as that concentration which gives
a fluorescence intensity reading equal to 10% of full-scale deflection on
the meter of the Aminco-Bowman instrument, at highest sensitivity,
using one-sixteenth inch defining slits (band pass = 6 mp) and the ~28
photomultiplier. Such a deflection represents approximately 20 t,imes
the value of the blank reading obtained for a pure sample of the appropriate solvent under the same conditions.
A number of compounds which absorb visible or ultraviolet light failed
6
DUGGAN,
BOWMAN,
BRODIE
AND
UDENFRIEND
to show measurable fluorescence. These included the pyrimidines,
phenylalanine, phenylpyruvic acid, h&dine, and all of the nonaromatic
steroids studied (the common androgens and cortical hormones).
The pH values and wavelengths of maximum activation given in Table I represent those conditions under which fluorescence intensity is
maximal. In several practical applications of spectrophotofluorometry
to qualitative or quantitative analysis, greater specificity may be
achieved by the use of other media or activating wavelengths. For example, folic and folinic acids both show maximum fluorescence at 450 rnp
when activated at 365 mp. While both exhibit considerable variation of
fluorescence intensity with pH, a satisfactory differential analysis cannot
be achieved at any pH under these conditions of activation and measurement of fluorescence. When activated at 290 rnp, however, both
show a somewhat weaker fluorescence, but the wavelength of folinic acid
fluorescence is shifted to 370 mp while that of folic acid remains at 450
ml.c.As shown in Fig. 2, activation at 290 ml.cof an acid or neutral solution containing both materials allows of a satisfactory determination of
each.
A similar differentiation between adenine and its nucleoside and
nucleotides may be made by proper choice of medium. Adenine, adenosine, adenylic acid, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) all show a maximum fluorescence in the region 375-395
60
50
g?40
z
= 30
&
Q 20
F
0
200
300
400
500
600
200
300
400
500
600
200
300
400
500
600
w
FIG. 2. All curves are fluorescence spectra. Solid lines indicate spectra obtained upon activating at 370 9; broken lines, upon activating at 280 -. Concentrations of the pure samples are each 1.0 pg./ml.; concentrations in the mixture are 1.33 pg. folic acid and 10.0 pg. folinic acid/ml. A: pH 1.0; B: pH 7.0; C:
pH 10.
SPECTROPHOTOFLUOROMETRIC
STUDY
7
rnp when activated at 285 rnp at pH 1. In 5 N sulfuric acid, however, the
nucleoside and nucleotides all show an approximately
equal molar
fluorescence at the same wavelength while adenine shows negligible
fluorescence.
Practical Considerations in Spectrophotojkorometry
A. Interference by the Sample. In addition to the obvious possibility of
interference in the form of blank fluorescence, other potential sources of
error, leading to either high or low results are inherent in fluorescence
methods. The ready availability of complete activation and fluorescence
spectra provide a convenient and reliable means of detecting such interferences and providing for appropriate correction. The most frequently
encountered interferences of this type are light scattering and absorption
by the sample.
Light scattering may be a true Tyndall effect due to colloidal material
or a simple reflection from particles of dust, paper, or glass suspended in
the sample. At higher sensitivities light scattering will invariably be
evident in the fluorescence spectrum but is easily distinguished from
fluorescence since it appears at the activating wavelength. Where the
degree of scattering is very great, however, it may lead to significantly
high or low results: high if the activation and fluorescence wavelengths
are too close together to be completely resolved; otherwise low, since
the fluorescent light is being deflected away from the phototube. Figure
3 illustrates an extreme case of light scattering due to colloidal silicate in
an alkaline protein hydrolyzate, and the notable concomitant increase in
tryptophan fluorescence upon decreasing light scattering by high-speed
centrifugation.
At higher concentrations of the material being measured or of other
materials absorbing at the activation wavelength, self quenching or
“concentration quenching” becomes significant and lead to low results.
This effect will be evidenced by one of several typical distortions of the
activation spectrum as illustrated in Fig. 4.
When the activating wavelength is in the region where the output of
the xenon source varies sharply with wavelength (below 300 mp), selfabsorption is evidenced by a shift in the activation spectrum toward
longer wavelengths as illustrated by adenine. In regions where the spectral output of the source is essentially flat, self-absorption causes a
depression in the middle of the activation peak, as evidenced by the activation spectra for pyridoxine. For compounds with more than one activa-
DUGGAN,
BOWMAN,
0
100
200
BRODIE
300
AND
UDENFRIEND
400
500
OF
TRYPTOPHAN
HYDROLYSATE
600
mw
ACTIVATION
SPECTRA
IN B-LACTOGLOBULIN
FIG. 3. Activation
spectrum of tryptophan in alkaline &lactoglobulin
hydrolyzate before and after centrifugation. Peaks at 280 are the activation peaks;
at 370, the scattering peaks.
tion peak, that activation peak at which the extinction coefficient is
higher will first be effected by concentration quenching, and as concentration is increased, eventually disappear. This is illustrated by folic
acid. As concentration is increased still further, the second peak will be
affected. These generalizations hold only for instruments in which the
geometry of the sample with respect to the activating and fluorescence
monochromators is of the type employed in the spectrophotofluorometers
employed in these studies, i.e., when the optical systems of both monochromators are focused upon the exact center of the sample.
The absorption of fluorescence by other compounds in the sample may
be a serious source of error, but low results due to this cause are readily
detected by the shape of the fluorescence spectrum and may easily be
corrected using absorption data. This is most readily accomplished by
SPECTROPHOTOFLUOROMETRIC
I
9
STTJDY
I
ADENINE
I
FOLK
1
I
ACID
60 ----
400
I .o y/ml.
IO ylml.
100 y/ml.
t
!‘Ii
fj
pI!!
t-1
I\,,
,/$.j
/
*I
il .,’ “.
200
300
FIG. 4. The effect of concentration quenching upon activation spectra. The
three curves for each compound were obtained at different sensitivity settings.
The 10 pg./ml. curve (- - -) and the 100 pg./ml. curve (.-.-a) were obtained
at sensitivities which were respectively 33.370and 10% of that used to obtain the
1.0 pg./ml. curve ().
determining the optical density at the fluorescence wavelength of a 1.Ocm. sample, dividing by two to correct for the depth of solution through
which the fluorescent light must pass, and converting this value to per
cent kansmission. The observed fluorescence intensity is then corrected
for the fraction of light calculated to have been transmitted by the sample. h similar correction may be made for the presence of any material
having a significant absorption at the activating wavelength.
Errors due to absorption or scattering of such magnitudes as indicated
by the foregoing examples are but rarely encountered in bhe course of
routine analyses. Where interferences of this t,ype become significant,
however, they may be most easily and reliably corrected for by the use
of internal standards.
B. Instrumental Variations. In addition to the easily correctable interferences due to the sample, a number of variables are inherent in t,he
design of the instruments employed in t,hese studies.
1. IVaveZength Data. The wavelength data cited in Table I are instrument,al values uncorrected for the over-all spectral response of t,he inskmrent. While correction curves for converting instrumental to absolute wavelength values may be obtained for any given instrumental
10
DUGGAN,
BOWMAN,
BRODIE
AND
UDENFRIEND
arrangement,8 for purposes of quantitative analysis or qualitative comparison of unknown to standard samples, instrumental values are entirely adequate. In addition, both qualitative and quantitative results for
a given set of instrumental conditions are highly reproducible from day
to day, and since all three instruments employ the same essential components, most qualitative data are reproducible from one instrument to
another. Distortions in activation spectra are determined largely by the
spectral output of the xenon arc source and to a lesser degree by the overall spectral transmission of the grating, lenses, and mirrors of the activating monochromator. Such distortions, however, except in the region
where the source output falls off very sharply with wavelength (below
280 mp) should be small. Barbital, for instance, whose absorption maximum is 240 rnp, has an apparent activation maximum at 260 rnp since
the intensity of the source at the latter wavelength is many times more
than at the former (12). Above 280 rnp, however, the spectral output of
the source is relatively flat, and instrumental activation maxima correspond closely to known absorption peaks.
In a similar manner, distortions of fluorescence spectra are determined
by the spectral response of the phototube employed and the over-all
spectral transmission of the optics in the analyzing monochromator.
Since the phototube response curves are somewhat sharper than the
source output curves and fall off at both ends of their usable range, the
displacement of fluorescence maxima is a more commonly encountered
problem. The magnitude of such instrumental displacements may be
illustrated by the interchanging of phototubes with slightly different response characteristics. Using the lP28 ultraviolet-sensitive photomultiplier for example, a fluorescence maximum of 320 rnp is obtained for
estradiol, while the same sample gives a maximum at 340 rnp using the
visible-sensitive lP21 photomultiplier (Fig. 5). The lP28 photomultiplier
was used to obtain all of the data in Table I.
Obviously, the indiscriminate use of uncorrected wavelength data for
purposes of selecting critical secondary filter systems for conventional
*A correction curve for fluorescentspectra may be calculated from
parison of the known spectral output of a standard light source with the
a comexperimental spectrum obtained by focusing this source upon the entrance slit of the
fluorescence monochromator.
This is a laborious
procedure necessitating
a prior
determination
of the spectrum of the individual
source used, and the curve is
applicable
only to the individual
instrumental
arrangement
for which it is determined. Studies directed toward the design of a stable and convenient
reference
standard
are currently
in progress.
SPECTROPHOTOFLUOROMETRIC STUDY
II
I
:-TOCOPH.
mu
FIG. 5. Effect of varying phototube responses upon fluorescence spectra. Vitamin Bre is 0.01 pg./ml. in phosphate buffer 7.0; a-tocopherol, 0.1 pg./ml. in absolute ethanol. Solid curves obtained using lP28 (ultraviolet-sensitive)
photomultiplier; broken curves, using lP21 (visible-sensitive)
photomultiplier.
fluorometers, or for comparison with data obtained on fluorescence attachments for spectrophotometers, may lead to inconsistencies where
the wavelengths concerned are in that region of the spectrum where they
are subject to significant variation from absolute values.
2. Sensitivity. The lower limits of concentration at which each compound in Table I can be measured is a somewhat arbitrary figure representing a compromise between sensitivity on the one hand versus resolution, minimizing of light scattering, blank fluorescence, and spurious
circuit noise. Theoretically,
ultimate sensitivity is determined by the
extinction coefficient of the compound at its activating wavelength and
the quantum yield of fluorescent light. Upon these theoretical limits,
however, several instrumental variables and practical limitations are imposed.
Changing from minimum to maximum resolving power, with the various slit arrangements possible, results in a variation in sensitivity of
several thousand times. The voltage at which the photomultiplier
tube
is operated largely determines sensitivity, but is limited to a maximum
of about 900 v. above which its random noise reaches impractically high
12
DUGGAN,
BOWMAN,
BRODIE
AND UDENFRIEND
levels. The age of the xenon arc source has a
though real effect upon sensitivity. In addition,
and spectral transmission of the optical systems of
result in varying sensitivity from one instrument
comparatively minor,
variations in aperture
individual instruments
to another.
DISCUSSION
The results of this survey allow of several generalizations as to the
structural requirements for fluorescence in solution. In most general
terms, these would appear to be the presence of either an aromatic nucleus substituted by at least one electron-donating group, or a conjugated unsaturated system capable of a high degree of resonance. On this
basis, each of the compounds listed in Table I may be classified as possessing at least one of several general fluorophoric structures:
1. Phenols: the estrogens, tocopherols, pyridoxine and its analogs, and
all of the phenol and catechol derivatives related to tyrosine and adrenaline.
2. Aromatic Amines: p-aminobenzoic acid, anthranilic
acid, kynurenine, and related compounds. The fluorescence of aromatic amines,
and of phenols, is completely lost upon acetylation suggesting that the
substituent requirement is not for nitrogen or oxygen per se, but for a
grouping capable of donating electrons to the aromatic nucleus.
3. Indoles: these include tryptophan, serotonin, and many of their
precursors and metabolites.
4. Polycyclic Aromatic Compounds: (a) Homocyclic; equilinine. (b).
Heterocyclic: the purines, their nucleosides and nucleotides, riboflavine,
kynurenic acid, and xanthurenic acid.
5. Conju.gated Polyenes: vitamin A and its precursors.
Notable exceptions to these generalizations are thyroxine and the
reduced forms of vitamins K1 and Kz which, although conforming to one
or more of the above categories, show no fluorescence at concentrations
up to 10 pg./ml. The inability of the natural K vitamins to fluoresce is
all the more anomalous since reduced menadione which is similar in
structure shows a strong fluorescence in aqueous solution.
In general, spectrophotofluorometric
assay offers several distinct advantages over other physical methods utilizing light absorption. The
fluorescent method where applicable offers a sensitivity at least two
orders of magnitude greater than spectrophotometry and the additional
specificity of two spectral requirements instead of one.
As compared to conventional filter fluorometers, the spectrophoto-
SPECTROPHOTOFLUOROMETRIC
STUDY
13
fluorometer extends the utility of fluorescence as an analytical tool in
several respects. The use of a continuous source and activating monochromator provides high-intensity activating light at the exact wavelength of maximum absorption, as opposed to the few wavelengths corresponding to lines of the mercury spectrum available in conventional
fluorometers. The detection of fluorescence is not limited to the visible
region, but is extended into the ultraviolet. Specificity is enhanced by the
greater resolving power of monochromators as compared to filters. The
ready availability of full spectra obviates the necessity of a prior knowledge of absorption and fluorescencewavelengths, as neededfor t.he proper
choice of filter systems, and provides a ready means of detecting the
presence of extraneous materials which may cause serious errors due to
light scattering, blank fluorescence, or absorption of either activating
or fluorescent light.
SUMMARY
An experimental spectrophotofluorometer and two commercial instruments of essentially the same design have been applied to a broad survey
of the fluorescence characteristics of organic metabolites in solution.
Useful ultraviolet or visible fluorescence was found to be exhibited by a
large number of the light-absorbing compounds of biochemical significance, many of which had not previously been reported as fluorescing in
solution.
For each fluorophor, the wavelengths of maximum activation and
fluorescence, the optimum pH for the development of fluorescence intensity, and an arbitrary measure of the ultimate sensitivity are presented.
Some practical aspects of the application of spectrophotofluorometry
to qualitative and quantitative analysis are discussed.
REFERENCES
1. DESHA, L. J., J. Am. C&em. Sot. 42, 1355 (1920).
Analysis in Ultraviolet
Light,”
2. RADLEY, J. A., AND GRANT, J., “Fluorescence
4th ed. Chapman and Hall, London, 1954.
3. HENNESSEY, D. J., AND CERECEDO, L. R., J. Am. Chem. Sot. 61, 179 (1939).
4. HODSON, A. Z., ANI) NORRIS, L. C., J. Biol. Chem.. 131, 621 (1939).
5. LUND, A., Acfa PharmacoZ. Toxicol. 6, 231 (1949).
6. WEIL-MALHERBE,
H., AND BONE, A. D., Lancet 264, 974 (1953).
7. BOWMAN, R. L., CAULFIELD, P. A., AND UDENFRIEND, S., Science 122,32 (1955).
8. UDENFRIEND, S., CLARK, C. T., AND WEISSBACH, H., J. Biol. Chem. 216, 337
(1955).
14
DTJGGAN,
BOWMAN,
BRODIE
AND
UDENFRIEND
9. DUGGAN, D. E., AND UDENFRIEND, S., J. Biol. Chem. 22s. 313 (1956).
10. UDENFRIEND, S., WEISSBACH, H., AND BOGDANSKI, D. F., Science 122, 972
(1955).
11. BOQDANSKI, D. F., PLET~CHER, A., BRODIE, B. B., AND UDENFRIEND, S., J.
Phurmucol. Exptl. Therap. 117, 82 (1956).
12. UDENFEIEND, S., DUQGAN, D. E., VASTA, B., AND BRODIE, B. B., J.
Phwmad.
Exptl. Therup. (ii press).