ON EXCITATION OF BIOLOGICAL SURSTANCh'P

BY RICHARD H. STEELE/' AND ALBERT SZENT-GY~RGYI

INSTITUTE FOR MUSCLE RESEARCH, MARINE BIOLOGICAL LABORATORY,
            WOODS HOLE, MASSACHUSETTS

Communicated April Id, 1967

It has been noted* that watery solutions of fluorescent dyes profoundly alter their
optical behavior on freezing: their fluorescent emission disappears. On further
lowering of the temperature, light emission reappears, but the emitted light differs
from the earlier fluorescent light in having a longer wave length and coming from an
excitation having a longer lifetime, answering, thus, to the formal definition of
"phosphorescence."  This change in properties is not limited to dyes. Certain
proteins, amino acids, coenzymes, and steroid hormones, for instance, show anal-
ogous behavior.

With regard to its possible biological implications, this phenomenon deserves
further study.  Two questions suggest themselves: (1) Does the phosphorescent
light originate from a triplet excitation or from a crystal phosphor?  (2) Is this
behavior a universal property of fluorescent substances?  The experiments reported
in this paper suggest answers to these questions.
There are several criteria which can be utilized in studies of luminescence to
characterize an emission as arising from a metastable triplet level.

1. Triplet emission is shifted toward the longer wave lengths relative to the
  fluorescent emission.
2.  Triplet emissions have a much longer lifetime than singlet emissions.


478         BIOCHEMISTRY: STEELE AND SZENT-GYijRGYI    PROC. N. A. S.

3.  Triplet-state emissions obey first-order decay kinetics.
4. Triplet-state emissions are usually only slightly temperature-dependent, i.e.,
activation energy is negligible.
5.  The molecule in the triplet state, having an unpaired electron spin, is paramag-
  netic.
G.  Triplet emissions induced by polarized exciting light are slightly polarized or
nonpolarized.
7.  The molecule in the triplet state, being a chemical species in its own right with
its own set of properties, will exhibit, its own characteristic absorption spectrum.

For the work reported in this paper we have used Criteria l-4 to aid in character-
izing the electronic states involved.

METHODS

CRITERION 1:  EMISSION SPECTRil

       A. VlSUAL

A knowledge of the fluorescent spectrum for a particular compound permits one
to observe visually whether a subsequent delayed emission has undergone a wave-
length shift.  For example, tyrosine fluorescence resides exclusively in the ultra-
violet, but when the compound is frozen in the presence of glucose, it exhibits, upon
proper excitation, a pale blue afterglow,  We recognize this shift of the emission
toward longer wave lengths as presumptive evidence for the occurrence in the
molecule of an electronic transition different from that responsible for the ultra-
violet fluorescence.  In this study most of our observations of wave-length shift
have been visual.

B. INSTRUMENTAL

1.  Beckman DK-1 Spectrophotometer.-We have occasionally measured fluores-
cent and phosphorescent spectra with the DK-1 spectrophotometer by a technique
described by Gemmill. 2  Spectra obtained by this method are subject to consider-
able error unless the spectral response of the prism-photomultiplier assembly has
been calibrated against a standard lamp.

2. Leiss Monochromator-Red-sensitive Phototube Assembly.- Emission spectra
have been obtained with a Leiss fused quartz monochromator coupled with a Du-
Mont red-sensitive photomultiplier tube, type K 1292, sensitive to 1.3 p. The
photomultiplier signal was piped into a Beckman Model V micromicroammeter.
which was reversed to take a negative input signal.  Spectral emission intensity was
measured as a linear response of the ammeter-galvanometer deflections.  The
monochromator-photomultiplier-ammeter assembly was calibrated for spectral
distribution intensity in relative units against a standard lamp calibrated for us by
the National Bureau of Standards.  Monochromator slit widths were 0.5 mm.
Samples under study were contained in flat-faced Pyrex cuvettes of 4-mm. inside
width and were frozen in liquid nitrogen contained in an unsilvered Pyrex Dewar
flask.  The frozen system was illuminated from one side .with a Hanovia Compact
xenon arc, and the emission from the opposite side was introduced into the entrance
slit of the kiss monochromator.  Judicious use of liquid and glass filters permitted
proper separation of exciting and emitted light.


VOL. 43, 1957   BIOCHlZMISTRY: STEELE AND XZENT-GYORGYI          479

CRITERION 2 : LIFETIMES

Since fluorescent lifetimes are of the order of 10es second in duration, any emission
which is apparent after removal of the exciting source, especially when accompanied
by a shift toward longer wave lengths relative to the fluorescence emission, is in-
dicative of a metastable-state involvement.  Increased lifetimes are obvious visually
and may be quantitated from kinetic studies.

CRITERION 3: DECAY KINETICS

The Beckman DK-1 automatic recording spectrophotometer was used for kinetic
studies.  With the instrument set to record energy, the wave-length drum was set
to an appropriate wave length, and the slits were set to maximum width, namely,
2.0 mm.  Aqueous solutions of the systems under study were placed in 25 X ZOO-
mm. Pyrex test tubes and immersed initially in a methanol-dry ice bath for prior
freezing.  Systems studied at 194" K. were permitted to remain in this bath for
a minimum of 15 minutes to attain temperature equilibrium.  Samples to be studied
at 77" K., following freezing in the dry-ice bath, were then transferred to Dewar
vacuum flasks containing liquid nitrogen.  Samples were allowed to remain here
for a minimum of 15 minutes to insure temperature equilibration, following which
they were placed for 5 seconds in the unfiltered beam of a Hanovia high-pressure
mercury lamp.  The glowing sample was then immediately transferred into the
lamp-housing compartment of the DK-1 and positioned at the light-entrance port.
Following an immediate deflection response of the potentiometer, the magnitude of
which depended on the intensity of the afterglow, the decay was tracked auto-
matically, with the recorder chart set to roll at 12 inches per minute.  Energy
recorded in this manner on the DK-1 was read off the graph in relative linear units.
The curves so obtained were examined for fit to kinetic equations.

In making kinetic studies with the DK-1, it should be noted that the instrument
itself has a finite decay time.  For example, in recording on energy with a light
source inducing a 100 per cent energy reading on the potentiometer, shutting off
the light causes the potentiometer to "decay" to 0 per cent energy.  By running
the chart at 12 inches per minute, we were able to obtain an instrument "decay
curve."  For our instrument this "decay curve" was found to fit a first-order kinetic
equation and had a rate constant of approximately 1.  Kinetic data obtained with
the DK-1, therefore, represent mixtures of two rate constants, that of the instru-
ment and that of the sample being studied.  We express our thanks to Dr. William
Arnold for deriving an equation for us which permitted the two rate constants to
be resolved from mixed kinetic data.  Using this equation we have checked the
accuracy of our experimentally determined rate constants.  The reported rate con-
stants are accurate to within t 5 per cent; most within f_ 2 per cent usually on
the low side.

In a communication from the Beckman Instrument Company concerning the use
of the DK-1 spectrophotometer for rapid kinetic studies they pointed out that one
should be able to determine the time constant (reciprocal of the rate constant) for
a phosphorescent decay if it is not less than 10 per cent of the time constant of the
DK-1.  In our studies no phosphorescences were observed with rate constants
exceeding that of our -DK-1.


480         BIOCHEMISTRY: STEELE AND SZENT-GYORGYI    PROC. N. A. S.

It should be emphasized that in the text figures recording our kinetic results, the
ordinates represent phosphorescent emission intensities in relative energy units.
No significance is to be attached to the relative vertical positions of the curves on
the logarithmic scale.  The positions were selected for a.rbitrary convenience in
plotting the data.

A statement accompanying the figures to the effect that the emission for a par-
ticular rate study was unfiltered means that the RCA lP28 photomultiplier tube on
the DK-1 was exposed directly (the prism system was not traversed) to the glowing
sample under study.  This technique was possible only at the temperature of dry
ice, for at lower temperatures nonspecific emissions interfered.  The technique also
required extra precautions to exclude outside light from the photomultiplier tube.

CRITERION 4 : ACTIVATION ENERGY

Activation energies were measured in the usual way, i.e., from the slope obtained
by plotting the log of the rate constant against the reciprocal of the absolute tem-
perature. The temperatures used were those of dry ice (194" K.) and liquid
nitrogen (77" K.).

RESULTS

We have obtained and studied delayed emissions for the following compounds
(source given) : bullock lens, intact and homogenized; tryptophan and tyrosine,
H & M Chemical Company; adenine HCl and adenosine, Schwarz Laboratories,
Inc. ; adenylic acid (muscle), adenosine diphosphate, and adenosine triphosphate,
Pabst; desoxynucleic acid (sperm) and ribonucleic acid, Nutritional Biochemical
Corporation; progesterone and alpha-estradiol, Ciba; quinine sulfate, Merck;
quinidine sulfate, Fisher; acridine orange and acridine yellow, Nat'ional Aniline
Division, Allied Chemical and Dye Corporation.  We will now consider the result,s
for the individual materials separately.

                       1.   BULLOCK LENS
For the study of the optical properties of structural proteins, the lens, being

devoid of pigments, offers a unique material.3  Lenses taken from different animals
-bullock, rabbit, and various fishes-show a brilliant fluorescence when illuminated
with ultraviolet light.  When fresh bullock lenses were examined for fluorescence in
the dark at room temperature, an intense blue emission was observed which had a
lifetime shorter than low3 second, since no light was observed in our phosphoroscope
which had this resolution time.  If the lenses were permitted to attain tempera-
ture equilibrium at 194" K. (dry-ice temperature), an even more intense emission
was exhibited, which persisted, with progressive fading, for about 20 seconds after
the exciting light was removed.  Such a long-lived afterglow and the shift from
fluorescence to phosphorescence on cooling are characteristics of compounds which
can undergo singlet-triplet intercombinations.  To resolve the question whether
the observed phosphorescence was emitted from a triplet state or by a crystal phos-
phor, we examined the decay kinetics and the activation energy of the emission.
Emissions of light by crystal phosphors involve activation energies and are thus
strongly temperature-dependent, while the emission decays from triplet excitation
states require no activation energy and are temperature-independent.  In addition,


VOII. 43, 1957   BIOCHEMISTRY: STEELE AND SZENT-GYORGYI          481

the first-order decay kinetics characteristic of triplet emissions is not to be expected
with crystal phosphor emissions.

The temperature dependence of the emission lifetime for bullock lens was first
qualitatively studied in the following manner.  Two Dewar vacuum flasks were
placed side by side, one containing dry-ice freezing mixture, the other liquid nitro-
gen.  A test tube holding the bullock lenses was immersed in each.  After tempera-
ture equilibrium was established, the two test tubes were illuminated side by side
for 2-3 seconds with ultraviolet light and then returned to their respective Dewar
flasks, and the gradual disappearance of their emissions was observed.  The after-
glow faded out in both tubes at about the
same time, although the temperature dif-
ference between the two systems exceeded
110" C. This temperature-independent
lifetime strongly pleads for the assumption
that the observed phosphorescence comes
from a triplet excitation state.

These qualitative  observations were
quantitatively corroborated from kinetic
studies carried out as described under the
technique for Criterion 3, decay kinetics.
Figure 1 depicts the results. It is to be
noted first that the curves fit a first-order
kinetic equation, i.e., the decay rate is a
function only of the concentration of the
residual  metastable,  triplet,  species.
Second, since the decay-rate constant is
temperature-independent, the activation
energy is negligible, and we have further
evidence for triplet-state involvement.

The structural protein of the lens forms
a fairly solid gel in vivo and can thus be   sec. 0       5       IO      15

expected to be fixed in space.  This ma-
trix should also impede the diffusion of   FIG. l.--Phosphorescent decay kinetic
                         data for lens systems.  Ic = rate constant.  I,
dissolved protein molecules.  This assump-  intact ox lens, 7' = 194" K., emissions unfil-
tion must hold even more strongly for a  tered; 2, intact ox lens, T = 77" K.., emis-
frozen system.  The change from fluores-  sion measured at 420 p,; S, homogenized ox
                       lens (diluted 1 to 30 with water), T = 77"

cence to phosphorescence with cooling can  K., emission measured at 420 ml*.

thus hardly be ascribed to dimerization or to higher polymerization of protein
molecules but rather must be attributed to a change in the solvent and its relation
to the protein.

If the lenses are minced in a Latapie mincer and suspended in 30 volumes of water
(our lens homogenate), the suspension shows no striking fluorescence and no phos-
phorescence at 194" K.  The same is true for less dilute suspensions.  If, however,
the suspensions are cooled further to 77" K., they emit a phosphorescence with an
afterglow duration indistinguishable from that of the intact lens,
It was noted earlier that the addition of glucose to aqueous solutions of fluorescent
substances in many instances enhances the phosphorescence of the frozen state.


especially at 194" K.  This effect was ascribed -to a more intimate contact of the
luminescent species with water, glucose disfavoring a sharp separatior~ of wat,er
crystals from the solute,  This effect was observed also with the lens suspension.
If the suspension contained 0.5 per cent glucose, freezing to 194" K., with subse-
quent illumination, caused the appearance of a strong blue emission resembling
that of the intact lens and persisting for about half a minute after removal of the
illu~nation. This afterglow was fairly independent of glucose concentration,
being equally strong at 0.25 per cent and at 5 per cent.  The light intensities were,
under the conditions of our experiment, also independent, within wide limits, of the
concentration of the suspension, the afterglow being about equally strong whether 1
part of lens was suspended in 30 or in 300 parts of water.
The fact that the materia.1 of the lens loses its fluorescence and phosphorescence
by being minced and suspended in water indicates that t'he proteins in the native
state of the lens are kept in a specific condition which favors these light emissions
and the underlying excitations.  This condition in all probability involves some
specific relation to the solvent water, which relation can be restored by the addition
of glucose.
The phosphorescence of the frozen (194" K.) lens suspension was strongly
quenched if, in addition to the presence of 0.5 per cent glucose, the solution was
made to 3 per cent (v/v> with glycerol or 0.25 per cent (v/v) with methanol prior
to freezing.  Acetone was found to be inactive even at a concentration of 8 volumes
per cent., The phosphoresceI~ce was also insensitive to the action of oxygen.
Equally inactive were classes of "quenchers" such as KI ( 10V3 ii/l), NaSCN ( 10W3 M>,
2,4-dinitrophenol (lo+ H), and 2,4-dinitronaphthol (5 X 1O-G LV).
Figure 1 also gives the decay curve for homogenized lens phosphorescence at 77"
K., which was no different at 194" El;.  In addition, its should be noted that at> 77" K.
the decay-rate constant for homogenized lens phosphorescence was the same with-
out as with glucose.
The question arises whether the phosphorescence of the lens is due t,o some super-
structure or to smaller units like amino acids making up the molecule.  The paper of
Debye and Edwards4 appears to be the first systematic attempt to examine protein
phosphorescence.  They observed that most proteins emitted a brilliant, blue phos-
phorescence at liquid-nitrogen temperature.  The visible spectrum was found tjo be
pII-dependent, the protein phosphorescence from an alkaline medium exhibiting a
minor maximum at 418 rnp and an intense maxin~um at 440 mp.  In an acid medium
the results were less consistent, the main maximum appearing at 417 mp.  The
phosphorescer~t spectra of tyrosine and tryptophan `were found to lie in the same
spectral region as the protein phosphorescence.  Debye and Edwards found t.he
amino acid phosphorescent, emission decays to be exponential and suggested that the
exponential decay phosphorescence of proteins was due to their content of tyrosine,
tryptophan, and possibly phenylalanine.  From their finding that, the phosphores-
cent decay was temperature-independent, they felt, that an electronic transition was
involved.  Grossweiner, using flash photolysis techniques, has complemented this
work of Debye and Edwards and has presented spectra for the metastable states of
ovalbumin, tyrosine, phenol, tryptophan, and indole. The wave-length shift,
temperature independence, and metastable absorption spectra obtained by these
workers suggested triplet-state invol~~e~nent.  Grossweiner further concluded that3


VOL. 43, 1957    BIOCHEMIiWRY: STEELE AND SZENT-GYORGYI           483

the metastable absorption spectrum for ovalbumin principally represented the sum
of contributions from tyrosine and tryptophan, with negligible effects from the
peptide linkages.

The lens phosphorescence which we have observed (see Fig. 1) appears to have a
slightly longer mean lifetime (5 seconds) than the protein phosphorescence studied
by Debye and Edwards (approximately 3 seconds).  In addition, we have observed
slight differences in the kinetic data for amino acid phosphorescences when com-
pared with the data of Debye and Edwards, but these are probably differences in
degree only and not in kind arising from differences in technique.

                           2.   AMINO ACIDS
The following amino acids were tested: dl-alanine, Z-glutamic acid, I-histidine,
dl-phenylalanine, I-tyrosine, and dl-tryptophan.  None of these in aqueous solution
showed any striking luminescence on visual observation under the ultraviolet lamp,
frozen or unfrozen.  Of these amino acids it might be anticipated from absorption
spectral data that tryptophan and possibly tyrosint: would be the only compounds
which could possibly emit a visible fluorescence.  This fact becomes apparent when
one examines the fluorescent spectral data for the aromatic amino acids reported in
the recent publications of Bowman, Caulfield, and Udenfriend,6 Sprince, Rowley,
and Jameson and Teale and Weber.8  Phenylalanine and tyrosine emissions lie
exclusively in the ultraviolet, while tryptophan emission, with a maximum in the
near-ultraviolet, has a long-wave-length tail which "spills" over into the visible
out to about 440 rnp.  For this reason, therefore, a tryptophan solution of proper
dilution, upon exposure to an unfiltered ultraviolet lamp, emits a distinct, though
weak, violet fluorescence easily observable with the unaided eye.  The failure to
observe any appreciable fluorescence in a 0.01 M tryptophan solution, while a solu-
tion of 10M4 M emits a distinct violet light, indicates that the negative results at
higher concentration may be due to self-quenching.

This emission picture changed markedly for the aromatic acids when the solutions
were made to 0.5 per cent in glucose prior to freezing.  Tyrosine and tryptophan in
10e3 molar concentration gave short and prolonged afterglows,. respectively.  These
emissions were of considerable intensity.  The behavior of tryptophan appeared
similar to that of the lens, being blue and lasting for about half a minute.  The
wave-length shifts for these two compounds in going from fluorescence to phos-
phorescence were visually apparent, the colorless emission of tyrosine shifting to blue
and the violet emission of tryptophan shifting to blue.

The marked enhancement of phosphorescence in t'he frozen state displayed by
many compounds in the presence of glucose (other sugars were equally as effective)
motivated us to examine this effect in more detail.  Studies were made with trypto-
phan as the luminous species.

It was found that the phosphorescent decay-rate constant for tryptophan-glucose
systems in ice was practically independent of temperature9 and tryptophan concen-
tration.  In addition, we have observed an influence of glucose concentration on the
phosphorescent emission intensity.  When the tryptophan concentration was held
constant and the glucose concentration varied, the emission intensity was observed
to pass through a maximum, while the decay-rate constant remained essentially
unchanged.  A preliminary examination of this effect made with a tryptophan con-


484        UIOCHEMISTRY: S1'EELE AND SZENT-GYiiRGYZ   PR0C.N. A.S.

centration of 5 X 10m4 molar resulted in a maximum phosphorescent intensity at a
glucose concentration of 0.27 per cent.  Table 1 summarizes the results for the
slight in~uence of temperature and tryptophan coll~entration on the phosphorescent
decay kinetics.

Substitution of the rate data from Table 1 for the IOU3 molar tryptophan system
into the equation,

gave an activation energy of only 76 calories.  In this equation 1~~ and B1 are the
rate constants at the high (7'2) and low (T,) temperatures, respectively; AH, is the
activation energy; and R is the gas constant.

Figure 2 illustrates the influence of temperature on the phosphorescent decay-
rat,e constant for a lo-" molar tryptophan con~er~tratior~ system containing 1 per
cent glucose.  It should be observed that the tryptophan phosphorescence was in-
sensitive to oxygen.

TABLE I

THE INFLUBNCE OF TEMPERATURE AND TRYPTOPHAN CONCENTRATION ON THE DECAY-
          CONSTANTFORTRY~OPHAN~~~OSP~OR~SCENCE*

    TECYPKXPHAN                                 TMWWPHAX
   CONCENTRATION   ~~4~T~~coNsl~~~~,~-.        CONCXNTRATION     --ltATI$ COiSSTAX'T k-
  (ii'$OLES PER LITER)   0.40'   0.39'      (MOLES PER hTER)      194O K.   7j" K.
                                    JO-5         0.26     0.17
                 0.27     0.20          IO-         0.24     0.17
    10-4           0.21     0.18
* The glucose concentration was held constant at 0.25 per cent.  Rate data were obtained with the spectro-
photometer set at 420 mp and with 2.0~mm. slit widths.  Systems were contained in 25 X ZOO-mm. Pyrex test tubes
and, following a minimum of 15 minutes at the proper temperature to allow temperature equilibration, were illu-
minated with the unfiltered k&am of a high-pressure Hanovia mercury arc for 5 seconds.

Figure 3 contains the phosphorescent decay kinetic data of the tyrosine-glucose
system. The intensity of the emitted light in the absence of glucose was too low
to permit its measurement by the t)echnique used in this study.  At `194" K., even
in the presence of glucose, the intensity of the emitted light was so low that it was
difficult to obtain a satisfactory decay curve, and the reported rate constant should
be considered accurate within ? 5 per cent.  We have made no effort to obtain
kinetic data for phenylalanine.

Wave-length shifts, long-lived afterglows, first-order decay kinetics, and negligible
temperature dependence of the rate constants may be presented as presumptive
evidence that tyrosine and tryptophan afterglows are emitted from a metastable
triplet state.

3.  An~NIN~, AD~NOSI~~, ADI~NOSI~II: PIIOSPHhTE, ADENOSINE DII'HOSI'IIATE, Axl>
          ADENOSXNE TRIPHOSPHATE (ATI')

Euler, Brand& and Neumiillerlo and Stimson and neuter" presented fluorescent
data for several purines and pyrimidines and their derivatives and gave the color
of their emissions as a function of pHl,  Since we have been interested in t'he energy
~on~guration of ATP in relation to its possible fullc,tional par~.icipation in the energy
transformations in which the molecule participates, we have limited our study to
the purine adenine and its derivatives,


VOL. 43, 1957   BIOCHEMISTRY: STEELE AND SZENT-GYdRGYl          485

`t----J
sec. 0       5      IO      15

Fro. 2.-Influence of temperature on
the phosphorescent decay rate constant, k,
for tryptophan-glucose system; tryptophan
= low4 M, glucose = 1 per cent.  1, system
approaching temperature equilibrium at 194"
K.; 8, system at temperature equilibrium
at 194" K.; S, system at temperature equilib-
rium at 77" K., emission measured at 420 rnp.

sec. 0       5       IO      15
FIG. 3.-Phosphorescent decay kinetic
data for tyrosine-glucose systems. k =
rate constant.  l, lO-3 M tyrosine + 0.5 per
cent glucose, T = 194' K., emission unfil-
tered; 2, 4.4 X lo-* M tyrohne + 0.3 per
cent glucose, T = 77" K., emission measured
at 420 me.

In view of the fact that the absorption bands of the adenine compounds lie rather
deep in the ultraviolet, it might be anticipated that only slight or no visible fluores-
cence would be observable from them upon proper excitation.  Stimson and Reuterll
reported a faint green fluorescence for adenine sulfate in strong alkali and a faint
blue emission in weak alkali.  They observed no emission for adenylic acid solutions
at any pH.  In our work we have never observed a visible emission from adenine,
adenosine, or any of the phosphate derivatives at any pH at room temperature.
Adenine compounds of 0.01-0.0005 M concentrations, when frozen to 194" K., dis-
played a very faint blue short-lived afterglow,  This -emission could usually be
enhanced in intensity by the addition of glucose to 0.5 per cent prior to freezing.
ATP, for example, with glucose added, displayed an intense afterglow lasting for
about 15 seconds after the exciting light was removed.  ATP, in contrast to trypto-
phan, displayed no self-quenching in 0.01 M concentration.  This may have bio-
logical significance, especially in muscle, where ATP exists in rather high concen-
tration.  While the ATP phosphorescence was insensitive to KI, NaSCN, 2,4-
dinitrophenol, 2,4-dinitronaphthol, and acetone (8 per cent v/v) it was abolished
by 5 per cent (v/v) glycerol and 0.1 per cent (v/v) methanol at 194' K.  These later
quenching effects were not observed, however, at 77" K.


486        BIOCHEMISTRY: STEELE AND SZBNT-GYORGYI   l'RW. N. A. 8.

Figure 4 summarizes the phosphorescent decay kinetics for all the adenine sys-
tems studied.  Kinetic data at 194" K. were obtained for ATP only and indicated

sec. 0

5           IO

FIG. 4.-Phosphorescent decay kinetic
data for adenine (I), adenosine (Z?!,
adenosine-5'-phosphate (S), adenosine-5'-dl-
phosphate (Q), and adenosine-5'-triphos-
phate (5). All concentrations 0.001 M;
pH's adjusted to 5 with acetate buffer prior
to freezing.  1' = 77" K. All emissions
measured at 420 mp.  k = rate constant.

that the rate constant was practically in-
dependent of temperature. Though the
emission intensities were found to be mark-
edly sensitive to pH, the rate constant,s
remained essent!ially independent of this
variable. An important feature observed
in studies of pH effects on the emission in-
tensities was the finding that, t'he amino
nitrogen at position 6 on Ohe adenine ring
must, be dissociated for luminescence to

occur.  Varying the pH of adenine-com-
pound systems from 2 to 7 by units of 1 gave
a progressive increase in the phosphores-
cent intensity from nil to brilliant.  Since,
according to the work of Alberty, Smith,
and Bock,12 the amino group has a pK, of
approximately 4, we visualize the increased
alkalinity as dissociating the "adenin-
ium" cation.  This dependence of luminous
intensity on the degree of dissociation
of an a.mino group is not a unique find-
ing.  Ley and von Engelhardt13 noted that,
whereas the aniline molecule was fluores-
cent, the anilinium cation was nonfluores-
cent.  By contrast, Ley and Grife14 and
Dickson15 found that t'he blue fluores-
cence of alpha- and beta-naphthylamine
was not lost by proton addit'ion but,
instead, was shifted into the ult.raviolet,
with, however, a considerable diminution
in intensity, Ilewis and Kasha"j have
given phosphorescent emission data for ani-
                                _ _

line and alpha- and beta-naphthylamine, but we have found no reports on the phos-
phorescence of the anilinium cation or of the alpha- or beta-naphthylammonium
cations.

For the adenine derivatives studied, therefore, the wave-length shifts, i.e., from
no visible emission to an intense blue afterglow, the long lifetimes, and the first-
order kinetics plead for triplet-state participation in the emissions ohserved for
these compounds.

                            4.  NUCLEIC ACIrE
Nucleic acids show a long-lived phosphorescence which might be due to t,he col-
lective action of the column of cyclic structures constituting, according to the model
of Watson and Crick," the core of the molecule.  The central cyclic compounds
would thus, according to this assumption, be analogous to Scheibe's pseudoiso-
cyanine dyes, l* which polymerize to columns in the shape of "money-rolls" through
which excitons are freely propagated by resonance.


VOL. 43, 1957   RIOCHE:MIS1'RY: S2'EELh' AND SZENT-GYtiRGYI          487

The phosphorescent light emission from aqueous solutions of DNA and RNA
preparations, 0.1-1.0 per cent, frozen to 194" K., could be prolonged by the ad-
dition of 0.25-4 per cent glucose.  This prolongation was abolished by small con-
centrations of glycerol (3 per cent, v/v) or methanol (0.25 per cent, v/v), while
acetone (16 per cent, v/v) was found to be inactive.  The phosphorescence was
found to be insensitive to KI, 2,4-dinitrophenol, 2,4-dinitronaphthol, salicylate
(0.002 &Q, or pyrrolazote (2 X 10M4 n/r).  Morphia and aconit,ine (10V3 M) were
equally inactive, while colchicine in 2 X 10M4 M concentration quenched DNA
strongly, the effect increasing somewhat on storage.  Since the nucleic acids were
present in rather high concentration, 0.5--l per cent, there was a disproportionality
between the molarity of the alkaloid and the mass of t'he nucleic acids or the number
of nucleotides building the nucleic acid molecule.  This pleads for a collective
action of the nucleotides in the production of phosphorescence.  ScheibelQ found
the energy transmission in his dye polymers decreased by relatively small concen-
trations of hydroquinone, the conduction within the "money-rolls" being cut by
the interposition of a "dud," a hydroquinone molecule. Hydroquinone at
0.001 M also strongly quenched the phosphorescence of DNA. The first pre-
liminary experiments repeated with high-polymer DNA indicated that the quench-
ing action of colchicine was about ten times stronger with this material t,han with
the low-polymer DNA, the alkaloid being active in 10e5 LV concentration.  This
concentration is near the level which is effective in vivo.  If the alkaloid acts by
cutting the energy transmission through the row of central bases, then it should be
more active the longer the molecule.

The carcinogen benzpyrene also showed a fairly st,rong quenching activity.  In
these experiments the carcinogen was dissolved in acetone (0.1 per cent). One-
tenth of a milliliter of this solution added to 5 ml. of 0.5 per cent DNA almost
completely eliminated the long afterglow enhanced by glucose.  In this instance,
too, the action seemed to be more marked when high-polymer DNA was used in the
experiment.  Benzpyrene, as is generally known from the work of Brock, Druckrey,
and Hamper& 2o emits a blue light when molecularly dispersed and a yellow-green
light in colloidal emulsion when illuminated by ultraviolet light. This can be
strikingly demonstrated by adding a drop of acetone solution of henzpyrene to
water.  The resulting colloidal solution emits a yellow-green fluorescence which
changes dramatically to blue if the solution is shaken up with a little solid caffein.
The purine forms a complex with the carcinogen, bringing it into molecular dis-
persion.  Analogous behavior can be demonstrated with DNA, indicating that the
carcinogen complexes with the bases of the nucleic acids.  This might have been
anticipated also from the work of Booth and Boyland, who demonstrated that
the variably carcinogenic bicyclic dibenzacridines formed well-defined crystalline
complexes with purines and nucleic acids.

Figure 5 depicts the phosphorescent decay kinetic results as measured for DNA
and RNA at 77" K. Decay kinetics are not reported for these compounds at
194"K., for the emission intensities were too low to be recorded by the technique
here employed.  The rate constants given should be considered only as indicating
the order of magnitude of the decay rate, for the purity of these preparations was
uncertain.  The rate constant for DNA appears to be complex, and until highly
purified preparations of reasonably well-known polymer composition are avail-


488

BIOCHEMISTRY: STEELE AND SZh'NT-GYijRGYI   PROC. N. A. S.

      A
sec. 0           5
FIG. S.-Phosphorescent decay kinetic
data for desoxyribonucleic acid and ribonu-
cleic acid.  T = 77" K.  k = rate constant.
I, 1 per cent DNA + 0.25 per cent glucose,
480 p; 2, 1 per cent DNA, no glucose, 520
rnp; S, 1 per cent RNA + 0.25 per cent glu-
cose, 400 rnp.

secu

FIG. 6.-Phosphorescent decay kinetic
data. 1, quinidine-SO+ IOF M aqueous
solution + 0.25 per cent glucose, green
emission measured at 510 rnp 7' = 77' K.;
2, progesterone, 7.3 X 10e4 ~yi in 95 per cent
ethanol + 0.5 per cent glucose, blue emis-
sion measured at 420 rnp,. T = 77" K.; S,
alpha-estradiol, lOpa M m 95 per cent cth-
anol, no glucose, violet emission measured
at 410 rnfi, I' = 77' K. k = rate constant.

able, we will restrict our comments to one observation, namely, that while a 0.1 per
cent DNA solution (glucose present) displayed a blue phosphorescence, a 1 per cent
DNA solution (also in the presence of glucose) gave a green phosphorescence.  We
have noticed a somewhat analogous phenomenon with adenosine.  Whereas ade-
nosine in the absence of glucose emitted a distinct green phosphorescence, in the
presence of glucose the emission reverted to the blue characteristic for adenine and
the adenosine phosphate derivatives.  The effect was most marked at low pH val-
ues, and none of the other adenine compounds showed the effect.  Therefore, the
difference observed in the rate constant for DNA may reflect a mixture of these two
emissions.  The data predict that the green emitting system, if real, will be found
to have the higher rate constant.  No such wave-length shifts have been observed
for RNA.


VOL. 43, 1957   BIOCHEMISTRY: STEELE AND SZENT-GYiiRGYI           489

First-order kinetics and increased lifetimes are the only criteria we have obtained
so far for the nucleic acids which suggest triplet-state involvement.

5. MISCELLANEOTJS

Figure 6 contains rate data for quinidine sulfate, progesterone, and alpha-
estradiol phosphorescences, all measured at 77" K. and at the wave lengths given.
Quinine sulfate gave practically the same rate constant as quinidine sulfate, and
glucose addition was necessary for both compounds in order to obtain sufficient
luminous intensity to permit decay curves to be run.  The emission was a distinct
green in both instances and of rather intense brightness, though of very short du-
ration.  The blue emission of progesterone and the violet emission of alpha-estradiol,
though of low intensities, could both be measured without glucose addition; in fact,
glucose addition had little influence on their emission intensities. It should be
noted that the solvent for the hormones was 95 per cent ethanol, not water.  The
very low emission intensities at 194' prevented rate-constant, measurements by the
technique used in this study.

Quinine and quinidine sulfate fluoresced blue, while the phosphorescence was
green.  We have measured the fluorescence of alpha-estradiol (unpublished) and
found it to be exclusively ultraviolet, with a maximum at approximately 310 D.
We were unable to obtain any indication of progesterone fluorescence.  With these
compounds, therefore, wave-length shift (with the possible exception of proges-
terone), first-order kinetics, and increased lifetimes represent the criteria suggestive
of triplet-state involvement.

6. DYES

In Figure 7 are presented a few preliminary kinetic studies made for the dyes
acridine yellow and acridine orange.  The break in rate curves at the higher dye
concentration and elevated water content suggest that polymer involvement may
influence the rate constant.  Zanker22 has made a detailed study on the influence of
solvent, concentration, temperature, and pH on the absorption and emission char-
acteristics of acridine orange that may prove to be pertinent here.

We have measured the following emission shifts for these two dyes in going from
fluorescence to the phosphorescent state.  Acridine yellow fluoresced at 511 rnp and
phosphoresced with a maximum at 614 mp.  Acridine ora.nge gave a fluorescence
band with a maximum at 530 rnp and a phosphorescence-band maximum at, 660 rnp.
These wave-length shifts, first-order kinetics, and long-lived afterglow, we cite,
therefore, as evidence for triplet-state participation in the phosphorescence of these
dyes.

                     7.   WATER
A finding which we can mention only briefly at this time was an observation con-
cerning the emission from ice following ultraviolet excitation.23  The "afterglow"
was not visible in an undarkened room, and we have made no attempt to visualize
it with dark-adapted eyes.  The intensity appeared greatest when the emission
from ice at 77" K. was permitted to impinge unfiltered directly onto the RCA
lP28 photomultiplier tube set electronically at maximum sensitivity. Precau-
tions were always taken to insure that this ice emission did not interfere with the


490       BlOCHEMIS1'RY: S1'EELE AND S%ENT-GYORGYI   PROC. N. A. S.

a-
6-

4-
3-

2-

sec. 0       -5            IO

_ FIG. 7.FPhosphorescent decay kinetic
data for the dyes acridine yellow and acri-
. .           -    --^ -- .

    I
sec. 0

5       IO      15      1

dine orange.  II' = 77" K. lc = rate con-
stant.  I, 10m4 IM acridine yellow in 90 per
cent ethanol + 10 per cent water (v/v), emis-
sion measured at 550 rnp; d-S, 5 X 1O-4 il!l
acridine yellow in 50 per cent ethanol + 50
per cent water (v/v), emission measured at
530 rnp; 4-5,5 X 10W4 M acridine orange in
50 per cent ethanol + 50 per cent water
(v/v), emission measured at 600 m/l.

FIG. S----Phosphorescent decay kinetics
for the unfiltered afterglow from ice at 77"
and 194" K.  The ice was frozen around the
outside of a hollow aluminum bar (freezing
mixtures inside) and exposed for 5 seconds
to the unfiltered beam of a high-pressure
mercury lamp prior to the kinetic run.  k=
rate constant. I = 77" K., 2 = 194' K.

kinetic measurements.  Rate data for this emission at 77" and 194' K. are given
in Figure 8.  These results were fairly reproducible.  It is interesting that the de-
cay appears to obey first-order kinetics and has a rather low activation energy.
Unfortunately, the intensities were too low to permit us to obtain reliable informa-
tion about their spectral distribution.  The rate constant, however, appeared to
be about the same whether the emission was permitted to traverse a Corning 5860
band-pass filter (maxinum wave length passed at 360 mp) or a Corning 3486 cutoff
filter, cutting out light below 510 mp.

DISCUSSION AND STJMMARY

Fluorescent and phosphorescent spectral data, kinetic studies (including order of
reaction), lifetimes, and activation-energy considerations have been present'ed for
a wide variety of biological compounds and the dyes acridine yellow and aeridine
orange.  Results were obtained which we submit as evidence that electronic transi-
tions involving singlet-triplet intercombinations are functional in these compounds.
The marked optical changes observed in freezing aqueous solutions, namely, the
shift from fluorescence to phosphorescence, appear to be a universal property of
fluorescent materials.

* This work was supported in part by a grant from the Commonwealth Fund, the National
Heart Institute (H-2042 R), the Muscular Dystrophy Associations of America, Inc., the National
Science Foundation, the American Heart Association, the Association for the Aid of Crippled
Children, and the United Cerebral Palsy Foundation.


VOL. 43, 1957          BIOCHEMISTRY: H. J. VOGEL                   49 1

t This author, as a visiting investigator at the Institute for Muscle Research, was aided in part
by a grant from the Muscular Dystrophy Associations of America, Inc., and by a grant from the
National Institutes of Health (RG-4643).

1 Albert Scent-Gyiirgyi, Science, X24,873-575, 1956.
2 Chalmers L. Gemmill, Anal. Chem., 28,1061-1064,1956.
3 Albert Szent-GyGrgyi, Biochim. et Biophys. Ada., 16, 167, 1955.
4 P. Debye and J. 0. Edwards, Science, 116,143-144,1952.
5 L. I. Grossweiner, J. Chem. Phys., 24, 1255-1256, 1956.
6 Robert L. Bowman, Patricia A. Caulfield, and Sidney Udenfriend, Science, 122, 32-33, 1955.
7 Herbert Sprince, George R. Rowley, and Dorothy Jameson, Science, 125, 442-443, 1957.
* F. W. J. Tealc and B. Weber, Biachem. J., 65,476-482,1957.
9 The change in the rate constant never varied by more than a factor of 2 for a change in the
temperature of more than 100" C.  Consequently, the activation energy for the tryptophan-
glucose system may be considered negligible.  It was found, nonetheless, that slight variations in
the rate constant could be controlled when more care was exercised in controlling the temperature
((x.g., insuring that temperature equilibrium was established) and tryptophan concentration.
10 H. Euler, K. M. Brandt, and G. Neumiiller, Biochem. Z., 281, 206-214, 1935.
11 Miriam Michael Stimson and Mary Agnita Reuter, J. Am. Chem. SW., 63,697-699, 1941.
12 Robert A. Alberty, Rex M. Smith, and Robert M. Bock, J. Biol. Chem., 193, 425-434, 1951.
13 II. Ley and K. von Engelhardt, Ber. Deut. them. Ges., 41,2988-2995, 1908.
l4 H. Ley and W. Grtife, 2. wiss. Phot. 8, 294-300, 1910.
15 Ernest Dickson, Z. wiss. Phot., 10, 166-180, 1911.
16 G. N. Lewis and M. Kasha, J. Am. Chem. SW., 66,2100-2116, 1944.
17 J. D. Watson and F. I-1. C. Crick, Nature, 171,737-738, 1953.
18 G. Scheibe, 2. Elelctrochem., 52,283-292, 1948.
19 G. Scheibe, A. Schontag, and F. Katheder, Naturwissenschuften, 2'7,499-501, 1939.
20 N. Brock, H. Druckrey, and H. Hamperl, Arch. Exptl. Pathol. Pharmakal., 189, 709-731, 1938.
21 J. Booth and E. Boyland, Biochim. et Biophys. Ada, 12,75-87, 1953.
22 Valentin Zanker, i. physik. Chemie, 199,225-258, 1952; 200, 250-292, 1952; 8, 20-31, 1956.
23 Leonard I. Grossweiner and Max S. Matheson (J. Chem. Phys., 20, 1654-1655, 1952; 22,
1514-1526, 1954) have reported observing an X-ray-induced fluorescence from ice at low tem-
peratures.  The emission at -183' C. had an almost symmetrical band with a peak at 385 p.
They observed no phosphorescence whatever.  That energy was trapped, however, was made
evident from glow-curve studies following X-ray excitation.  The difference between their ac-
tivation energy (0.25 ev.) and ours (0.02 ev.) indicates that different mechanisms are involved in
"trapping" the energy.  This would be expected, since energy was stored in the `one instance by
X-ray excitation and in the other instance by ultraviolet light.  We have made no study of the
influence of oxygen on the emission, and dissolved oxygen was not excluded prior to freezing.
Grossweiner and Matheson observed a suppression of thermoluminescence by oxygen.