Professional Documents
Culture Documents
m
Marine Biological Laboratory Library
Woods
Hole, Mass.
S3
*/*^'=s.
Presented
r>y
S^^^^^^^^^B^^^S
Bioenergetics
Bioenergetics
ALBERT SZENT-GYORGYI
The
Institute
for
Muscle Research
Woods
Hole, Massachusetts
NEW YORK
1957
Copyright , 1957
By
Academic Press Inc.
Ill Fifth
Avenue
New York
3,
New York
no part of this book may be reproduced in any form, by photostat, microfilm, or any other means, without written permission from the publishers
Library of Congress Catalog Card Number:
57-8382
Foreword
There
is
\\'ay
to avoid mistakes: to
do nothing
or,
at least, to
itself,
may be the greatest mistake of all. The selected, who are able to open new roads to science without erring, are very few and the author, certainly, does not belong to them. The unknown lends an
insecure foothold and venturing out into
it,
more than
One
istry
is
of the most characteristic features of present-day biochemthe coexistence of highlights with darkness,
knowledge
we can perform
reactions that
amount
to a
we
can-
many
We
why
specific
and nucleoproteins.
We
so. The same holds true for nucleic know most hormones, and many of
living body. In a
few
we
can even produce more active agents than nature did. But
how hormones act, what they do on the molecular level, we do not know; we have not gone beyond symptomatology in the analysis of their action. The same holds true for most of our drugs. The same duality exists also in our knowledge relating to the
high-energy bonds, the main representative of which
is
the high-
We
know how,
at the
VI
endergonic bond
is
established.
We
know how,
to
in fermentation,
till
the
ADP,
can support
endergonic syntheses.
We
the processes in which our foodstuffs are used to build our body,
erect the edifice of life, construct
its
machinery; but
is
how
energy
is
moving
life,
this
machine,
how work,
tu,
done, be
it
motion, meis
how
energy
driving
we do
we even
forget to
ask.
This
''chiaroscuro,"'^ "dear-obscure,"
is
acteristic traits
some
basic information
is
its
nature.
this duality
is
There
urgent
human
suffering, at present,
is
tive diseases"
the
name standing
gap
in our basic
knowledge. Possibly,
these gaps,
may
they
relate to
normal
function or to disease,
have one
common denomi-
some process which, hitherto, eluded detection. Some fundamental fact, if not a whole dimension, is missing from our biologinator,
cal thinking.
and the
basic
is
that
on the
Vll
approach.
Woods
Hole, Massachusetts
Albert Szent-Gyorgyi
July 19^6
ACKNOWLEDGMENTS
When
supported
letting
this
book go
my
its
who
my
risks,
my
associates.
gamble.
many
years
and so were the Heart Association, the Muscular Dystrophy Associations, the Association for the Aid of Crippled Children, who all greatly helped the
development of the views presented here by not
by strings attached to their grants.
by the National Heart Institute through
t>'ing
down
the author's
mind
Much encouragement
its
Grant
My
thanks are due to Dr. William Arnold for his invaluable help in shaping
thanks are due to Dr. Oscar Hechter for the patience with which he
My
listened to
my
Contents
Foreword
Abbreviations
PART
General Considerations
1.
The Problem
Is Stated
2.
3.
4.
5.
6.
7.
Theory of Energy Transmission The Mobility of * and Organization Absorption, Fluorescence, and Phosphorescence Triplets and Water Excitations and the Biological Matrix Quenching and Quenchers
Miscellaneous
Observations
10
18
25
32 41
8.
47
PART n
Biological Structures
9.
and Functions
59
10.
The
ATP
64 74
80
11. Riboflavine
13.
On
Uncoupling
90
99
14. Depolarization
the
Cell
Membrane
PART
m
and Disease
107
On
15.
Drugs, Hormones,
16. 17.
18.
19.
On On On On
112
Myotonia
the
120
127
133
Thymus
20. Conclusion
138
IX
72018
Abbreviations
A ADP
2,4
z= Angstrom =z 10"^
cm
2, 4-Dichlorophenoxy acetic
acid
E*
()
Excitation energy
= Bond energy
:= Inosine diphosphate
IDF
MW = Molecular Weight
^P r= UV =
A
= Wavelength = Millimicron
lOA
z= 10"^
n =r 0.001 millimeter
m/i,
mm
PART
General Considerations
^'There are
more things
in heaven
and
earth,
(Hamlet)
'^\OAl
1.
The Problem
Is
Stated
The problem
is:
how
life?
How
it.
does
it
move
no answer
to
So
is
it
is
possible
due
to
our
in-
answer
this question.
we have to I made a
and
a
this
experiment
few days
I
in the refrigerator
and for
few weeks
at
suspended
it
in 0.1 Al
KG
room temperature, added a little Mg, and added ATP in the same concentration as the muscle contained it in vivo. The muscle contracted and developed the same tension as it developed maximally in the living animal. If
could say: the muscle came to
we
motion we
life again.
ATP
it
was
a
split,
losing
its
by
P. Since
we know
a so-called high-energy
it
moved
we can narrow our problem down and did the energy of the '^P move the muscle? Progress in the chemistry of muscle made it possible to
tl^ie
ask
how
simplify
that
problem even
further.
is,
contraction in muscle
ATP
and
ions.
Of
is
mentary
act of contraction
how
the energy
studies of Edsall
sin molecule
tails
de-
about
it,
we
enters into
some chemical
local
which a
its
change
is
to
folding.
An ATPsplits up,
to
ADP, and
many
with symbols of
classical chemistry.
is
The
alternative picture
ATP
molecule does not enter into any such local reaction, but the bond
energy of
its
^P's becomes
is
released in a
more
active
and mobile
moves
and produces
vv'ake
of quantum mechanics.
Compared
if
to the
first,
this picture is
vague,
ask
may
pictures
we
can
make
deep
knowledge?
The inadequacy of the earlier classical pictures was brought out by the advances made in the chemistry of myosin. The more we
we understand it, which suggests that we are looking at it in the wrong way. Continuing some studies made by Gergely, Perry, and Mihalyi, Andrew Szent-Gyorgyi
learn about myosin the less
showed the myosin molecule to be built of two kinds of subunits, "meromyosins" which, within the molecule, stand in a row in
series
(Lauffer and
Andrew Szent-Gyorgyi )
is
If Laki
and Caroll's
correct,
one molecule of
if
Weber's value
is
Assuming the smaller value to be correct, would look something like Fig. 1 Of the two kinds of meromyosins one is somewhat plumper and sediments faster and has been called H-meromyosin, the H standing for
contains six.
"heavy." In Fig.
1,
arbitrarily,
it
is
The
s
<
m m
a
Fig
Fig.
1.
1
b
Fig 2
one
Fig.
a:
rearrangement of protomyosins
in contraction.
called L-meromyosins,
stant.
What makes
difficult to
is
interacts
is
with ATP,
re-
its
the energy.
to get,
difficult to see
how
do
to
this.
There are
still
possibilities for
line
One
could suppose,
the
ATP
on
it,
it
good ones. newer knowledge should support If a theory is good, then any and contribute to clarification, as was the case with intermediary
metabolism.
With myosin
Andrew
The protomyosins
is
are of equal
that
MW
Waal's and
electrostatic attractions. If
we
call a
molecule a
no molecule
at all,
only an aggregate.
in Fig. 2a. It
is
The
structure
is
way
it
difficult to see
how
is
seems more
more rounded,
short-
ened form, as symbolized in Fig. 2b. In order to produce such a rearrangement, many weak forces must be disturbed which keep
the protomyosins together.
ergy, enclosed in a
if
It is
impossible to see
how
bond
en-
^P,
that
We
sum up
we do
to
not
contracts,
how
it
uses
its
bond energy
at the
produce
under-
we know
to
about
we
We
edge of the
may be
responsible for
2.
A Theory of
Energy Transmission
It
my
due
bookkeeping.
actions
When making up
we
bond
and
so,
unconsciously,
between
illustrate
we
can
by comparing
atomic
it
bomb while
energy
is
it
potential
is
a potential,
its
its
and
its
locked up inside
remain sitting on
tial
when
for
more
active, kinetic
may
we
will sense
The
situa-
-^P
is
A-bomb. While
as a potential,
it
its
energy
is
showing a
were more
such,
little
if
our balances
as
sensitive
from molecule
to
go
produce work or
more
active
some form of molecular excitation, be it electronic, viSo what we biologists can safely do without getting into an argument with statistical mechanics is to use different symbols for bond energies which are linked to molecules and have no outward action, and excitation energies which are
else than
brational, or rotational.
will denote *. So
tlie
() of the
ATP
is
not
to
go
and pro-
duce contraction.^ Group transfer reactions of intermediary metabolism could be symbolized by writing:
(En)
ergy of reserve food as fat and carbohydrate while (Ex) stands for
the energy of the substance which
directly fed into the
muscle
energy
stance.
is
transferred
from bond
Bond
symbols of
ity to
classical chemistry.
is
The
question
is
whether our
inabil-
understand muscle
further belongs to a different group of reactions which can no more be described by these symbols, in which (E) is turned into E*? This duality may hold for all reactions in which work, w is produced, be it mechanical, osmotic, or electric work, etc. While () may be the core of reactions in which substances are synthetized and the living machinery is built, E* may be the core of reaction in which this machinery is driven and work is produced. This could explain why our notions, derived from intermediary
When
life
is
we
are not
all
is
lost in the
built is
world to drive
This E*
is
it.
is
captured by dyes,
shifted
it is
stored
The
hv-^E*-^ (0
The
stance, the firefly emits light:
(n)
-^E* -^hv
its
Looking
at this
row of
is
reversed. If
we
only as upon an
two
In muscle 1, which
nism,
is
is
mechahis
the () of
ATP, and
the recent
ATP
first
be reversible.
what
is
called "interthis
book
whether (1) - * -^ w does not represent the reaction which drives the living machine and, belonging in the realm of quantum
mechanics, can be expressed only in terms of the
latter.
lines can
make such
suggest
new
experiments.
3.
No
if
there
is
nothing to conduct
it So if
we
which could
to consider
we have
itself,
conduct
it.
In this chapter
energy and discuss the qualities demanded of the medium, leaving open the question of which of these mechanisms play a role in living systems. That such transmissions do occur was shown by
photosynthesis in which
the reduction
many chlorophyll molecules collaborate of one CO2 molecule (Arnold and Meek)
v ELECTRONS,
in
CONJUGATED SYSTEMS,
then
also has
AND
n,7r
TRANSITIONS
electrons
which
are
no longer bound
such a
to
any
single
electron
excited to a higher
energy
level,
then
its
E* belongs
changes
at
to the
any of
its
The purine
in
ATP
"nonbonded" "lone pair" of electrons which can be excited to the levels and thus contribute to the pool of ir* electrons. Those so-called n,ir excitations discovered by McMurry and Mulliken,
have
specific qualities: their lifetime
is
10
11
that of
TT-JT*
excitations
and
their absorption
is
is
quenched by strong
H-bonding,
acids.
The
hetero-
with
its
increases
the
dissociation
COUPLED OSCILLATORS
If
then
they
make "coupled
oscillators"
and the
If,
for instance,
two penduli
of the same periodicity are suspended on the same wire and one
of them
is
it
over the motion. After a while the second pendulum will pass
and
are the
its energy, and so it will go on. two penduli and motion is *, then
A''+B-^A-\-B*-^A*-i-B-^A
The
point about this simple experiment
is
+ B*
etc.
com-
oscillators
divided between the two and thus degraded to lower values, but!
is
periodically transmitted in
this transmission of
its
entirety. If there
were no
loss,
friction!
and
motion
The
tions,
oscillators.
all
communicated
may appear
at
any point.
12
ELECTROMAGNETIC COUPLING
An
tion.
upon
as
an
oscilla-
Two
distance
is
wavelength )
The
transmission
of
excitation
is
energies
between molecules
phenomena,
such as the "concentration depolarization" of fluorescences are explained. It has been found, as a general rule, that
10"*
if
a dilute (e.g.,
M)
is
peratures to
form
a solid glass in
if
its
however,
this
experiment
is
re-
(e.g.,
10"^ Af), then the fluorescent light emitted will not be polarized.
The
is
this:
owing
is
to the
high con-
centration,
now
field,
close to
the
E*
is
The
different
molecules visited by
JB*
molecules
is
transfer of excitation
possible. Figure 3
shows the
relation of
concentration to the
statistical distance
13
solution.
lost,
By determining
is
action
A 200
(80
160
140
120
80
60
40
20
M
Fig. 3.
10'
10'
-3
^-2
10"
two
about
70%
of the values
which corresponds
diameter of
M. The
E* can
many macromolecules
of this magnitude; so
One
point
we must mark
is
are involved. It
14
cule.
The
process
is
photon
free,
due
occur not
The
is
most favorable
if
there
spectrum of molecule
B, which has to take
the lifetime of
A
*
integral,"
A*
result.
The
distance through
depends on these
shown
to exist.
transmitted
to the chlorophyll
with a
|
high
rest
efficiency.
The
chlorophyll, in
its
turn, transmits
it
to thej
with
also
b in
blue and red alga and from chlorophyll b to a in the green alga
Chlorella.
arrangement of
energy
atoms (as
is
orbitals, the
common,
that
is
is,
practically continuous
is filled,
contains the
maximal
,
number of
is
the
then
its
direction
electricity.
how
metals conduct
If
them
15
it
will be
tlie
an insulator, a
dielectric.
However,
if
may be
raised
may
suffice
produce
this change, in
which
is
a "semicon-
may do
is
may have an
that of semiconductors.
to the
The
distance
to
filled
needed
to raise
first to
Parfitt, Perry,
and
edestine.
filled
electrons over
it.
W.
showed
recently that
sorbed photons, which energy they could again shoot out later in
the form of light,
if
other
The
there in "wells,"
tion.
still
The
empty
16
at the confines
is
of a protein
intimate enough,
may
common
system.
iff
an experi-
The
first
Bucher and Kaspers, who found that the light absorbed by the
protein in myoglobin caused the dissociation of the
CO
bound
by the heme.
The energy
CO.
had
the
to travel first
heme
to reach the
is
and not
as electronic excitation.
who
100%
efficiency.
to a resonance coupling
between the
would not be the protein but the electromagresponsible for the energy transmission.
I
which
is
As
tion open,
which
(if
columns
^
Watson and Crick model, forms the core of DNA (and/ possibly RNA), may conduct energy by electrostatic coupling in;
analogy to Sheibe's dyes, the moieaiies of which form columns!
and conduct *.
I have found earlier (1955) that certain proteins have a stronger fluorescence than corresponds to the additive
17
fluorescence of their constituents,
showing
its
rendered inoperative
hydroquinone molecules.
4.
it
through
it
messages in relation
sion.
If a
to the
photon
hits a
up
its
energy, then
turbance behind. If
In order to transmit
a substance
must be able
to accept
is
it.
The
what energies
a molecule
capable of
accepting.
The fate that the E* thus communicated to a molecule in vitro may meet varies according to the qualities of the excited electron
and the molecule which contains
it.
bonded
tion
heat.
to
its
nucleus. If such a
bonded electron
around
its
is
excited,
it is
equilibrium posias
and spend
its
E* which,
eventually,
becomes dissipated
unfit to act as
would be
an
energy transmitter.
to single
atoms but
to the
whole
18
19
excitable than the
to any
However, if the molecule contains any weak bonds which can be broken or distorted by * (if the dissociation energy
lower than the excitation energy), then the * communicated
molecule will be used up in breaking or distorting that bond.
is
to the
would be equally
its
unfit to function as
weak
colli-
and
is
unable to dissipate
it
E* (and
if
there
its
was no
as a photon,
for molecules can hold E* only for a very short period of the order of 10"* to 10"^ seconds. This means that the molecule will
tells
is
ca-
act as
may have no
direct biological
meaning, fluorescent light not being different from any other light. Naturally, the biological role of these molecules will not be
to emit but to transmit energy
and so
at all or
spilled)
it
mean
The
fluorescence
may be
may
dif-
20
system of conjugated double bonds.
One
made up of
is
independent of
pH
lies
mostly in the
UV.
is
formed by
The
is
sensitive to
pH
XOOH
Fig. 4.
RJiodamin B.
and redox
group.
is
mostly
at a
longer wave-
One
of the classic
members of
this
group
is
the dye
its
oscillates
between
in the
if
UV
and
maximum
at
554
rrifi.
So
irradiated by
UV
it
fluorescent light
5, in
band. This
symbolized in Fig.
The arrow
arrow S2 ->
excess energy
its
The arrow
Si ->
symbolizes
the shooting out of the energy in the form of the orange fluorescent light, whereby the electron drops back to the ground level,
joining
its
pair.
As
is
generally
known,
electrons, as a rule,
occupy
in opposite directions.
21
no more than
only
if
t^'o
and
this
the two
W
//
G
Fig. 5. Schematic representation of an electronic excitation.
level;
Si:
G: ground
S2:
Such fluorescence
pressure mercury
as that of
near
UV
light to pass.
The
UV
excites the
The
maximum
band
is
at
554
rcifi,
the
maximum
of
its
fluorescent
578
nifi.
is
simply raised to a
is
called a "sing-
22
let excitation"
it
generates in
tlie
may
spin,
parallel
to
that of
its
earlier partner.
This
called the
"triplet state"
for
quantum mechanical
vice versa.
and
"Forbidden"
just
But once
spin,
it
earlier partner
is
which
is
spinning in the
same
direction.
The
excited electron
triplet level
corresponding singlet
If
in the process.
is
small, heat
Si,
agitation
may
may drop
its
What
is
excess energy
The chances of
from
the triplet level directly to the singlet level, emitting a photon, are
Ti -^
in Fig.
is
a rare occurrence.
which corresponds
dropping
triplet to the
ground
state is
N. Lewis and
phorescence, to distinguish
it
direct transition
(G
->
is
termed
triplet absorption.
23
The
The
In
many ways
If
it
we want
we have
to stabi-
lize
or liquid
N2
to a rigid "glass."
By doing
triplet
so
state.
we can increase the number of electrons present in The more electrons in this state the greater
fall
the the
ting "phosphorescence."
To show
is
phos-
comes from a
is
triplet,
we
an instrument
which
cuts
ofli
is
The author has used two phosphoroscopes one with a time constant of 10"^ seconds and
light to pass only if
a slow one
which allowed
coming from
There
is
triplet state
is
which might be
The
24
paramagnetic molecules, as Oj, or of heavy atoms with a high
atomic number, as iodide, the nucleus of which can create an
electromagnetic disturbance in
enters
it,
its
vicinity. If
an excited electron
it
may
reverse
its
spin.
beautiful
example of
this has
been given by Kasha (1952), who showed that the almost pure singlet spectrum of dichloronaphthalene went over into an almost
pure
triplet
is
spectrum
if
But what
it
to intro-
this
change.
was
sufficient to
effects
add
to
it
form of
ethyliodide.
is
These
seem
me
is
one of the
normal
state, is
most
inti-
life.
The
is
^P is of the order
as a
photon of
this
thus
the biologists.
electronic
has an interest of
its
own.
It is
transition
between
the two
is
why
the
biological quant
this spectral
region, too,
no man's
land, lying
beyond the
may hold
guarded by technical
the lack of
in
5.
Triplets
and Water
much
which
mands energy quanta which, as a rule, are too high to be available in biological systems. The longest visible wavelength corresponds
to
^P,
about 10 Calories.
difficulties are
is
These
triplets,
of which
the lifetime
may
lie
seems also to
make
it
An
excitation
no use
to the cell.
we
are left
empty-handed.
There
is
in this.
strong dipole
The biological solvent is many queer and unique character. The reasons why the
shunned water
On
freezing,
having a tendency to
The
biologist,
however, 25
is
inseparably linked to
it.
26
Life originated in water,
is
its sol-
It is
the matrix of
as the
means of
at
bio-
we have
to
have a look
water and
repeat
some of the
it
classical
ever unfit
eliminate
stance,
may be
we
cannot
for indis-
its
shortcomings
we
We can,
try to
which includes
walled
test
with dry
ice
suspended in
it.
We
can
start
of the
substances
first
in glycerol.
filter
and other fluorescent Viewing these solutions under the UV which allows only UV to pass, we can
test
make much difference. The intensity of may somewhat increase, some "delayed fluoresthe color, that
is
cence"
emitted
the
be
same
we
use a
10%
water)^ solution
of glycerol or
2%
methanol.
if
we
shift to
pure water
At room temperature we
but on freezing
it
we
is
changed
in color.
This
is
illustrated
by Fig.
which
UV
contain in their lower half the dye solution in frozen state (cooled
in dry ice)
and in
their
liquid condition.
The
first
solution.
The upper
known
Fig.
6.
All
tubes
From
0.001
B and
M KI.
27
brilliant
orange fluorescence of
this dye,
Ample
evidence
show that this change is not due to the dye molecules having become inexcitable nor to their dissipating the energy of their singlet excitation. They are just as excitable as before (as shown by the almost unchanged color) and do not dissipate their energy (the low temperature and solid state also
disfavors such dissipation).
The change
is
due
to the excited
The
peculiar
make
only an allowed one but the most probable one, and so the electrons
go
into
it
wholesale.
the left in Fig. 6 contained the same
first
solution as the
solved in
it.
The unfrozen
as before.
light emission,
comparable
emission
is
red, of longer
due to the
turn of the excited electrons from their excited triplet into the
ground
state.
this transi-
ground
state.
emerging from
water
triplet transitions
stances.
Thus the
and
if
these excitations
would play
28
The
is
usually
singlet levels.
bolized in Fig.
by our
UV
Si,
to
sion" to
emitting
fluorescent light.
What happens
to
its
second
tube in Fig. 5
returns
is
from
gets
S2 to
Ti and then
in the
from here
ground
light.
state
G, emitting energy
it
form of phosphorescent
Si or
Whether
from
S2 to
Ti via
T2 can be
left
an open question.
On
liquid
min-thiamine mixture
found
to
one on
top, a phosphorescent
a zone
This intermediar)'
We
nonluminous phase
On
becomes
100C
colder.
when we go from dry ice to liquid N2 which is about One would have expected radiation to become
What happens
is
the
showing
activation energies.
in Fig.
flavine-
'-phosphate.^
The
is
the
first
known
shows no
^
light emission at
all.
It is
The
riboflavine
is
even stored
minishing
dark refrigerator
it
loses its
phosphorescence without
its
fluorescence.
of the molecule.
29
prior to cooling by bubbling through N2. If the cooling
is
effected
is
the one
shown
The O2
present
made
no difference
the frozen part emit an orange phosphorescence. the triplet state unstable,
cited
electrons to
state,
The O2 rendered making it emit light, "allowing" the exreturn from their lower triplet state to the
ground
The
last
atmospheric O2 and
shows, the phosphorescence was completely "quenched." Transitions are accessible to regulatory influences both ways, as
shown
triplet
it.
I'.
If the
T -^ G
Such antagonistic
motes
it
M concentration.
correctly as a phos-
The
Dhere and
Castelli,
and interpreted
phorescence.
We
profound change
two
fluorescent
substances studied,
making forbidden
transitions
into probable
rhodamin
my
experience goes
we
be-
be quoted: on freezing of
its
30
yellow fluorescence of acridine orange gives place to an orange
phosphorescence.
acridine yellow.
As we
light
all
On
50C.
In acridine yellow
it is
tube
is
is
moving phosphoroscope. If the test of the UV lamp and then the illumination
after-
glow
an orange-yellow
rhodamin)
emission as soon as
No
even
at the
tempera-
However,
1%
glucose
is
present,
below
40C
all
shown by
show
similar
sudden changes
on
freezing.
A saturated watery
room temfreezing.
The blue
on
Atebrin (0.0005
M), which
shown
to be
composed of a yellow
fluores-
1%
glucose to the
On
is
found
to
excitation. In quinine
no
light filters
31
means a mixed
tensified
color, the yellow component of which can be inby freezing the solution of the alkaloid in the presence
of 10"^
M)
the blue
of
1%
glucose.
Such changes are not limited to fluorescent substances. Isopentaquine, for instance, emits under the
UV
low fluorescence (which can be greatly increased by 10% glucose) and a red phosphorescence which can be seen in the fast phosphoroscope and can be somewhat increased by 10"^ glutathione.
dyes:
on freezing
its
red fluorescence.^
In
all
on cooling, had a
known
to
have a tendenq^
tion was, to
my
They demand no paraphernalia, except an UV lamp and some dry ice. Doing an experiment is different from reading about it. These experiments make the impression of a new, colorful world opening up unexpectedly and promising a
deeper insight into the mechanisms of with their matrix, water.
^
life
and
their interaction
New York
was
used.
6.
Excitations
and the
Biological Matrix
The
first
The
make
it
clear that
the effect of water and cooling was not additive but the two did
something
specific together.
Water, in
itself,
room temperature
if
10%
is
also
damin or
or liquid
riboflavine
essentially identical
ice
N2
for cooling,
though there
is
differ-
new and
specific
had
to be
and cooling. This could hardly be anything else than the formation of ice. That this is actually so is borne out by the temperature
dependence of the observed phenomena.
and
its
on temperature
lowed
to
rhodamin
stance, cooled to
78C
warm up gradually, the light emission fades out around 40C. From 40 upwards there is no phosphorescence and
does not melt there
all.
is
no fluorescence
either.
There
the ice
no
light emission at
when
starts to melt.
Thus the
does not
melt.
We
33
periment to produce
cient,
ice
triplets.
Freezing
at
1C
would be
suffi-
volume of mother
liquor.
The deep
freezing
is
needed
at the
melting point.
and
riboflavine.
The
orange
around
0.001
20C,
in riboflavine
around
3C;
So the
KI
20C.
where the
ice
optically less
inhomogeneous.
is
is
not
of
Ice
is
As
is
modits
its
move
relative to
form regular
the relative
which
and
is
possible only
if
position of particles
is
fixed
by
rigid
its
at
room
solid.
and so
is
looked
upon by the
physicist
more
as a fluid of
The same
structure
is
low tem-
though
it
readily changes
may endow
their
tion
is
that the transition of our excited molecules into the "fortriplet state
bidden"
34
cules
and
lattices
of this
This explains
why
at
there
was no
may
form a
rigid
mass
crystals.
why an admixture
it
10%
glycerol or
2%
of
its
specific action
on
excita-
water
When
we
on high
ices.
pressure, could
go
and Fowler,
ice
classic
ture
like.
which
different
from
that of
common
which
is
tridymit-
Though
somewhat been
HgO
The
the
towards
other
side,
contributing
to
the
dipole
character
(Pople). The two protons can be shared, through H-bonds, with two other water molecules, while the lone pair of electrons may attract two protons from two other HsO's, so that each water molecule can link
up with four
state,
is
still
due
be broken, and, as
agi-
down
to
permanent
lattice
to
form such a
at
there,
to
seems
which
the water goes from a rigid crystal, so to say, into a liquid one.
35
The
is
at
which tempossible^
around 37C
tures of their
own
choosing.
still if
The
situation
we
consider water
structures built
lem
cation.
collected,
as early as
1947, 175
come
"deep-surface orientation
is
many
liquids.
The
commonly assumed."
clear
re-
and
the dielectric
showing
These
ice.
were found
to
means
a cer-
its site.
Such a binding
especially strong
as those of ions.
which
The building
36
to
and probably are formed around any dissolved molecule. Frank and Evans introduced the picturesque expression
structures can,
As
de-
is
different
from
lattice
common
hexagonal
ice,
"This
One
the
dis-
was the
effects
when
at
temperatures as high as
is
ice
We
may have
a different crystalline
water
crystal
forms
or, if
complex.
we
can expect that the dimensions and the nature of the icebergs will
decide
how
membrane.
ices
Two
may hold
for
of different nature.
37
B. Jacobson has studied highly polymer deoxyribose nucleic acid
its
as-
"lattice
of rotational ellipsoids.
The
great
number of
electropolar groups
this
molecule
may
it,
so that
it
avoid formations of icebergs which would interfere with moSince structural proteins are fibrous and closely packed,
is
it
bility.
Bernall and Fowler introduced the idea of "structural temperature" which they defined as the temperature at which free water
as the
We
The
pure water
when
the temperature
is
cell,
may
not be
random water
it
No
made
knowledge of
triplet excita-
to the solution.
low concentration and so we can expect that at the high protein would be favored still
more.
I
if
suspended in
38
0.1
yVl
KG
on illumination
underlying
cence of the dye, while the dye seeping out of the muscle into the
filter
was brought
and
The
pressed out by such a muscle also indicated that the physical state
KG
disfavored phosphorescence.
As will be shown later Na disturbs water structure. The study of water, in its relation to biological structures and
electronic excitation processes, opens a fascinating
field
and promising
water struc-
of inquiry which
may
new
outlook on
less, as a
life itself.
neutral
medium,
cell.
In the outlook
The
protoplasmic
may
tures,
water being part and parcel of the living machinery and not
its
merely
structures
One
new
As
is
organization,
in a specific
way form
may be connected
39
sentences, etc., so atoms can join to molecules, molecules to organelles, organelles to cells, etc., every level of organization having
new meaning
of
its
own and
bilities.
matter,
cogwheels do in a watch.
Water
is
it is
understanding
the:"
focused
its
attention only
on
the;
from
its
One
critical
prove
trip-
were actually
excitation. Triplets
known
In
may
fuse to
ground
level
to be a
excitation,
alter
little
E*
this
would
may assume an
un-
usual long-lived form which might be of prime import for biological energy transmissions.
The evidence
which
also
40
book, that the blueprint of the energetics of the living world consists essentially
its
reversal.
Increasing evidence
synthesis,
is
available to
show
same way
as other
going thus,
in all probability,
on
excitation.
So
triplets
seem
to
the
two processes
closer. It
made
possible
structures.
and water may also open the way to the understanding of mechanism by which the * of chlorophyll is used to decompose water into its elements, which is at the core of photosynthesis.
the
As will be shown later, triplet excitations in ice are stabilized by SH, which may help to fit Calvin's thioctic acid into the picture. I have considered here only changes which may be induced in
the solvent, water, by
its
may be induced
On
the water crystallizes out, which m.ay lead to increased local concentrations of the solute with consequent self-quenching or poly-
merization which
light emissions.
may profoundly
alter
excitational
states
and
These
possibilities will
be studied
later.
7.
When
we
We
we
could also
Light emission, be
electronic excitation
and
a substance quenches
it,
it
has to inter-
E*
is
involved in a biological
it.
how
to find
them?
that certain
It is
known
quenching
action.
Such an atom
SCN
and
NO3
known
as
quench-
make
quenchers; O,
N, and S
are such
The
activity of these
itself also
UV.
much, partly because
action.
ture.
Such a
specificity
demands
struc-
we
can
make
a
introduced into
the molecule
more complex
its
fluorescent molecule.
They make
dissipate
the jE*.
own
41
42
*, but
may
it
and
not
do with
In this way
we
at
molecules which
may have
may have
to be
also selective
To
is
known
weakly
fluores-
cent but if
NO2
is
introduced into
its
even more
its
easily
strong ab-
sorption in the
UV
as a
"chromophore"
easier
still
and
to
with
its
surroundings by electro-
NO2
thus
By introducing
additional
NO2
groups
at the points
most
fit
for
OH,
or by
we
abilities as
well
fascinat-
When
These considerations
puzzles
also lead us to
of contemporary biochemistry:
(Fig.
substance
7a)
presence,
go on oxidizing
foodstuflFs,
increased rate, but waste the energy thus liberated as heat. This
caused dinitrophenol, a few decades ago, to become a most fashionable drug for reducing body weight. Unfortunately,
its
use in
43
ously,
it
fat or
hungry
to being blind.
is
The mechanism of
puzzling because
it
the
more
to
acts not
seems to
act
and put
and
os-
activity" of cell
is
membranes,
in
which
that
it
was impossible
to bring
them
to a
common denominator
OH
N02
(a)
N02
(6)
in terms of chemistry.
The only
point at which
is
all
these reactions
agree
is
involved, in one
way
is
or anin-
other. This
makes
it
seem
not
some
physical principle, as
is
may be
is,
essentially, dini-
dinitronaphthol even
more
so.
44
could not be accounted for by deactivating collisions only and had
to be, partly, a resonance
collision,
the whole,
But fluorescence,
lem. If
as such,
is
of
E* plays a role in biology it has to be in the form of triplets, as shown in the previous chapters. I found triplets most sensitive to quenchers, some of v^hich suppress phosphorescence in
Fig. 8. Phenothiazine.
light.
were the
case,
as well. plied,
The quenchers
quenching
activity
can have
better re-
frain
a suppression
E* and an
* has an important
One
is
0"'*
M concentration.
SC=CN
is
which
it
is
45
perimentation, owing to
its
9a)
is
more
soluble.
acridine,
this
10"*
That
due
to the
combina-
tion of the S, C,
and
^O^N-J^^
I
CH2
I
CH2
I
a
(a)
(6)
cycle
(Fig.
in
Pyrrolazote
activity,
is
not
known
to
it
have any
specific
pharmacological
which
indicates that
interferes with
E* and *
is
important for
life,
then
it
should
kill
the animal
if it
reaches the
if it
concentrations in which
kills the
it
interferes with
should quench
in the ani-
mal by a
in
lethal dose.
100
mg injected
mg killed the animals; 200 mg per kilogram corresponds to random concentration of 6.10"* Af. The phosphorescence of rhodamin and acridine were quenched by 3.10"* Af. It would be interesting to know how far the pharmacological
200
N
46
activity of other substances containing S, C,
imity
is
actually
due
NC
6-Mercaptopurine,
action
II
known
for
its
on
excitation of
rhodamin or
M concenspeci-
It is
the nucleic acid cuts the energy transmission along the column of
DNA
in the
which
the
N=N.
azo
we
among
compounds. McLaughlin and the author, while finding diazobenzene a poor quencher, found aminoazotoluene (toluazotoluidine)
highly active.
The
latter substance, as
difficult to evaluate,
owing
Both seem
to
with rhodamin.
8.
Miscellaneous Observations
I
In this chapter
seemed not
to be without interest
this
main theme of
NARCOSIS
Since Overton proposed his theory, narcosis has occupied
research workers.
their
many
none
new
theory of
that
we
still
do not know
it
what narcosis
pected to
light
is.
If our theory of
is
correct then
can be ex-
make
new
narcosis.'^
UV
lamp
fluorescence
No
long-
moving phosphoroscope.
If instead of
solution of cortisone
we
The
sud-
illumination
is
The
interaction of water
modified by the sterin which was present in a very low concentration, cortisone
'i-O'^
M).
47
48
sterin, the
number of
being
specific affinity
known
to exist
between
sterin
due
and
is
their electrons
with
Chloroform
the
same
if
%o
added
to the
served
on
freezing,
in-
reasonable to
relation
to
with
it
less, it is
Vv'ith
the interrelation of
The
total
phosphorescence
drastic actions
is
may
may be
responsible
symptoms produced
we
A*
and B.
seems likely that for an understanding of drug actions an have to be considered in analog}' with
form, in which the drug did not seem to act directly on the dye,
but seemed to act by modifying the relations of a third substance
to water. Naturally, this does not preclude that the excited molecules themselves should be also directly influenced by a narcotic,
if
it.
affinity, as
shown by the
49
form
as a colorless anhydride. If one-tenth part of a saturated watery
is
solution
added
to a watery
quent freezing and illumination the rhodamin will not go into the
triplet
and show
is
its
change
the chloroform
shown by
considerably
affinity
long-life phosphorescence
is
Yoo part of a
added
corti-
sone system.
ASCORBIC ACID
We
know
know
is
why, what
the role
it
fulfills. If
allowed to autooxidize
it
before,
as
far
as
experience goes,
all
go
this oxidation
is
BaOH
is
and
formed which
water
it
the
Ba
salt
is
dissolved in
it
shows no fluorescence
shows a
of
The oxide
it
ing
its
optical reactivity. It
forms such
as pros-
When
lymph glands, both contain yellow pigments. the author worked two decades ago along this line, he had
50
the structural proteins and were not
all
due
com-
show
after
after-
UV
lamp
a strong
and long-lasting
which
is
colored matter.
in a test tube,
is
placed
is
the air
ice
is
replaced by
N2 and
if
the tube
im-
mersed
in
the dry
freezing mixture,
held
(after
tem-
perature equilibrium
for a second
it
Hg
lamp
is
found
which
lasts for
lasting for
that
is
O2, no
such phosphorescence
such emission at
show any
room temperature
mamis
A 1%
(serum albumin
gelatin
cryst..
Armour),
as well as
1%
ovalbumin or
is
show
it
too,
though
of shorter
duration.
my
knowledge, not
owing
to the
note that this phosphorescence, which they observed at the temperature of liquid N2,
is
51
ture
at the
temperature of dry
.
ice
(which
is
ascribe
tempoelec-
tron's return to
original
ground
state.
PERTURBATIONS
shows
two solvents would be still bigger if, more nonpolar solvents could be used. This, unfortunately, cannot be done because in such solvents rhodamin forms a colorless internal lactone and the lactone formation makes resonance impossible and so eliminates the color (Lundgren and
the difference between the
instead of methanol
Ninkley).
What
olic
is
the methan-
substances
known
One
can
and so a similar
shift in
may
disturb the
tested by
McLaughlin
results are
summed up
in
Table
spectrum
to-
in
sodium
52
Table
I
Shift of the Specthum of Rhodamin B (Dissolved in Methanol) IN A Units tow.\rds the Longer Wavelength on Addition of Various Substances at 0.01, 0.001,
AND
0.0001
Concentration
53
The table shows most substances to be inactive, but also shows some of them to be highly active and to cause a maximal shift in 0.001 AI concentration and still be active in 0.0001 M. Among
the most active ones
we
find dinitrophenol
and dinitronaphthol,
one another
sug-
might
actually
to
is
known
to uncouple.
Owing
to
its
O-CH2-COOH
,C1
a
(a)
0>)
insolubility,
The
is
uncoupling
activity, also
action
is
on rhodamin.
A
and
acid
(Fig.
very different,
/"
intriguing to find
(
them
1
vitro
experiment
see Chapters
rhodamin molecule,
built of
nitro-
an atomic model,
active nitrophenol
and
NO2
of the dye. The same holds for the carboxylic O" and one CI (4)
54
of 2,4D, while the 2,4-dichlorophenol cannot be
the
made
to touch
its
OH and one of
is
its Cl's.
The
moment
of
compounds, though
less active.
A heavier
chloro
cule active,
to the insecticide
RHODAMIN COMPLEXES
During these experiments,
phenol
a fascinating reaction of
rhodamin
added
to
maxi-
mum
ing,
at
578
rcifx
however,
The change
is
due
to the
so.
R. Steele
(unpublished)
compound
its
evidently a "molecular
complex."
He
On
it
rendering
readily disinterest
There
to this reaction
sluggishness.
The formation
of the complex,
(bor-
The
that
first
eight curves
actually the
went through an
isobestic point,
showing
it
was
its
sedimentation
which caused the gradual change, the reaction having taken hours
55
1.00
0.90
0.8
0.70
0.60
0.50
0.40
0.30
0.2
0.10
0.05
PART
II
Biological Structures
and Functions
"Research
is to
see
and think
tvhat
9.
number of
a
biological
structures
and functions
order to see
them
in
re-
new
light by looking at
glasses of *.
To
main
faithful to
my
traditions
is
Most of
physics,
these theories
were
impact between
The
is
at
known
ele-
phant himself.
involves
If
we
fail to
function
ously
E* then our experience with * should fuse spontanewith older knowledge into a new theory of contraction. An
important place has to be given in this theory to the fact that the
strong expansion, the formed ice having a cubic lattice and a very
low
ing
density.
stances, like
ATP, bound
to myosin,
may
up water structures.
far as
As
we know,
is
per-
built of a greater
num-
59
60
Unlike covalent bonds, such links have no fixed valency angles.
This makes the whole structure comparable
to a string of beads,
of resistance to deformation.
to
The
forces be-
fibrous
form
(Fig. 2).
If, all
the same,
we
find the
myosin
particles in solution
must
The problem
of contraction
is
makes the
at rest, or stretches
It
is
which the
due
particles build
The
latter
energy of
ATP.
to
ATP
would have
The
myosin
theory proposed
particle
is is
is,
in
its
traction
lishment of which
relaxation.^
The question
facts
is
how
far
is
this
known
explain them.''
Myosin,
^
in a
The fibrous molecules of deoxyribose nucleic acid, dissolved in water, known to shorten by 30% or so on addition of salts, Jacobson found under these conditions (using NaCl) that the water structure collapsed.
are Possibly,
is
61
that
its
No ATP
is
thus
needed
keep them
in this form.
The
myosin
to water are
it is
shown by
water.
plex. In fact,
difficult to
97%
The high
viscosity
shows the
parti-
What ATP
is
does to this
its
to deprive
parti-
make them
contract.
The
contracted gel
its
to destroy the
shown by Varga.
it
The denaturing
action of freezing
volume
in excita-
which
entails
volume
contraction. This
Our
theory
demands
that
should be such a
struc-
volume contraction
tures
expanded water
a
volume
is
particle in
extended
state
then
it
hand
this
in
is
Brown who
also
62
it
considerable.
The contraction of muscle exposed to high presknown for a long time as ""Ebbecke's phenomenon."
such as
I~,
Bulky
ions,
fit
into
lattices
state" of
later analyses
of his observations by
The
action
in
found
that
to
is
depend on
on
their
position
the
"Hofmeister
series,"
may have
E*.
is
not merely to
at
when
contraction
is
needed, and do so
when
the
done. Accordingly,
we
in
myosin
role.
a triggering
mechanism
which
prominent
According
tains
to the experience of
my
side,
and the
ATP
its
is
four
de-
whereupon actin and myoform actomyosin. In the actomyosin thus formed the terminal
of
ATP
becomes
split
and
its
ADP
into
ATP
which, with
the free myosin particles rebuild their water structures and stretch
new
contrac-
The
shown
beautifully in
63
the pictures taken by Spiro)
is
a thickening.
At higher degrees of
should give
rise to
new
lines.
The
theory proposed
may be
it
10.
Looking
12), one's
at the
first
ATP
(Fig.
impression
Nature does
O- P link is all that is needed. A a complex molecule if a P much simpler inorganic polyphosphate should do just as well. The molecule has two ends: a phosphate-end and a purine-end. The phosphate-end represents the energy store, (); one may ask whether the purine-end may not represent *, thus providing the
The purine
its
nonlocalized
tt
electrons
and
five
N-atoms,
later,
under conditions,
also
become
demands of an E* The purine may thus be instrumental in transforming the (E) of ^P into *, when this (E) has to go into biological action and drive the living machine. The whole ATP molecule
strongly fluorescent and thus conform to our
transmitter.
What
is
12
is
how
electrons.
this structure.^
ATP
Are we
made by
a structural formula,
^ The contents of this chapter were presented at the International Enzyme Symposium, Ford Foundation, Detroit, November 1-3, 1955, and pubHshed in "Enzymes: Units of Biological Structure and Function," Oliver H. Gaebler, Ed., Academic Press, New York, N.Y.
64
65
the correctness of
which we
fail to
we have
C,
seen
it
too often?-
The
ATP
and one
O
NHp
I
N, one C
link
(marked
N^^.,^
^ ^ ^N
OH
OH
OH
/ /
OH OH
\
\
I!
II
II
Fig.
12.
ATP.
a free rotation
freedom
to
change
its
visual pictures
also offers an example for such an influence of which inhibit our thinking in certain directions. Relations between chlorophyll and carotenes have been sought for a long time without
"
Possibly,
chlorophyll
definite results.
The Mg-porphyrin
part of chlorophyll
is
is
reproduced
in
Fig.
13a, while
shown
in Fig. l4a.
The two
structures
seem
to
II
CH
*^,^3
CH3
CH ^
C^
/\ /"\ /\ CHt-CH, / \
C
I
C
I
CH, ^
/
HO,
C=N
C
^.
MQ
N G
\ .CH
\X / HC^
C
/\ /\ /\
C C G
C^
OCX
\
/ \ J:H
^C
N
I
N
II
C^
V
I
yC
^/
I
\ GH3 .C
\g^
CH
^c
HG
I
G=0
COOGH3
Fig.
13.
a:
chain, b:
Same
Mg-porphyrin part of chlorophyll without the phytol side as "a" with Mg, N's, and longer side chains eliminated.
66
is
open
that by rotation
E of the '^P which bond energy which has no outward action. For the transference of (E) there must be a close fit, point counter point. The atoms which have to touch one
ends, an "approach"
is
has to be transferred
is
an (), that
is,
show no
the latter
by a metal, while
built of isoprenes.
However,
is
if
we
eliminate the
Mg
with the
are left
four N's, as well as the longer side chains from the porphyrin,
we
this is
fact that
we
CH
HC
< y
C
OH,
\
C-CH3
CH3
HC
HC
>
\CH
HC
C P
67
another are two 0~'s of the dissociated
OH
The two
^P, which
give
up
its
probability, the
of the
NHo
in
group
at position
6 (since this
and
its
second neighbor
However,
mean
to
free motion.
in
Such a
it
not
bend
any way
pleases,
is
The motion
the
same
axis.
ATP
make make
that
molecule
a very limited
statistically,
the chances
that the
NH2
a close
a close
fit,
would be probable
that this
meeting
possible
ATP
molecule
to be that
way.
an atomic model which keeps account of atomic radii and limitations of freedom, the rigidity
and small
flexibility
of bond angles.
Such a model
is
ATP
molecule built up of
model, in
its
conventional linear
is
form corresponding
If the
to Fig. 12.
(The molecule
here in
its
dis-
OH's on
the phosphates.)
molecule
is
now
O bonds,
is
C,
N,
and
the O's of the terminal and middle phosphate just touch the N's
is
shown
in Fig.
16. If
some
sort of a
formed now between the 0~'s and N's, then the P O which has to be broken in contraction and supply the energy
Griffin
Scientific
Boston
10,
Mass.
68
Fig. 15 {above).
ATP
Fig. 16 {beloiv).
Same
69
for
it,
would have
to
and N's might do, having been shown by Gergely and Evans that
H-bonding can
tt
orbitals overlap. It
is
ATP
from one
This, however, does not explain the role of the bivalent ions,
Ca and Mg, both of which can accelerate the ATP-ase activity of myosin. The possible answer to this problem w^as given by a chance observation. As discussed in Chapter 4, we may expect
energy transmitters to be fluorescent. Since there are violent shifts
in energ)'
UV
if
Mg,
The
fluorescent substance
was
found
to
ATP
by the
loss of
its
terminal phos-
phate and
its
hydrolytic deamination.
structure of the metal
complex
is
elucidated by
known
to
form with
Mg
(
stable,
strongly
fluorescent
chelate
Fig.
17b).
Evidently,
an
Mg
to
form very
stable, coordinative
is
com-
ATP
ture
is
is
illustrated
just
there
enough room
and
70
r
in Fig.
attracts
an
from the quinolinol (Leverenz). The same can be expected to happen also in the complex of inosine, and if the metal
/
0^
I.
Mg
Mg
I
\
a
Fig. 17. a:
w
b
b:
Mg
complex of oxypurine.
H
Mg
complex of oxyquinolinol.
OH
P-0
HN
V
N=
\
\
H
I I
\_j:i.::s^o\p=o
K.
H
I
H
I
H
I
CH-C C C C
I
/f^=o
-r-.
OH
OH OH
Fig,
18.
Possible structure of
Mg
complex of
ATP
(interatomic dis-
may
from the
latter.
thus
from phosphate
So the
it
Mg
may
of a molecule;
single,
common
nonlocalized
71
electrons
its
P,
in
which represents the (), could merge thus with the adenine one extensive system of mobile electrons.
This opens the possibility that
when
is
it
attracts electrons
V^v^
Fig.
19.
Mg
OP bond,
its
which
then
falls
energy appears in
E* completing
,
* transformation.
That
Mg
one substance
this
system the
greatly speeded
up by Mg,
reaction
ATP-ase
is
activity of
without
Mg,
but
The
One
it is
ATP-ase
activity
is
that
activity.
72
A
bility
serious objection
may be
Mg
isonine, in water,
not
great
enough
to hold the
is
we
ATP
to
be present in muscle
Mg complex,
much
Mg in muscle as there
in Fig.
ATP,
Mg
16 only says
that
it is
formed. However,
splitting in
is
we have
muscle,
to
is
remember
by
that the
is
"activated"
We
a
Probably
it
means
it
consecutive
deformation. So
may be
ATP molecule
between
activa-
this
is
actually
what we mean by
One
if
say that
high enough to bring the two ends of the molecule together and
make
the
~P
occur,
^P
would be
split
and
its
It is
spontaneous reacin
hand. If
run
down
senselessly as a watch
to
released
from
in
its
regulators.
We
it
have
ATP
molecules,
bound and
when
^P
word may be
member
of the
ATP
mole-
73
cule, the pentose
which has
to connect the
phates in such a
position.
to
It
it.
way
that they
come together
may
not be
the
all
there
is
Nature often
same
stone.
United States
Patent Office has granted two patents for the hardening of gelatine
which
may very well play an important role in the "activation" of ATP. One last remark may be made about Ca. It greatly promotes
the
"ATP-ase
activity"
as "superprecipitation." It
One
tentative explanation
behavior
Mg, forms
its
chelates
~P
Ca
but
is
unable to transmit
Ca and N. Accordingly,
also unable to
we
more
to a possible additional
While reading
[B'wchJm.
et
book
my
attention
was
W.
James
Its
summary may be
"The
AMP, ADP,
such a
and
ATP
to
show manner
is
that the
that
ATP
is
molecule
is
folded
bonding
permitted between
ATP.
CTP
and
UTP
and
*USA
(Nov.
(Nov.
3,
21,
1939), the
first
Kodak
Co., Jersey
Cit>'.
11.
Riboflavine
As
is
generally
I
riboflavine- 5 -phosphate
is
(which
to act as intermediary
to
This function
pairs
in accord with
off electrons
ability to
its
and giving
one by one,
must be able
to
form
a free radical.
The
reducing
it
in a strongly acid
greenish-yel-
red-brown color of
not merely to
is
serve as a bridge for the H's or electrons to pass over, but to convert the energy, released in
its
problem here
is
to find out
whether *
As has been
*, may thus
act as
an energy transto
be able to go
yellow fluorescence of
agement.
The
that of
ATP.
We
ATP
its
ATP
it
compound with an
74
75
at
in a
is
ATP. While
the function of
ATP
is
spend the
to
invest
phosphorylation and
stabi-
ATP
accepts phosphates
a triphosphate
in
the
form of
OH
CH2-C-C-C-C-O-P-OH
H
I II
H
H
R
1^
VO
^^
(a)
(6)
Same reduced.
(as
chain,
then breaking
its
down
this
chain,
converting
we
sup-
posed)
esis
oxido-reduction in
up
it
a triphosphate chain
on
its
itself.
mechanism of these reactions we supposed that ATP connected its two ends (on the enzyme) by forming a coordinative metal complex between the active groups of its purine and the triphosphate end. So, when studying ATP, our first question was
As
to the
is
sterically possible. If
ATP
and
riboflavine, then
is
we might
possible,
sterically
as
its
76
own phosphate
tion in
its
alloxazine.
To do
an
would have
to
have
C=0
in
at position 2,
NH
is
at 3,
and another,
it is
C=0
at
4 (Fig.
bring
whether
sterically possible to
The answer
is
by
=0, NH,
and
=0
ATP,
the sta-
more
distant here
say that the structure pleads for the assumption that the riboflavine
is
made
We
NH
at
may
NH.
For
this function
we
Once
tral
is
structurally possible,
possible energetically.
The
spec-
The
riboflavine has
445
So
if
the molecule
is
excited by
UV
its
one of the
UV
conversion" to the lowest singlet level and emit from here the well
known
sion
band
540 m^M
(Si ->
in Fig. 5).
maximum To excite
of
its
emis-
electrons to
77
V c
78
this
540 m/A
level
64
calories
are not
likely to
which has
maximum
which
to
at
is
to
an energy of 47
calories
still
fulfills
somewhat closer to biological values but seems be too high. However, we must not forget that riboflavine its role in the cell "activated" by the protein and we do
far
its
not
know how
its
being
We
know
bound
it
fluorescence (which
may
its
mean
that
energy
is
needed for
its
excitation )
How
we do
needed
possible
not know.
It is
To
state.
return once
in th frozen tube,
the riboflavine
into the triplet
Even
if
light.
its
singlet excitation
a transition
and
was formed
from the
tint
Ti in Fig.
The
tube containing
KI showed
if
a strong
brownish
the tube
to
was removed
to
warm up
some
made
form of thyroid hormones, is one of the main regulators of metabolism and its action on E* is independent of
its
is
which
is
M conit
There are
first
tvv'o
circumstances which
make
this effect
On
freezing,
the peripheral
up making
more
difficult to
see
what
is
On warming up
the opposite
happens.
79
remarkable.
tissues,
The one
is
that serotonin
is
a constituent of
normal
The
is
that serotonin
is
to plant
indole seem to be
among
promote the
direct transition
10~^ AI serotonin
added
to
a solution of riboflavine
induces a
this
brown
is
The
change
due
to the
which according
to
its
position cor-
responds to the
Serotonin
changes.
is
triplet.
Lysergic acid
similarly,
1-
last
two
am
deeply obliged
Dr. D.
W.
to increase the
state into the
and
shown by
O2
solution
acid.
M serotonin or lysergic
how
How
on *
shown by more
deserve.
^
detailed studies
certainly
The
similarity of action
was surprising
since
(1955), however,
12.
and Alcohols
of
It
While a E* only
single experiment
might
suffice to
make
it
acceptable.
new
yet
which have
at all.
found no
satisfactory
Such
IONS
The main
intracellular
monovalent cation
cell
is:
fit
is
K^ while Na+
so.'^
is
why
is
this
Buswell and
Rodenbush
bigger than
K+
so.
ions
K+ do not do
The K"
ions
fit
of a defective hexagonal
to those of the
lattice.
water molecules. So
we
can expect
K+
to cause
no
than
this
K"^,
can be expected to do
so.
According
to the contents of
cell life
book
bigger than
K+ because
its
nucleus
is
less
which leads
to the
we
can
so then excitation
presence and
we
understand
ing
why
Na" out
as far as possible,
mak-
K+
its
main
80
81
In phosphorescent dyes
we have an
to demonstrate
Na
which would
in
it
be incompatible with
life
* played
itself in
major role
and
sensi-
to disorder
which declares
and thus
seconds, and
in
is
The
in
somewhat longer
acridine orange.
The
excitations,
The behavior of
22.
excited
rhodamin
RbCl
is
shown
in Fig.
The
frozen watery
weak
light emission
and
may
its
rescence or in
phy-
concentrations
emission
seen which
(as
judged by
results
on riboflavine)
might be due
to the CI anion.
In contrast to this
strong disturbance.
The
difference between
The
more
in
82
UV
lamp, three
test tubes
containing
at the side
salt at all.
The
latter
NaCl shows an
o
I/) I/)
Nl
0.001
aqueous rhodamin
solution (10^^
M)
in
CsCl.
The
weak
with no
below
means quenching.
It is
glow
at the
very impres-
The hydrate
shell of Li^
is
still
Na+ and
still
so
harm caused by
have to be
graver.
induces
mon-
in
at
low concentrations of
itself in a
In rhodamin
light emisis
quenching of
rhodamin
rather
weak
83
the difference between Li^ and
Na+ we
M concentration to a
UV
lamp, the
first,
(O.IQ-^
M)
show
practically
no
light emission.
\
The
its
may be
The dimensions of
K".
It
the
ammonium
that
NH4CI,
similarly to
KCl,
caused no disturbance in
GLUTATHIONE
One
is
is
glutathione which
concentrations as one
mM. Though
it
real biological
meaning
is
unknown.
its
It is
assumed that
acts as
with
own SH
reduced condition.
it
because then
certainly
we may
a
ask:
SH
in
to
its
do
in proteins? It
plays
most important
function.
Many
enzymes are "SH-enzymes," the intactness of the SH group of which is indispensable for activity. There are even S-containing
their
we
meaning of sulfur
its
Glutathione,
when
tested for
influence
on the
excitation of
rhodamin
clue.
in the to
Added
ways described in the previous pages, yielded no the solutions of this dye it produced the same
which
is
is
84
One
may have
major
is
biological
importance
is
lifetime.
The
lifetime of
rhodamin
greater
number of
sub-
stances have been tested for their ability to prolong this lifetime,
with negative
results.
it
to be cait
pable of extending
showed
moving phos-
M concentration the
can be
life excitation at
the low
Oo concentrations of tissues. That the lengthening of lifetime was actually due to the SH group could be shown by the fact that glutathione lost activity on oxidation of this group and that the same effect was produced by cysteine but not by cystine. There was only one difference between
the action of cysteine and glutathione: while the former acted in
acid solution only
at
(dissolved as cystine
inactive
neutral
reaction,
glutathione
at neutral
show^ed
opposite
pH
de-
but not
at acid reaction.
Cysteine
was found
the
SH
COOH
group and
its
dissociation.
Pos-
and produced
glutathione to
make
the substance
more
SH
to
do
its
The lifetime of triplet excitations varies within wide limits. Rhodamin has a short one, quinidine and acridine orange a long one. The longer the lifetime originally, the easier it is prolonged
into the 0.1 or 1 second dimension. Accordingly
it
while this effect could be achieved in rhodamin with three substances only, a greater
number of kno\\n
or
unknown
cell
85
stituents
effect
extracts
to
produce
excitation with in
quinidine
high dilution.
this for
There
is
periments.
When
how
far
is
biological action
* can
we
the phosphorescent
We
in-
may
is
due
to
an
state.'*
These sub-
making
the
a
stable
return of the excited electron from the triplet into the ground
state
(Ti -^
in Fig.
5).
It
is
stances
triplet
greater the
the
more stable allowing a saturation of the triplet level. The number of the electrons in the triplet state, the greater probability that some of them will drop back under light emisground
state.
In this case
a delay
we
photon wdth
long-lived excitation
may
declare itself in an
moving
so-
so that
it
was due
^ G transition.
86
an afterglow was produced by the addition of
1-2%
glucose to
mono-
as
ribose,
galactose,
mannose, fructose,
action.
A
if
tentative explanation
is
ment:
(e.g.,
a dye solution
it
placed in a
tube and
at
is
frozen slowly
a sheet of
is
by placing
is
in the
deep freeze
20C),
sugar
is
colorless ice
formed
in the periphery
concen-
If,
is
prior to freezing,
1-2%
added
to the
found more or
less
homogeneously distributed
between
ice
The
and dye,
it
intimate. If
triplets,
is
the
then the
The
own
"ice-
formed on freezing, or
would
better than
pure
While
at
at all, in
presence
the
1-2%
which
The
higher
the
sugar
concentration
the
stronger
the
fluorescence.
is
While
in the presence of
1%
red-orange,
in the presence of
10%
glucose
scope which does not allow the fluorescent light to pass. So glucose
way
it
seems
to render
87
it
also
more
state.
unstable, preventing
triplets
tiie
triplets
ground
its
biological bearing.
Deoxy-
and phosphoric
energy then the water structures built by the sugars and phosphates
may be
transmitting with
also
DNA.
Solutions of
DNA
None
RNA)
show
rescence on freezing.
colsysis,
long-lived
phosphorescence
stabilizer,
such as glucose, a
verj'
compo-
The weak,
short-lived
is
orange solution
due
to
two
ATP
larly
its
that of
nine,
though
also
in the
icebergs
may
play a
major
It is
icebergs
order
to
produce
long-lived
excitations
in
dyes.
88
Whetlier such an
effect will
mono- and polyvalent alcohols. As mentioned before, no triplets are formed in ice containing 5-10% glycerol. Similar is the effect
of monovalent alcohols, such as methanol or ethanol which in
2%
a remarkable parallelism
triplet excitations
on
and
this latter
may be due
Two
per cent
dum);
it
vitro.
Rho-
2%
alcohol,
0.2%
make
us heavily
drunk. In vitro
cuts
down
eliminated even by
0.06%
ethanol. In
reflected in
The
in presence of glutathione,
which
is
fairly insensitive to
0.6%
ethanol,
is
cut
down by mere
is
traces of
methanol; the
triplet state
of chlorophyll
summed up
in
Table
II
89
lifetime
may have
a certain specificity, as
shown by
life
the example
of
ATP.
only in relait
tively
ATP
produced
in a
LoNG-LiFE Phosphorescence in the Presence of Glucose, Glutathione, and ATP in Frozen Solutions of Acridine Orange, Rhodamin B, Riboflavine and Quinidine
Glucose (2%)
Acridine orange
ATP
(0.002 Af)
13.
On
One
is
cell
oxidative energy to
if
ATP,
stabilizing
it
in the
is
it
we
can
expect
approach
this
directions.
We We
could
could
known
to
on *
which they
to act
uncouple, and
we
known
on
* do uncouple
oxidative phosphor)'lation.^
When
we
will have to
go back for an
no
light, as
shown by
is
in
strict
making
in
its
transitions possible
is
which had
practically
no probability
is
absence. If there
so
also
O2 and
we
can expect
an altered
reactivity.
The O2
90
91
tron acceptor but at the same time also tunes the reactivity of this
central
member
O2
it
to
perform
its
function.
atmosphere is low, 0.01 Af, and its concenmust be considerably lower, owing to the poor
that while bringing the
O2 does not
decrease
equilibrium with
it.
The
action
maximal throughout
this
wide range of
O2 can be expected
to exert a
maximal
tissues.
re-
in the
animal
Most chemical
reactions
is
action partners. It
O2
inde-
O2 present
to
diluted
air,
had
in the solution, in balance with the be considerably lower than 10"^ Af, the concen-
we can exclude a direct interand O2; we must look out for an un-
usual
mechanism of action, based on some exceptional quality of O2 which can be brought into a direct relation with the triplet state. O2 has such a quality, being one of the very rare substances which are paramagnetic in their ground state, and paramagnetic
molecules produce a magnetic perturbation of the electromagnetic
field
which
is
known
triplet excitations
of electrons.
is
comes
an O2 molecule,
we
can
The
O2
which
it
ceases to
have a maximal
this radius
effect.
My
me
to state that
must be
100 A.
92
Summing
up,
we
life occurs in a
perturbed
a prothis
may have
for
then there
is
new
possible
mechanism
act also
drug action
which we have
the riboflavine
stances
to consider:
drugs
may
by eliminating, in
If this influence of
O2 on
an important one, then we can expect that subthis influence will interfere
which eliminate
with oxida-
an important role in
we
will have to
owes
we
afifect
triplet
excitations
its
then this
triplet
form, plays an
specifi-
on
is
riboflavine
excitation.
its
triplet
IODIDE
One
of
the
substances
phorescence of riboflavine
10"^, incompletely in 10"*
iodide.
It
quenches completely in
Its action
Al concentration.
fades out on
M. Middlebrook
rat liver in the
isolated mitochondria
from
usual way, and measured their oxygen uptake and phosphorylation in the presence of beta hydroxybut)Tate.
The
chloride in the
The
P/O
up
The
results of the
summed
As
this figure
9^
uncoupled completely by iodide in a 0.03
corresponding to
in
M concentration, while
a similar action, but
weaker quenching
abilities
had
to be applied
effect.
These
who found
^ 100
80 -
c o
60 -
40
20
c
.2
3 E
-20
94
tion
While O2 seems
to
make
transition of electrons
from the
opposite
sions
ground
state,
making the
G -^ Ti
(Fig,
M. At
higher concentrations
begins to acts as a
salt,
These
effects
its
charge or binding.
Bound
the
had
same
isomolar concentrations.
It
emission and changed the color. Tliis indicates that iodine does
not produce these changes by a direct chemical interaction but by
electromagnetic coupling.
to act as they
were
it
known from
the
The quenching
not
specific.
emission of rhodamin
perature in 10"*
solutions
(0.0001
M)
at
dry
ice
tem-
M concentration.
its
This
is
in
activity to its
and
fat ion,
by which
is
meant
all
that
it
has a great
number of
between
electrons
sorts of transitions
energy
cules.
levels,
nucleus
may
also
owing
to the
this ele-
ment and the correspondingly high positive nuclear charge. The assumption that iodide suppressed the phosphorescence of
95
riboflavine (and, possibly, also suppressed oxidative phosphoryla-
of 0.1
The white
its
color intint
yellow
showed
this actually
was the
case could be
shown by observing
no added
unstable.
NaQ. What
The
the
NaCl
no longer go
if
as
and
they did so
more
KI
is
added
in 10"^
AI
2,4-DINITROPHENOL
The
classic
uncoupler
is
2,4 dinitrophenol as
It
shown
in the
studies of
may do
agent
It is this
made
nitrophenol
reducing
is
(used
reducing body
weight).
Its
medical dose
1.2
specific weight). But even smaller single doses, such as 25 mg, can cause a rise of the basal metabolism by 30%, which would
mean
a concentration in the
is
body of 4
^^'^
^- The molecule
of
dinitrophenol
we
can suppose
to
have no
its
constituents
and
to exert
if
pharmacological action at
random
concentration. So
we want
96
uncouples oxidative phosphorylation by interfering with *, then
we have
10'^ Ai.
to
show
that
it
flavine solution
is
M dinitrophenol and
/"
4.10"
still
had a marked
So our experiment
vitro agrees
vivo,
specifically, interits
* of
function
by the O2 present.
In a way, the quenching action of dinitrophenol on the phos-
specific.
In 10"*
M con-
its
in this case
is
not a "true
quenching."
2,4-DINITRONAPHTHOL
This substance
is
Accordingly,
we
can expect
it
trophenol.
The experiment
times
in vitro
more
is
which
Her
results are
summed up
As can
97
more
tion.
it
at a
lower coficentra-
also
show
low concentration,
increases the
effect
150
too
o 50 o
5>P
98
cal eflfects different
is
more
water soluble.
2,4-DICHLOROPHENOL
Clowes and Krahi found
effects in
that
2,4-dichlorophenol
produces
phenol, which
higher
cell division.
This suggests an
mechanism
However,
its
action
is
less specific
and
it
had no appreciable
in a lO"*
was found
to
quench
concentration.
thus be ex-
same time,
showed
same time
it
cuts
have
to
be a powerful
to
and
uncouple
1^"^
^ concentration
(Smith
cor-
Membrane
The
cell
membrane
its
coming from
its
surit
roundings; with
specific permeability
on which
function depends.
tive inside
It
potential energy,
it
which
it
spends in "excitait
To
build
up
this potential
need
needs energy to
perform
its
pumping
action, to
move
needs
its
from
a physical point of
cell
contraction,
eye.
summed up
membrane
potential.
Whatever the
be, the fact
membrane
potential
say,
Ling and Gerard found that the resting poto a certain critical
was around 55
MV.
was reached
99
100
the potential collapsed altogether and contraction ensued. This
offers
good opportunity
to
compare
to 55
MV under action
answer.
If jE*
is
Duff, in England,
made
was due
to
its
interference with
*, then
it
E*
same
effect
and produce
with *; the
latter action
can be measured
in vitro.
The results obtained are illustrated by Fig. 25 taken from Hajdu and the author's paper. It shows the kymographic record of a rat diaphragm muscle, excited electrically at two different frequencies. At the arrow, 2,4-dinitro-l-naphthol was added to
the saline bathing the muscle. This addition had no immediate
effect, but, as
started to rise
The high
still
was
plenty of
ATP
available.
As
the comset-
rate of stimula-
The
Relaxation,
i.e.,
took
place with a normal speed even after the contracture began to set
in.
The energy
101
of the
the
membrane following
excitation
affected by
dinitronaphthol.
down by
energy supply.)
The
Fig.
a:
25.
rat
diaphragm muscle,
Same
with one stimulus per minute. At arrow 2.57 dinitronaphthol were added
per milliliter.
memmay
is,
brane or
pumping
action.
we
it
acts in
produces contracture
value, or produces
if it
critical
no action
dini-
per milli-
while 2.5
/j^g.
102
Five
tion
fig.
2,7-dinitrophe-
results
between the
According
to these data,
more
Comparison of
their action
on the
quenching could be obtained with a concentration of dinitronaphthol one-fourth as great as that of dinitrophenol. 3,5-Dinitrosalicylic acid
was found
was com-
Ten
fig.
sponds to a concentration of
10"*
^- ^^ ^^
on further
actually
dilution.
due
to the de-
and the author, who measured the resting membrane potential in isolated single muscle fibers and found it to disappear under
the action of dinitronaphthol, reaching the critical values at ap-
when
contracture began.
To summarize
the results,
we
E*
is
involved in maintain-
The
action of dinitronaphthol
on the membrane of
this
isolated
drug
if
ingested
mg
it
oUy solution)
shows
Then
few seconds later, all its muscles are found in violent contracture. Death occurs, evidently, when the membrane potentials reach the
103
aitical value of
was
insuffi-
our assumptions *
is
activities; so the
symptoms
in the
compound which
on the question
the greatest
depend
to
which
drug has
affinity.
According to the experience quoted, dinitroaffinity for the cell affinity for
membrane.
mitochondria
and so
picture.
its
compounds
is
knov.n
slowing
down
cell.
According,
it
was
when
same
intensity as
biological activity
be proportional to
free dinitrophenol:
its
it
in vitro action
so with unspecific
symptoms
few days
PART
III
On
"God made
If
the
little fly,
you squash
it it
will die."
15.
What
characterizes
is
modern medicine
as
compared
to the earlier
empiricism
that
it is
we have
to
first.
This
is
why-
medicine has to lag one step behind the advances in basic knowledge.
To compensate
if
knowledge can
natural consequence.
But conversely,
there
is
gap
in basic
such a
it
in the
hope
that having
is
in
mind, he will
adverse to
Most of
book
The
that there
on
and com-
adapting
In spite of the
great variations in
appearance
life is built
number of
meet
But
it;
and
in
there
no
real difference
if
appear-
variety of
also for disease, and a great symptoms can be caused by disturbance of single basic mechanisms, and the way in which disease declares itself may have no direct relation to the underlying cause. Vitamin Bi, for in-
stance,
is
all cells,
animal or
vegetable, but
the same,
its
107
108
diet of the rat, then the animal's tail drops off
It
would be erroneous
is
these acids
to
keep
tails in place.
The
The
or
is
turbance of which
diseases.
may
symptoms
The
last part
to find connections
ful to
my
traditions
for example.
The
difficulties
in
its
formed
in
tures
the latest
achievements of science, the interaction of the two necessarily belongs to the blank spaces on our
map
of knowledge. Secondly,
even
if
we
we
how
how,
for instance,
change
in stability of
an * should
So in our
first
approach
we
showing
that
which
affect
affect
function affect
E*
have exerted
that the
its
is
it
function by affecting
* we
will have to
show
drug
capable of affecting
its
E*
in vitro in the
same con-
centration as
exerts
row
have no
specific structures
can be expected
ac-
random
The higher
109
have
to
being random in
We
difficulty in
its
two * then it on
have
or
to
NO2
SCN
effect
by which
it
Our drug
duce an
in question,
if it acts
in vitro at the same concentration as related compounds which have no specific affinities, lacking the specific structural details to which specific affinity is due, but having the same active nucleus. To quote an example: if thyroxine owes its biological action to its iodine, then we can expect it to act on *
on *
in vitro in the
same concentrations
as
The
other
way
to
circumvent
this
would be
to
less specific
is
symptoms of
.
entirely specific)
If
E*
life
then
we
on *
some
extent.
unspecific
symptoms with
a specific drug,
to
needed
produce
specific action.
We
which
it
produced
its
To
illus-
specific effect
on
affects the
whole
action
basal metabolism. If
pharmacological activity
it
is
due
to
its
to affect
E*
in vitro in the
same con-
metabolism in vivo.
active concentrations in vitro
and in
vivo,
we
making
its
final statements
tube with
frozen contents.
Water has
may
be, there
is
random
distri-
110
Before closing
this chapter
I
would
like to return to
my
point
underlying
disturbance.
It
likely to
To
more
Thermodynamically,
tions, that
is,
on
a substance an
in this case
make
it
go "uphill," but
"down-
hill," that is
mem-
branes of the muscle fiber spend free energy in function, they have
to
be reenergized after and then kept in the high-energy-restingbe ready for renewed action.
is
state to
reenergization
at ease,
done
after
as
the recharging
The slower "uphill" work of completed action, when it can be done of an accumulator. The active state
state,
low energy
state,
spent in that action. So, for instance, the low energy state in
membrane
state.
An
muscle membrane
membrane
potential
may
fire
Ill
case of a poor energy supply
100
MV
shown
may
cause
making
16.
On
Iodine
and Chlorpromazine
The
list
is
not understood,
is
long one. So when attempting to correlate pharmacological activity with E* we can hardly do more than to pick one or tT\'o examples and see whether * leads us to a better understanding. I will pick "something old and something new": KI and chlorpromazine.
KI
When
was
form of KI was
it
it
did, but
did somethe
We
students used to
sum up
rhyme:
nicht weisst wo, was,
"Wenn Du
warum,
know
why
Prescribe ye then
K and
Our medical
instru-
eyes
They were
we
can
expect
in the
it
to act
on *
which
it
reaches
it
provided that
owes
its 1
KI
is
gram, which
112
113
centration of 2
is:
can
I" affect
in
M?
It has been shown earlier that iodide strongly quenches the phosphorescence of riboflavine in 10"* M. It was also indicated as
probable that
it
KI
is
not
known
(if
to affect
effects
of
its
medication
of
The experiment shows that it acts on the * of a variety substances. For instance, KI strongly quenches the yellow phosat
M.
Its action
on quinidine
is
more
having a colorful behavior. As mentioned before (Chapter 5), 10"^ KI greatly favors the transitory yellow phosphorescence. At
making the
the solution of the alkaloid has been "washed out" with pure
fast
N2. In the
is
an increased luminosity,
still
yVlKI.
KI
still
What
in
lends interest to
action
on *
is
major role
atom,
we
as free iodide
is
and do
this
/"
same concentration
to
as the
equimolar
I'.
tested
were found
bility the
Owing
^^-
to their insolu-
in higher concentrations,
10*^
Owing
to their
114
specific affinities, in the
has
come
one of
say,
nuin drugs
Needless to
CHo
I
CH2
I
CH2
I
N(CH3)2
Fig. 26. Chlorpromazine.
if
it
does
in
to
E*
then, evidently,
E* must be involved
is
in schizophrenia.
centrations in
which
it
drug action
in
is
a phenothiazine
and a member
we
E* while
As
is
known from
zine, this
tion. In
in the animal symptoms akin to hibernaorder to be able to compare this action with actions on *
drug produces
was measured
in
mice by the
author and
S.
L. Baird.
The
results,
115
originality, are
summed up
in
Table
III.^
As
tlie
depends on
to
from 8.5
140
*
mg
effect, de-
group
quantity of "thoraiine"
controls,
injected subcutaneously.
The
showing the normal basal metabolic rate. The COi production was measured individually in each animal. The numbers in Table III are the aver-
was
baryta
(F in
Fig. 27). It
was passed through a filter disk with fine pores was found that even this way CO? was lost, the air bubbles
Fig.
27.
in
116
pressing the basal metabolic rate by about one-half.
dose of 8.5
mg
effect as a
ing that within this range the drug suppresses a process necessary
for the maintenance of the normal metabolic level but not indis-
pensable for
life.
more
vitally
mg
g body weight equal to 1 ml) 0.0013 had roughly the same 0.00073-0.000044 strong effect, while the action began to fade out around 0.00002 M. As the table shows, the temperature of the animals dropped
concentration (taking 1.2
:
in basal
metabolic rate
being too large and passing too rapidly through the liquid. This difficulty
was overcome by adding 5% butyl alcohol to the baryta, which makes the bubbles smaller and makes them rise to the surface much slower. The whole apparatus consisted of six units, making it possible to measure the CO2 production of six mice simultaneously. Its essentials are shown in Fig. 12, in
which only one of the six units is represented. The experiment was performed as follows: 200 ml of 5% watery -butanol solution were placed into the glass cylinder (T), about 60 cm long and baryta 6 cm wide, closed on both ends by rubber stoppers; 10 ml of 0.2
10%
alcoholic
Then
the animal
its
was enclosed
in a small cylindrical
closed on both ends by rubber stoppers. Air was sucked through this con-
CO: in a soda lime tower (SL). The air was led from through a rubber tube and through the stainless steel filter disk F (A. H. Thomas, Philadelphia, Catalogue No. 5151 S). This filter disk was mounted
into
way
pass
it
only
was sucked out of the cylinder T with a constant negative pressure of 20 cm Hg. The air, passing through the filter, formed a fine foam which filled tube T to about its middle. The rate of airflow was regulated by the clamp-screw R. After everything was set, the baryta was allowed to run into T, the container
through
air
rinsed. The measurement began with the running in of the baryta and ended with the complete discoloration of the solution, the interval being
117
could have been the consequence of the decreased temperature.
this case
we have
only
drug exerted
its
action
its
own
excitational states,
its
own
Table III Effect of Varying Amounts of Thorazine (Chlorpromazine) on THE Basal Metabolic Rate and Body Temperature of Swiss Albino Mice
%
Thorazine Milliinjected
Decrease
in
Over-all
BMR
Time needed
for
grams
per
concentration
in the
three hours
after injection
complete
(mg)
kg
animal
recovery
10
118
molecules.-^ In 2
^^'^ ^^
^^
emission of riboflavine. In 2
it
rhodamin B,
also
if
the
as Ritaline or
thiamine. So
it is
exerts
it
pharmacological
is
activit)'.
new group
it
of
substances, to
sible that
it
is
posit
Many
and
structure to quinidine
that
it
is
0.125% methanol)
at
it
was strong
0.00017
M and
noticeable at 5
it
'^^'^
^-
riboflavine
favored the
triplet.
All this shows the high and colorful reactivity of chlorpromazine in relation to
its
/ vitro in concentrations in
which
it
exerts
its
molecule
actually exerts
If
it
its
influence
on biologi-
influences schizophrenic
metabolism
this
may be due
either to
its
accumulation in certain
We can expect, accordingly, oxidative phosphorylation to be uncoupled by chlorpromazine. This to be the case has been shown lately by Berger.
^
Strccker,
and Waelsh.
119
KI and chlorpromazine, were picked at random. Their strong action on E* and the similarity of concentrations in which these drugs acted on * /w v/tro and exThe two examples
discussed,
such as actions
which were
M.
was found
to
be even more
ac-
completely quenched
10~*
in 10"^
from whitish-blue
deep blue.
17.
On Myotonia
fascinating
for
the experimental
re-
man
Owing
to the gener-
Mr. R. Lombardi, of Philadelphia, Pennsylvania, the auhappy owner of a small herd of such animals in which myotonia is transmitted from generation to generation. G. L. Brown and Harvey showed that this myotonic behavior is due to
thor
is
the
nerve
is
one wave of depolarization, the end plate fires a whole train of them. Qjrresponding to this, if a resting myotonic animal wants
to
make
a sudden
then
start
stiffens
up
for a while
and will
to get
balance and
up
As
in
human
symptoms
its
starts
moving
it
symp-
No
elicited
Brown
The
method
ground.
then
is
consists of
The animal
to
was trained
find food
could
and
is
measured
up and
121
My
goats needed 7 to 20
Under standard conditions, the experiment yields for one and the same animal surprisingly reproducible results, the running time differing on various days by no more
than
to 3 seconds.
to the attractions of
myotonia
that the
symptoms of
this disease
by the administration of a drug, 2,4-dichlorophenoxyacetic acid, commonly called 2,4D. Bucher has discovered that this drug produces myotonic symptoms in laboratory animals such as mice and
rats.
showed
that the
electric
is
similar to that of
waves of
excitation.
most
interest-
also
What known
is
2,4D
is
is
widely
used as a weed
showing the
on
which the animal and vegetable world are built. When trying to connect myotonia with * one could
trying to
start
with
show
that
same concentration
itary
its
elicits
essentially, a disturbance of
*, related in
2,4D per kilogram ingested parenterally to rats is lethal, while 200 mg causes grave myotonic symptoms. The author repeated these experiments in Swiss albino mice and
earlier
mg of
shows the
COi..
summed up in Table IV. The production. The methods used here were
As can
the
same
be seen, the myotonic symptoms were accompanied by a drop in the basal metabolic rate depressed by 200 mg to about one-third
of
its
mg
it
to one-
122
quarter, keeping the animal for days in a comatous condition in
which signs of
ture to
life
The drop
in basal
22C
or so, the
The drop
in the
difficult to say
how
far
The
gravity
Table IV
Basal Metabolic Rate and Myotonic Symptoms in 2,4D Poisoning in Mice
2,4D injected
123
ergy relations of that system which declared
itself in
the decrease
may
may prevent
if
from the
usually
is
which
is
somewhat lower. So
insufficient to
two
states
may
become
We
was surprising
On
possessed by 2,4-dichlorophenol.
One
As has been shown beof rhodamin from 10"^ While a pure rhodamin
showed
M glutathione
it
showed
a strong phosphorescence even in the slow moving phosphoroscope and showed a strong afterglow. Addition of 10"^
2,4D completely abolished this long life phosphorescence. In its presence the tube showed even in the fast moving phosphoroscope no increased phosphorescence; 2,4D thus completely eliminates
the long life excitation caused by 0.01
cose. Since
M glutathione or 2%
glu-
less
2,4D had
the stability of the triplet state. If such states are important for the
cell
it is
2,4D has
profound disturbance
that
in en-
M concentration; 2 X
124
the triplet state.
As has been
is
emission of rhodamin
cence
sion
is
red. It
was noticed
to
light emisit
from red
orange in
concentrations in which
its
reduced
able to do so
absence.
the
an improbable one.
riboflavine,
is
emission orange.
2,4D turned
from orange
into greenish-yellow,
came
to a great extent
from
which indicated
cence was not a "delayed" one, did not occur because the excitations
but occurred
still
went
into
is,
a de-
was
that 2,4D-dinitro-l-naphthol,
jB*,
There are
not localized
for in-
It is
known,
stance, that
also
my
goats
though no
125
records have been kept
on
this.
There
is
man,
too,
myotonia
is
some
is
As
is
generally
known, myotonia
The
dis-
worth noting in
this
the
more remarkable
tains
patients
showing a
idiocy.
Myotonia
is
and
has, consequently,
it
may
be.
What may
it is its
close rela-
The symptoms
is
opposite:
what
is
positive in the
one
is
The two
it
more than
myotonic one
is
worst
when
starting activity
out with exercise, while the myasthenic muscle does best after rest
and
tire rapidly.
dis-
The experiments
^
This cataract
is
known
for
its
it
it
appeared
while
after a
prolonged and
numbers from the slaughterhouse, I found most of them colorless. A small number, however, about 2-3%, were yellow, which color seemed to be due to the oxidized state of the flavines present. This color may indicate a sluggishness of the energy household, which might be in connection with the ease by which cataract is contracted under influence of a dinitrophenol
medication.
126
molecular mechanism of myotonia unsolved.
It
supports the
as-
in this dis-
owing
may be due
case, the
2,4D. In any
ther experimentation.
They
more stable and transition into the more probable. There are substances which act this way. Some of them have been mentioned before: iodide, or iodine in its organically bound form. There are other such substances, even among normal cell constituents. As has been shown, serosubstances rendering triplets
triplet state
tonine
tal
is
work
possible
on these
lines
and
this
lead not only to a better understanding of the degenerative diseases of muscle but also
may
reveal
on
this line
may
find
One
known
to
to
have a beneficial
eflfect
on myas-
be true even to a higher degree in other drugs, having a similar but stronger action in *.
relieved in
man
by quinidine.
It
L. Baird
(unpublished)
found the
It
in myotonia, induced in
mice by 2,4D.
may
18.
On
the
Thymus
The
tients.
There has
to be thus
some
relation
between
this disease
and
tonia
is
its
between
this
may be connected
size of the
it
There
is
a curious disproportionality
between the
its
function. If
at all,
it is
knowledge about
its
function
and Thimann, have not even a chapter on it. This lack of interest is probably due to the fact that the thymus can be extirpated without harmful
it
effects. If
it
it is
by letting
its
atrophy in adolescence. If
we
it,
then
ble.
impecca-
her
The question is only whether nature does not follow a logic of own? Histologically, the thymus is a lymphoid tissue, part of
it
the lymphatic system, together with the spleen, bone marrow, and
if
the thymus
is
may
lymphatic system
one, although
One
protein,
is
which substance
on most
is
in greater
demand when
So
the building
going
actively,
in early childhood.
in this
127
128
phatic system
lescence,
retires.
when
why
function
important
because
the
be (for
all
we know)
all,
that the
one big gland of internal secretion, may be even the biggest and
most important of
a possibility
it is
too obvious.
also explain
why thymus
extirpation
is
due
to
far this
would
An
adult
dog
will out-
run almost any other animal, but a three-year-old child (which has
nearly the same
amount of muscle
lift
for
body weight)
going on,
tires in a short
walk and
it
is
unable to
a heavy weight.
is
to
should proceed
kills
it is
possible that
which
is
The
thymus (and,
possibly, other
synthesis finds
some support
129
terest gradually
The hormonal
more
recently in experiments of
in
also
showed
While
While
produced by the
latter,
There
is
to
sum up
subject, this
One
possibilities
decade
of a substance, in
itself,
What
on
its
is
unknown
The
we have
test.
some-
This
is
a circle which,
somehow, has
to be broken.
constituents of the
if
effects in the
animal only
myo-
test
hormone par
130
Fig.
28.
goats. Abscissa:
days. Ordinate:
extract.
Arrows
tained in 5
ml
g frozen
calf
thymus,
was
down by methanol but was precipitated by an excess The results of this experiment are reproduced in Fig.
Kg
The
^
Prep.
liters
16
28
4l4 ml of
water.
liters
of
The
131
as
constant as in previous
work with
The
The
results
seemed positive
in
the
case
of
"Bent Horn,"
of myotonia.
The
extracts
had no acute
effect
and developed
their
and disapthe
peared gradually.
The
when
their concentration
is
The myotonic
goat
is
not an ideal
test object.
The
size
and smell
are time
any, will
of the animal are bigger than desired; the expense and labor in-
volved in keeping
it
are considerable.
The measurements
if
consuming. So the
final isolation
the clear liquid concentrated in vacuo at 20-23 C in two steps to 1.4 liter. The fluid was stored overnight at 20C, on thawing the precipitate removed on a small Sharpies centrifuge. Four volumes of methanol were added,
the precipitate separated, the fluid stored at
precipitate
20C,
to
separated
again.
to
The
fluid,
when
liberated
its
from
its
methanol
content,
was poisonous
mice, probably
owing
potassium content.
Concentrated sulphuric acid was added gradually under strong mixing, whereupon a white crystalline precipitate formed, presumably consisting of K2SO4. H2SO4 was added until the addition of 10% sulphuric acid caused no appreciable precipitate. The pH at this state was about 5. The solution was concentrated in vacuo to 500 ml. Two liters of acetone were added, the precipitate separated, and shaken out with acetone. The supemates were rejected and the semi-liquid precipitate redissolved in 500 ml water. This fraction was injected after neutralization, a small quantity of
penicillin being
'
added
to prevent infection.
On
the
first
was measured
had no
was concluded
acute effect and subsequently the running times were measured only before
132
have to await the development of a simpler
test,
or the setting
up
The
thymus
is
the
more
urgent because
is
There are
also a
few observations
thymus
may
be, in
and thus
The
extraordinar)' wealth
this.
of the thymus in
DNA
may be taken
as
an indication of
My
thymus
extracts
if
added
in a higher concentration.
The
under the
UV
lamp)
shifted
was quenched
my
thymus
The
acridine-dye comit
was not
sur-
Goland found
An
attempt
seems to be indicated to
as therapeutic
is
suspected to
be the culprit.
especially
isolated earlier
19.
On
Of
to
the
was given
book
is
is
no reason
suppose that E*
essentially,
in coupling
re-
arranging the
H
P
and
in such a
way
"low-energ)^"
OP becomes
is
~P
which
is
ATP, and
Fermentation, essentially,
a series of
which belong
to
and
to
and transferred
tion.
form of
electronic excita-
We
were
also led to
suppose that
has specific forms which were probable only within the water
structures
itself,
the elec-
We
electro-
magnetic
the transition
probabilities of
Transition probabilities of triplets greatly depend on the perturbations of the electromagnetic field
and so have
to
depend on the
triplets.
Aerobic
cell life
has thus as
133
134
its
field,
the perturbation
summed up
is
By
this
meant a
two
cal
example of which
found
in yeast.
if
Under anaerobic
is
conditions
it
yeast lives
on fermentation, but
oxygen
admitted
shunts
over to oxidation.
The mechanism
its
many
theories have
explanation.
If O2,
Our
its
by
mere presence
new
that
then
it
new
paramagnetic O2 molecule.
has
No
shown
is
it
has
its
biological
meaning and
is
not accidental.
interrelation
is
Another intriguing
studies of O.
that of pathological
is
growth
known from
the classical
Warburg, cancer
change
cells
on
pri-
cells,
mary
role to this
and
production then
it
is
difficult to see
why
The
trans-
by the energies of
triplet excitations
135
bation
is
started
up by oxygen and
priming
action.
is
formed under
magnetic
ment.
its
Aerobic
instru-
Warburg
has
structure, oxidation
is
not.
We
state,"
with periodicities.
In this book
liness
we have extended
and order-
and shown that the two structures form one single unique system.
It
is
The water
the excited electrons and can thus be expected to play also a funda-
more
direct connec-
and
it
need not necessarily be invested into '^P's before they can be used
by the
cell
may
also be coupled
more
directly to oxidation
and use
its
excitation energies.
The immediate
on removal of
used also
excita-
its
So
if
to collapse, structures
must
disinte-
which the
cell is
conditions,
is
demand no
136
structures, neither structures of water
perturbed electromagnetic
aerobic one,
field.
The
way of
living,
the
demands
It
and
its
seems reasonable
to
suppose that
triplet excitations,
and energy transmissions which presuppose an orderly structure, play a role not only in oxidation but also in the most varied biological functions
and
their regulations,
structure
which
is
triplets
may belong
to these
orderly mytotic
to order, structure,
and what
goes with
it.
Looking
at cancer
relation to oxidation
and fermentation
from
clearer. If
O2
is
with-
however, O2
we
demand
structure
It
would be
living,
and respiring
and compare
it
as well as dead, cells. If the suggestions contained in this chapter are correct,
the
first
should be diamagnetic.
cells.
shift in
The
should show
less
frequency dependence
and with
it
No
flavine
light
in
in
illuminated
a
solutions
it is
by
placing
them
magnetic
of
about
20,000
oersted. So
more involved
inter-
O2 molecule and
137
gization.
The
cell will
thus revert to a
life
with
its
not dependent on
ordered structures, which also lacks the subtle regulations and has
thus
(as
in yeast)
its
charac-
teristics,
tion.
20. Conclusion
After
chapters,
details
through nineteen
we may
was about.
Going back
should see
its
we
not
outlines
somewhat
it is
clearer
now. Biochemistry
its
is
an independent principle;
ress
dependent for
advance on prog-
made
first
are definitely
The
that matter
was
built of
small,
indivisible units,
atoms.
What
how many
to isolate
about
hundred
The
dif-
means of
letters
bonds in
their
P O P), following up the various atoms and way through the maze of biological reactions.
step of the history of chemistry
was heralded by came of age in 1927 with Bohr and his orbits in Schrodinger's wave equation. In this theory, the atom is no more an indivisible unit, but a cloud of electrons, or more exactly, a
1913. The
theory
The second
cloud
of
the
probabilit^'-densities
of electrons-
Molecules are
138
139
dusters of such clouds of phantastic and changing shape.
great
all
is
these clouds
which may
be
in-
which take
place in dimensions
to the
its
unknown
to classical chemistry.
They belong
language.
yet,
still
has
made no
described
be
by
these
symbols.
"chiaroscuro."
This
is
The author
He
who know
He
backs on
him
becoming
insur-
mountable.
Lucretian biochemistry involves the assumption that no interaction can take place between molecules without their touching one
another. Support
interactions
is
which
The
140
material presented in this book pleads for the assumption that
water
itself.
is
more than this: it is part and parcel of the living matter One of the main functions of protoplasmic structures may
make forms
would be improbable outside these structures. The solid matter and the water of the cell form together that unique system which
has the queer property of being alive.
I
quantum mechanical biochemistry, built on top of the Lucretian one. This book may be but one of the early swallows of this
spring.
The
new
field,
can do so only
at at
many of
his interpretations
being found
He
left ajar,
researchers will follow him who are more qualified to deal with quantum mechanical problems and find him at fault at many
points.
at a future biochemistry,
letter-
us no
more about
saurus can
tell
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Nature of Life
A STUDY ON MUSCLE
By A. Szent-Gyorgyi
^\_;w
CONTENTS:
Histological, Molecular
The Muscle
Fibre.
REVIEW:
"The study of muscle metabolism and
ing muscle contraction and relaxation
the energy transformations durcloser to the physiology of the
come
basic processes of living matter than almost any other studies in the field
life."
Chemistry of Muscular
Contraction
SECOND EDITION, REVISED AND ENLARGED
By A. Szent-Gyorgyi
1951, 162 pp.. 55 illustrations,
$150
was
the
The
first
edition
new
vistas for
our understanding
of muscular contraction.
are
The experimental
now
new muscle
physiology.
have been
sifted
in the
United
States.
They
are
now
new
lines
such as electron
made
to corre-
The
rial.
work
is
a craving to understand
life,
Using muscle
a5 experimental mate-
CONTENTS:
Introduction
The
Structure of Muscle
Thermodynamics
Energetics and Mechanics
Whole Muscle
The
Physical Disintegration of
Muscle
of
"^^^^
Continuum Theory
Myosin
Actin
Actomvosin
Actin, actomyosin,
its
reactions with
ATP,
fit
all
and
his associates
were
reported in the
first
two
editions of
studies
CONTENTS:
The Molecular Mechanism
OF Motion:
Problems of Molecular Engineering Myosin and Meromyosins
Actin
Muscle
Fiber:
Actomyosin
The
Protofibril
The Action
of
Serum
^^^^""
ATP, ATP-ase. CP, and
Relaxation
Elasticity and Plasticity Thermodynamics, Isotonic and
Digitalis
Adrenaline, Acetyl
Cholme (ACH),
Isometric Registration
Interpretations
The Membrane
The Window
Field
(WF)
Biological Action