2 November 1956, Volume 124, Number 3227

SCIENCE

The most basic property of the heart is
that it is a muscle, and the chief property
of muscle is that we do not understand
it. The more we know about it, the less
we understand and it looks as if we would
soon know everything and understand
nothing. The situation is similar in most
other biological processes and pathologi-
cal conditions, such as the degenerative
diseases. This suggests that some very
basic information is missing. The story
of myosin may illustrate this point.

Energetics of Myosin

Myosin is the main contractile protein
of muscle. It converts the chemical
energy of adenosine triphosphate (ATP)
into motion. From the work of Edsall,
Weber, and their associates, we knew
that the myosin molecule was a rodlet
(I), as is indicated in Fig. la. We had
little doubt that contraction, essentially,
was a folding, as is symbolized in Fig. lb.
We supposed that the ATP produced this
folding by attaching itself at a point to
myosin and producing there a change
which, sooner or later, could be described
in terms of classical chemistry.

The first disturbance in this happy
state of affairs was caused by the studies
of Gergcly, Perry, and Mihalyi, who
found that myosin has a complex struc-
ture and is built of two kinds of subunits
(2)) which were isolated subsequently by
Andrew G. Szent-Gy6rgyi and called
meromyosins. The one sedimenting faster
was called H (heavy) and the other L
(light). There were twice as many L's as
H's, and they were arranged in series,

Dr. Szent-Gybrgyi is a member of the staff of the
Institute for Muscle Research at the Marine Bio-
losical Laboratory, Woods Hole, Massachusetts.
This article h based on a lecture given at the heart
symposium of the 20th international Physiological
Congress in Brussels OR 31 July 1956. The under-
lying studies will be published in their entirety
by Academic Press in a monograph of the same
title.

2 NOVEMBER 1956

Bioenergetics

Albert Szent-Gy6rgyi

in a row, as is symbolized in Fig. lc.
What was disturbing about this finding
was the fact that the L seemed to be
involved in contraction, while the 1-l
alone interacted with ATP. How could
the energy released on the H support
work done by the L's? How could a
bond-energy locked up in a chemical
link produce work somewhere else? It
was still possible to make thcorics to save
the situation and suppose, for instance,
that ATP produced some local change
on the H, whereupon the L folded back
on the H, but these theories began to
look rather artificial,

The real difficulty arose when A. G.
Szent-Gyorgyi and Borbiro (3) made the
discovery that the meromyosins them-
selves are built of a great number of
much smaller subunits, protomyosins,
which are held together only by second-
ary links, such as H-bonds, van der
Waals forces, and electric attractions. The
myosin "molecule" was thus no molecule
at all, if wc call a molcculc a structure of
atoms held together by covalent links.
The myosin particle was but a regular
heap of still smaller particles, as is sym
bolized in Fig. Id.

Contraction and Electronic Excitation

Secondary links have no fixed valency
angles, which must give a great pliability
to the structure. It bccamc difficult to
see how such a structure could "fold." It
seemed more probable that contraction
consisted of a rearrangement of proto-
myosms, which went into a more com-
pact heap, as is symbolized in Fig. le.
There must be strong attractive force be-
tween protomyosins to enable the myosin
particle to withstand strain, and these
forces must tend to pull the protomyosins
closer together. The force of muscular
contraction could thus be due to these at-
tractive forces, in which case we would
need force to stretch the particle out

again. This theory of contraction is
simple and attractive. The difficulty is
with ATP, which induces contraction
and foots the cncrgy bill. In order to in-
duct a rearrangcmcnt of protomyosins,
an ATP molecule would have to influence
many weak links, but how could a "bond
energy," locked up in a molecule, in-
fluence many links, especially if that
molecule is far away? To bridge this gap,
we would have to suppose that the bond
energy of the ATP molecules is trans-
formed into some more mobile and active
form of energy when it has to go into
biological action. Such an energy, on the
molecular level, could hardly be anything
other than the cncrgy of clcctronic cxci-
tation. Vibrational energies would, prob-
ably, be dissipated too easily. Practically
all molecules are excitable by light of
one wavelength or another, but most of
them immediately dissipate their excita-
tion energy and would thus be unfit to
partake in biological energy transmis-
sions. Only a relatively small number of
specifically built molecules do not dis-
sipate their excitation energy immedi-
ately. But, as a rule, no electron can stay
in the excited state longer than lo-* scc-
ond, and, if the molcculc is unable to
dissipate its excess energy, the electron
will drop back to its ground level in 10-s
second, shooting out its excess energy in
the form of a photon. This means that
the molecule is fluorescent. Fluorescence
thus becomes an indicator of qualities
that may have a major biological im-
portancc, indicating that the molecule
can accept energy without dissipating it.

It could be objected that such excita-
tion energies can play no role in biologi-
cal energy transmissions because 10-s
second is too short a time to allow their
utilization. However, there is always a
small but definite chance that the excited
electron may revert its spin, which would
greatly prolong its lifetime. With its spin
reverted, the electron is in the "triplet
state," from which it cannot drop back
to its original energy level, because the
single energy levels of an atom can be
occupied by no more than 2 electrons,
and only by 2 electrons of opposite spin.
The reverted spin of the excited electron
having become parallel to that of its
earlier partner, its return to its ground
level is "forbidden" by quantum rules.
So the clcctron has to remain in its ex-
cited triplet state till a thermic collision
dissipates its energy.

The physicist who wants to study trip-
let states has to protect his cxcitcd clcc-
                     873


trons from thermic agitation. This he can
do, to some extent, by enclosing the ex-
cited molecules in a rigid medium-for
instance, by dissolving his substances in
melted borax, which he then allows to
solidify. He can also use liquid solvents,
like glycerol, which he subscqucntly
freezes to a "glass" in Dry Ice, or liquid
nitrogen. The more electrons there are
in the triplet state, the greater the
chances that some of them will drop back
to the ground level, sending a message
to the observer in the form of an emitted
photon. Such light emission from the
triplet state is called, after the pioneering
studies of G. N. Lewis and his associates
(4)) phosphorescence to distinguish it
from puor~scence. It can be identified
as phosphorescence by measuring the
time that elapses between excitation and
light emission. This can bc done in a
phosphoroscopc, and if the lifetime is
found to bc of the order of 10m3 second
or more (instead of lOm8 or 1O-9), then it
is phosphorescence.

Excitations and Water

But here again the physicist may object
IO biological implications, because a
transition into the triplet state has a
very small probability of occurring, and
a transition that is improbable can have
no major biological meaning. I wasted
4 years in futile attempts to break
through these difficulties. Lately it oc-
currcd to mc that the physicists, in their
studies on this line, never used water as
solvent. They had a good reason to avoid
it, for ice at low temperatures cracks up
and becomes optically inhomogeneous,
making exact measurements impossible.
But the biologist is inseparably linked to
water, which is the matrix of life, and
water has many exceptional properties
that may have played a hand also in
the generation of life and might also alter
the probability of electronic transitions.
The most notable quality of water is its
strong dipole character with its resulting
high dielectric constant. I thus reinvcsti-
gated the problem, dissolving fluorescent
substances in water, freezing my solu-
tions in Dry ICC, and looking at them
under an ultraviolet lamp mounted with
a filter that passed only ultraviolet light
that could excite the fluorescent matter
without interfering with visual observa-
lion. All substances bchavcd in an cxtra-
ordinary fashion, going, if excited, into
the triplet state, opening up a new and
colorful world. This may be illustrated
by two examples.

If a glycerol solution of the dye rhoda-
mine B is frozen, freezing makes no
difference in the behavior. Frozen or
unfrozen, the solution shows the same
brilliant orange fluorescence. The situ-
ation is similar if the solvent is water
with lo-percent glycerol. However, if

874

pure water is used as solvent, then, on
freezing,  all fluorescence  disappears.
That this disappearance of the light
emission is the result of the electrons'
going into the triplet state can be shown
by cooling the solution to lower tempera-
tures. Below -4O"C, the system begins
to emit a red light, and the lifetime of
the underlying excitation exceeds 1O-3
second. If 3-percent glucose is present,
the light emission is very intense, and the
lifetime is around 1 second. IIpnce, if
the illumination is suddenly discon-
nected, the solution shows a strong after-
glow.

Riboflavin phosphate, one of the most
important picccs of the oxidative machin-
ery of the cell, is known for its brilliant
yellow-green fluorescence, which it dis-
plays under the ultraviolet lamp if it is
present in a watery solution or if it is
frozen in IO-percent glycerol. If the mo-
lecular oxygen present is eliminated and a
watery solution of riboflavin is frozen, it?
fluorescence disappears. If a trace of
oxygen is admitted, the system assumes a
red-brown phosphorescence (5, 6). Be-
cause there arc many more riboflavin
than oxygen molcculcs present, this
change cannot bc due to a direct inter-
action between riboflavin and oxygen.
What happened, probably, was the fol-
lowing: on freezing, the excited ribo-
flavin molecules went into the triplet
state, from which they could emit no
light, the transition from the triplet to
the ground state being forbidden. This

s
b

I

Fig. 1. The myosin molecule.

situation was altered by the oxygen,
which, as a paramagnetic molecule, pcr-
turbcd the electromagnetic field; in this
perturbed field the transition to the
ground state became possible, and thus
clcctrons emitted their excess energy in
the form of the red-brown phosphorcs-
cence. Oxygen thus profoundly alters the
reactivity of riboflavin and it is possible
that riboflavin fulfills its role in such
an altered state in viva, oxygen not
merely being an electron acceptor but
also acting by tuning the reactivity
of the molecules that cater to the elec-
trons it finally accepts. This may have
far-reaching biological implications, and
it may also explain the mysterious inter-
relations between oxidation and fermen-
tation (Pasteur reaction).
This simple experiment not only shows
that water can stabilize triplet states but
also shows that the further reactions of
the triplets are accessible to regulatory
influences. This can also be shown by
freezing a riboflavin solution in the prcs-
ence of air and 10-3&f potassium iodide.
Potassium iodide completely abolishes
the phosphorescence. This may evoke
biological associations, for iodine, in the
form of thyroxin, is one of the main
regulators of the energy household, and
Kasha (7) has shown that its infiuence
on triplet transitions is independent of
its state, being due to its high atomic
number---that is, to its heavy nucleus.
The great number of its clcctrons may
enable it also to take over cncrgies from
other excited molecules. In any cast, in
our frozen medium collisions of molc-
cules are very limited, and so we can
suppose that iodine did not exert its
action by a direct collision but par dis-
tance, being coupled to the excited mole-
cules by the intcrlying electromagnetic
field.

Structured Water

The main question is: Why dots frcczs
ing introduce such a change? The answer
is evident: Ice is not just solid water.
It is a crystalline solid with a regular
structure. Modern physics places less em-
phasis on the idea of "liquid" and "solid"
than on "random" and "regular." Glass
has no crystalline regularity, and thus
to the physicist it is a liquid of high
viscosity, like frozen glycerol.

The biologist may feel inclined to rc-
ject the biological implications of all
this, there being no "ice" in the body.
But probably there is. It seems rvcn likely
that our cells contain but very little
or no random water at all, but do con-
tain ice, or, more exactly, water which ac-
quircs an ordcrcd structure around sur-
faces or molecules. Observations on this
line were published, for example, by Pal-
mer, Cunliff e, and Hough (8)) who found
that around mica plates water behaved

SCIENCE. VOL. 124


as "liquid ice"  and showed dielectric
properties that pointed to an ordered
state. Dramatic developments were pre-
cipitated by the observation that gas
pipes carrying natural gas may freeze
up even in the summer. The studies of
Huswell and Rodmbush, summed up
lately in a fascinating article (9)) showed
that water forms cubic lattices around
nonpolar  substances. In Sweden, G.
Jacobson (10) showed that clcctropolar
groups on surfaces may also induce order
m the adjacent water. Hcncc, there is
every reason to bclicvc that most of the
water in the closely packed protoplasm
of cells is in an ordered state that may
render triplet excitation probable and
stable.

`i`he introduction of the electromag-
netic field and water structures into bi-
ology as a matrix could not fail to have
far-reaching conscqucnces, and so we
may do well to look around for evidence.
Naturally, such a theory cannot be
proved by any single observation and can
gain weight but gradually by suggesting
useful experiments or making biological
reactions appear in a new light, offering
an explanation for unexplained phenom-
ena.

Unsolved Problems

Where competitive inhibition or corn-
plex formation are excluded, the molec-
ular mechanism of drug actions is ob-
scure. Hormones and drugs produce vio-
lent reactions in the body even though
they are chemically inert, and the prob-
lem is how can a molecule that does
nothing chemically produce a reaction?
Hence, the question arises whether they
do not act by influencing electronic exci-
tations in one way or another. This as-
sumption could be supported by showing
that drugs are capable of influencing
electronic excitation in vitro in the same
concentration in which they produce re-
actions in uivo. Very specific drugs may
be accumulated by their target, and so we
do not know in what concentration they
actually act, but in the case of less spe-
cific drugs we can calculate with a ran-
dom distribution and thus compare bio-
logical action and in vitro activity. For
example,  2,4-dinitrophenol produces
marked changes in metabolism in man
in doses of 100 milligrams. This corres-
ponds to a random concentration of the
order of IO-siM. If this action is due to
an action of the excitation of riboflavin,
then lo-sA4 dinitrophenol should afiect
these excitations also in vitro. This is
actually the case, and it is easy 1.0 show

that 2,4-dinitrophenol actually quenches
the phosphorescence of riboflavin in this
concentration. Two more drugs have
been analyzed in an analogous fashion
with similar results-2,4-dichlorophen-
oxyacetic acid and chloropromazine. The
first is known to produce myotonia in
mice in doses of 200 milligrams per kilo-
gram. In this concentration it greatly
shortens the lifetimes of various triplet
excitations. Chloropromaxine has other,
very colorful actions on electronic tran-
sitions in concentrations in which it
affects the basal metabolic rate and in-
duces hypothermia in experimental nni-
mals. So have serotonin and lysergic acid,
while alcohol upsets triplet excitations in
concentrations in which it makes us tipsy.

We can extend such preliminary exper-
iments in various directions. A simple
experiment is the following: acridine
orange is frozen in water, whcrcupon it
shows a weak red-brown phosphorcs-
cence coming from a relatively short-
lived excitation. If a small quantity of
cortisone is dissolved in the water, the
lifetime becomes very long, of the order
of seconds. A trace of a narcotic, such as
chloroform or ether, cuts down the life-
time again to its original value, inviting
speculation about the nature of narcosis.
Similarly, the lifetime of the excitation
of rhodaminc in frozen water is rcla-
tively short, of the dimension of 10~~
second. Addition of glutathione in physi-
ological concentrations prolongs it lOOO-
fold, which may give a clue to the role
of sulfur in biological systems. One of
the oldest mysteries of biology is why, in
our bodies, potassium is kept inside our
cells and sodium outside of them. The
dimensions of the potassium ion are such
that it just fits nicely into the water
lattice, while sodium (which has a bigger
hydrate shell) does not do so and has to
cause a disturbance. So, if the regularity
of water structures is important for life,
then the presence of sodium in greater
quantity would be incompatible with it.
A watery rhodamine solution, if frozen
in Dry Ice in the presence of O.lM KCI,
shows practically no light emission under
the ultraviolet lamp, while, if it is frozen
with 0.1% NaCl, it shows a splendid
red glow, which indicates disturbed water
structures. It is very impressive to see the
difference of the two ions demonstrated
visually in such a simple and striking
manner. Muscle, if stained with acridin
orange, shows at room temperature a
red phosphorescence showing that within
the tissue the dye behaves in the same
way as it does in ice. (The dye seeping
out of the tissue into the underlying
filter paper shows the usual green fluo-

rescence.) These examples may sufhcc to
indicate that it seems likely that triplet
excitations play a major role in biology
and invite cxtensivc cxperimcntation.

To return to muscle, my point of dc-
parture, a theory of its contraction sug-
gests itself. It may be that muscular con-
traction, essentially, is a rearrangemrnt
of protomyosins,  consecutive to a dc-
struction of their surrounding water
structures, while relaxation is the re-
establishment of this hydrate envelope,
It is possible that the destruction is done
by the energy of ATP, released in the
form of a triplet excitation which intcr-
acts with the water structures (ZZ)

Conclusion

All this suggests that biological phe-
nomena, such as muscular contraction,
cannot bc dcscribcd in terms of classical
chemistry but belong to the domain of
quantum mechanics, to "quantum bi-
ology."

`l`he experiments described here indi-
cate that we will have to introduce three
new factors into our thinking if we want
to understand biological reactions: water
structures, the electromagnetic field, and
triplets or some other unusual form of
excitation made possible by water struc-
tures. As Chargaff says in a recent article
(12)> we have forgotten how to say
"don't know." The three factors men-
tioned may limit the number of ques-
tions to which we have to give this
answer.

References zmd Notes

1. H. II. Weher and II. Portzehl, Ergeb. Phyriol.
biol. Chem. exptl. Pharmokol. 47, 369 (1952).
2. A. G. Szent-GyGrgyi, Adunncer in Enrymoi.
16, 313 (1955).

3. -_ and M. Borbiro, Arch. Eiockem. and
Riofikyr. 60, 180 (1956).
4. Th. Fiilster, Fluorescenr Organircker Ver-
bindungen (Vandenhocck and Ruprecht, Gtt-
ingen, 1951).

5. The red-brown light en&ion of a frozen ribo-
flavin solution had been observed earlier by
DhCri and Caste% (6), who interpreted it
justly as phosphorescence.
6. Ch. DhPrd and V. Castelli, Comgt. rend. 206,
2003 (1938).

7. M. Kasha, J. Ckem. Pkyricr 20, 71 (1952).
8. L. S. Palmer, A. Cunliffe, J. M. Hough, Na-
Lure 170, 796 (19552).

9. A. M. Buswell and W. H. Rodenbush, Scien-
  tific American 194, No. 4, 77 (Apr. 1956).
IO. 8. Jacobson, J. Am. Ckem. Sot. 77, 2919
  (1955).

11. Such a rclensc of cncrgy would take place
  when the membrane of the fiber is depolarized
  and brings myasin-ATP together with the
  other contractile protein, actin. The theory
  does not necessarily involve electronic excita-
  tion and could be upheld even if contraction
  were only a precipitin reaction in which actin
  and myosin-ATP mutually discharge each
  other.

12. E. Chargaff, in Errayr in Biocfwmiitry, S.
  Graff, Ed. (Wiley, New York, 1956).

2 NOVEMBER 1956

875