98

On protoplasmic structure and functions.

(A survey)

(Leotare given before the X. anniversary meeting of the Hungsrien
   Physiclog. Soo. at Budepest on Mey 3Otb, 1940)

                       BY

A. SZENT-GYoRi%YI
With 3 plates and 54 figures.
   (~.VII.40.}

I. InWuetio~.

It is the tradition of this society find its pride in the honesty and ardour of the expe-
rimental work presented. Accordingly, I will celebrate this occasion of our tenth anniversary
by simply putting before you a part of the work done at my laboratory this academic year.
So you must not expect a survey of some well finished field; I will rather lead you into the
midst of the problems that keep our minds and hands busy.

To make myself intelligible, I will have to present, along with facts, 8lSO the theories that
have led us, but you are free to drop them at the exit taking with you only the solid results
obta'ined.

Let me start with my most profound conviction that has always led my research: that
Nature works only with a few basic principles, and the most varied expressions of life, like mus-
cular contraction and excretion of urine might be but the app~cation of the same fundamental
process. This is no pure speculation. Such ideas have immediate practical consequences. If you
accept them as your working hypothesis funny things might happen to you. You might start -
as happened to me some years ago -with the study of the function of the adrenal cortex, be
led to the study of oxidation and finish up with the isolation of a Vit8lXIiKI, or, as in the present
case, you might start with the study of muscular contraction and finish with a virus theory.
Also it will matter little to you which organ or whet special function you choose for study,
for you will expect that the basic processes of life will be the same in all. So you might start with
the study of an organ which Iends itself specially well to measurement and take muscle the
function of which is of a rough mechanical nature.

        II. On the chemistry of mnsoular w&a&ion.
Our work on this line bad as a starting point a little experiment performed last

year at Liege by BAUQ, GOFFA~XT and SZENT-GY~R~YI 1). We took two frog muscles,
perfused them with RINGER'S solution containing some veratriue. We made the fluid
pass through one musale and then put it through the second. There was no other oonnec-
tion between the two than the fluid perfusing both. Now if the first muscle was
made to contract the seaond muscle contracted too. Thus the liquid transmitted muscular
~ntraction from one muscle to the other and we had, what a physiologist would call a
,&bmordl trasnsmission of mtnuscular contraction". The train of thought which made us
perform this experiment was the following: if a muscle oontracts something has to happen
in it and when the muscle relaxes then the process must be reverted. What this ,,some-
thing" is, we do not know but it might be the production of a substance which makes
the muscXe contra&, the opposite prooess being the disappe~an~ of this substance.


ON PROTOPLASM10 STRUOTURIP AND FUNOTIONS           99

Singe contra&ion and relaxation take place very fast, within a few thousands of a second,
it is evident that this hypothetical ,,contractiOn-s&stance" must disappear just as fast
as it is formed, and we oan never hope to catch it in the fluid passing through the vas-
cular system of the musole. According to this all attempts to produce a ,,72umoral trans-
mSon of muscular contra&m" had hitherto failed. One could hope to demonstrate such
a humoral transmission only if one could slow down the disappearance of that hypothe-
tical ,,contraction substance". Now, if this slowing down would be done by means of a
pharmacological agent, what would be the effect of this substance on muscular oontrac-
tion? Evidently it would prolong the muscular twitch.

There is in fact a classical pharmacological agent, which has this effeot:
vera trine. If a muscleis treated with veratrineit willgive a prolonged contraction inst.ead
of a rapid twitch, which might be explained hypothetically by the assumption that
veratrine prolonges the persistence of the ,,wntractGm-substance". But if this is so we
might hope to catch the substance in the perfusion fluid and produoe by it a contraction
in a seoond veratrinised muscle. This, as I told you, aotually happened.

The first question, any chemist would ask, is: what was this substance which trans-
mitted contraction? The substance was potassium. With this observation the problem
became most exciting, for all bioohemists feel that potassium has something to do with
the most basic, simple cell function: excitation, - only we do not know what. BAOQ
and GOFFART a, 3, showed later that a great number of very simple sea animals give
the same reactions to veratrine, demonstrating in this way that veratrine attacks a basio
oellular function. That this is so, and veratrine influences some basic protoplasmio
reactions, is also clearly brought out by the fact that the effect of veratrine in the animal
body is not limited to musole. While it gives a prolonged contra&ion in musctle, in the
salivary gland it gives a prolonged saliva secretion. This effect might be taken as
evidence for my above statement that basic fun&ions of the cell, in spite of their varied
appearance, are the same, and muscular oontraction and saliva production have the
same primitive processes at their foundation.

/For :rne (personally, the'problem became especially fascinating, for my earlier studies on
metal complexes have led me to the conviction that the formation and splitting of metal com-
plexes might be the most primitive process of life. I strongly believe that the very first primitive
living molecule, formed somewhere in the ocean innumerable years ago, started its life by bin-
ding ions of its surrounding and using them as chemical tools. Now K, acoording to its distri-
bution, is the most important metal atom of living Nature.

  There are observations suggesting that potassium is intimately involved in muscular
contraction. Muscle contains a great deal of potassium and relatively little sodium and the
potassium is located, after all probability, in the contractile fibrill.
ERNST and FRIECKER 4) showed that the major part of this potassium is present in the
,,&u&" form and that K is ,&berated" in contraction. ERNST has also described a most
intriguing phenomenon: the ,,volume-contraction of muscle" (5). ,,VoZunzs contraction" means
that if the muscle contracts, its total volume decreases. ERNST and Kocetis 6) have shown
that this volume contraction is a very fast process, and lasts only a few u, i. e. a few
thousands of a second. It is also a very early process. I mean to say that if the muscle is
made to contract the first thing we can observe will be the action current and the volume
contraction, both processes being followed only by the shortening of muscle.
  A muscle can be looked upon as a watery solution of a certain structure. But now how
can watery solution decrease in its volume? There is only one way we know of: by the formation
of ions. If ions are dissolved in water they will bind water molecules, pull them together. This
will lead to a decrease of the volume of the fluid. Evidently the volume-contraction of muscle


100                   A. BZENT-GYiSRQYI

has a similar foundation. Thus this volume-contraction gives us a very aocurste means for
measuring the rate of production of ions within the muscle.
We can also tell which kind of ions might play a major part in this volume contraction,
for the number of ions possibly formed in sufficient concentration is very limited. In faat there
are only two such substances which could come into oonsideration: potassium and phosphate.
Thus we can limit our attention to these elements.
On my return to the laboratory last autumn my first action W8S to seoure the co~aboratioR
of E. ERNST and ask him to see what verattine does to the volume contraction of muscle.

The experiments clearly showed that the action of veratrine was no other than to
prolong volume-contraction i. e. prolong the existence of the liberated ions (plate I,
fig. 1). Evidently the prolonged presence of ions is the cause of the prolonged contraction
of the muscle, showing that production tbnd presence of ions is involved in contrao-
tion while relaxation must be connected with the disappearance, i. e. binding of these ions.
Further experiments of ERNST and M~ROCZ have also shown that under action of vera-
trine more potassium is ,&berated" and also given off by the muscle to the perfusion fluid.

          III. cfeneral ranarks on the shape of molecules.
As you know the mu&e fibre is built up of a great number of fibrills, roughly 1 p
in diameter, whiah are aotually the contr~t~e elements. Between the fibrills we find
the sareoplasm. If we make an extract of the miuoed muscle with a dilute saline
solution the soluble proteins, about l/S of the total protein, go into the extra&.
The fibrils are insoluble. They are built up of a specific protein, carefully studied
by EDSALL *) 8nd EDSALL and WJRALT 8). It is a globulin, but is, czontrary to most
other globulins, soluble only in strong, alkaline salt solutions. If the muscle is extraoted
with EDSALL'S salt mixture, part of the myosine goes into ,,solution". This myosine
is about 40-50 o/0 of the total protein. The extracted muscle, looking at it under the
microscope, has apparently retained its morphological structure but shows a strong
tendency to disintegrate into transversal discs, corresponding to the former transve~al
striation. The muscle has thus, by the dissolution of myosiue, lost its longitudinal
re8istance.

Myosine has, as compared to the greatest number of other proteins one very
peculiar property: its molecules are fibrous, i. e. long and thin, rodlike, while most other
soluble proteins are globular.

The rodshape lends very special physical properties to the molecules, which are greatly
different from the physical properties of globular proteins so that the division of protein mole-
cules into globular and fibrous molecules is very distinct.
H. STAUDINQER 0) and his collaborators, who have given much attention to the highly
polymer fibrous molecules, have shown that these molecules do not fold or roll UP but are
stretched out and behave like rods.

We can convince ourselves of the great difference of physical properties due to the rod or
globular shape by comparing the qualities of a heap of marbles aDd wooden tooth picks. The
marbles, if throw-n over a heap, will readily roll apart, and show great mobility. They will be
unable to form a texture or structure of any kind. We can make them into a solid lump only
by glueing them together but once the single balls part there will be nothing to hold them to-
gether and they will move freely relative to each other or to the medium. They will also be
u n 8 b 1 e thus to enclose a considerable quantity of water between them: to s we 11.

But if we throw toothpicks on a heap in a disorderly fashion they will form a three dimen-
sional, more or less solid texture (plate I, fig. 2). We can thus see that only this rodshape is


Tab. I.

  a.         L.
Fig. 1.  Volume contrucliun of
muscle. Single impulse. a. nor-
mal, b. veratrinised muscle
(frog). 3. impulse, 2. time in
seconds, 3. meniscus of fluid
marking the volumetontmct ion.
E. ERNST, unpublished rxprri-

mats.

Fig. 3.


ON PROTOPLASM10 STBUOTURBl AND FUNOTIONE          101

capable of giving 8 solid structure. The space taken up by this structure is many fold biggev
than the space actually occupied by the toothpicks themselves (plate I, fig. 2). If this structure
is immersed in water the quantity of water enclosed will be very big as compared to the bulk
of the toothpicks. In colloidal dimensions this would correspond to 8 strong ability of
swelling. If the structure is disintegrated, the single long toothpicks can be moved only with
difficulty through the fluid, as compared to the marbles and the single toothpicks, if not
taken widely apart, will easily inhibit each other in their motion. Again, in colloidal dimen-
sions, this means a high viscosity. Thus B w e 11 in g an d high vi s c o sit y at low concen-
tration, both can be taken as indication of the rod-shape of particles.

Rod-shaped particles, if standing in a solution, have often a tendency to aggregate in
such a way as to give, so to say, a scaffolding within the solvent, which resist deformation. In
this way solutions of rod shaped molecules might show a certain elasticity, and an abnormally
high viscosity. This is called thixotropy. This scaffolding can often be broken up by me-
ohanical agitation whereby the fluid suddenly looses its elasticity and great viscosity. Since
globular molecules are unable to give such a scaffolding thixotropy can also be taken 89 indi-
cation of the rod-shape of molecules.

Rod shaped particles have also the tendency to associate ,,end to end" forming in
this way, long and thin micells. We can demonstrate this by spreading matohes on a water
surface. (plate I, fig. 3).

If we dry down such a colloid the long particles will entangle and form resinous, elastic
masses while globular proteins give powders (STAUDIN~ER).
  From all this we can deduce that wherever nature needs a protein with a certain
mobility it will resort to the globular form. Wherever the protein will have 8 mechanical
function, command motion of water by its swelling or give a solid structure, Nature will resort
to the rod-shape.

In agreement with this we lind myosiue built up of long, rod-shape molecules which
form still longer mioells. In the muscle the molecules resp. micells are arranged coaxially, pa-
rallel to eachother as shown by their double refraction and X-ray spectrum. The single mice118
8re joined lengthwise giving the mechanical resistance of muscle to stretching. These long
bundles of myosine micells can be looked upon as primitive fib&, separated by intermicellar
spaces (WEBER lo)). One microscopic fibril is built up of a very great number, many millions of
such primitive fibrils.

  The double refraction of muscle shows that within the snisotropic discs these primitive
fibrils are arranged parallel to each other. In the isotropic discs the arrangement is less regular,
so that we can suppose that at the borderline of the two segments the relative position of the
primitive fibrils is fixed.

  While the intermicellar spaces allow a lateral displacement and accumulation of water
the fibrils resist stretching. Measurements of ERNST 11) and others have shown that resting
muscle needs 8 greater weight for its stretching than it is able to lift.

Iv.  &3ometric8l consider8tionS about muscuhr contr8ction.

If ions are produced at certain point,5 of 8 system like muscle, this must lead to 8 shift of
water either by the osmotic activity of the ions or by their influence on the swelling of colloids.
It is not a novel idea that such a shift of water might be the immediate cause of contrac-
tion. But how can a shift of water lead to a shortening of the system, to contraction?
We have agreed that the anisotropic part of the muscle fibril is built up of a great number
of relatively rigid, coaxially arranged primitive fibrils, micell-bundles, the ends of which are
fixed while water can be accumulated in the intermicellar spaoes. Now how will the length of
such a system be changed by the accumulation of water?
Let us consider this question first by taking into account geometrical considerations
only and let US, to start with, simplify the problem as far as possible. Let us take, in analogy


102                   A. SZENT-QT6RQYI

to the primitive fibrik, two threads (plate I, fig. 4a), which c8n not be stretched and the ends
of which are fixed but which can be pushed apart. To make it still simpler let us suppose
that they can move in the plane of the paper only. Now what will happen if water is accumu-
lated between these two threads? Since the threads do not allow stretching it is evident that
they will be pushed apart. In the extreme case they will be blown up into a circle (plate I,
fig. 4b) and will come to lie on the periphery of this circle and the new length of the system
will be its diameter. In the extreme case the system shortens thus to 2/u = 0,64, i. 8. 36o/o or
l/6. Evidently the same would happen in a three dimensional system also.

Though this simple geometrical consideration is indoubtedly correct it was desirable to
demonstrate its applicability in a model-experiment. In such a model we c8n replace the pri-
mitive fibrils by threads of cotton wool or Bilk, the problem is only how to accumulate water
between these threads so that it should not run out. This w&s done by E. ERNST, M. GERENDAEJ
and myself in three different ways.

In our first model (plate II, fig, la) threads of cotton wool were pulled through perforated
oelluloid discs. These discs serve to fix the threads at intervals. The space between the threads
was filled with liquified 50 o/o gelatine. After the gelatine has solidified the system W&B immer-
sed in water. It was expected that the gelatine would swell and thus attract and bind water
between the threads. It was found that in few days time the gel&tine actually swelled up and
the threads, whioh were parallel and straight before, now lay on the periphery of a circle
and that the whole system had shortened (plate II, fig. lb).

In our second model (plate II, fig. 2) thresds of artificial silk were wetted, pulled through
powdered agar-agar, receiving hereby a coating of this substance. After drying, the procedure
~8s repeated several times to make the coating thicker. Then the threads were joined to make
bundles and fixed at intervals by circular ligatures. It was expected that in this case the
water will be accumulated and retained by the swelling agar-agar.

On immersing the system into water the agar quickly swelled up pushing the threads apart
and lending a globular shape to the single segments. Within a few minutes the system shortened
more than 10%. If the time required for equilibrium is proportional to the dimensions of the
system it can be calculated that at the dimensions of the muscle fibril the shortening has to
ttlke plsce within 8 few u (0,001 sea).

In a third model (plate II, fig. S), especially fit for demonstration, a long rubber con-
dome was pulled through a series of celluloid rings. The rings were perforated and cotton
threads pulled through the holes. In this model the water is replaced by air. If blown up the
structure shortened 20%.

Only 8fter we finished playing about with these models did we find that as early as in 1897
MCDOUGALL 12) presented a theory according to which contraction of stri8ted muscle is due to a
shift of water from the sarcoplasm into the fib&, whicrh latter have 8 special structure which
shortens if distended. Later E. B. MEIoS 13 , who advocated this theory, constructed a model
almost identical with our model 3. This theory, t, owever, found no general acceptance (0. v. FOURTH 143
pertly because it admits only a shortening by l/3 while muscle is known to shorten more than 50%,
P artly because at that time the results of modern X-ray studies were not yet available and we have
earned only sinoe that the muscle fibrill itself is aotually composed of smaller, primitive fibrillse,
which structure makes such a function possible. So BERNSTEIN 15), who discussed these theories and
constructed a model very similar to that of MEIGS denies any physiolo~c&l ~por~ce to these
oonsideretions and calls his own model a useless toy. I will show presently how these considerations
admit also a contraction of more than l/3.

Naturally, whether muscular contra&ion actually has such a shift of water at its base
remains to be shown. What I want to make clear is only, that if we have a structure,
composed of relatively rigid fibres with fixed ends, and if water accumulates between these
fibres, the structure must shorten. Such a mechanism, if applied to the muscle, easily explains
many known facts. I want to mention here only two of these: the decrease of the intensity of
double refraction and of the distinctness of the X-ray spectrum of muscle in isotonic contraction.
Both, double refraction and X-ray spectrum persist in isometrio contraction. The explanation
of these facts by means of the theory presented is evident: in isotonic contraction the elements of


Fig. :$.


On protoplasmic structure CLH~ frructiorzs.

Fig. 1.

Fig. 2.

Fig. 4.


the mu&e 10~6 their parallel arrangement while this element persists in isometric contrac~on.

Theoretically there are different possible ways of applying the above geometrical oonail
derations to muso& It conld be thought that the primitive fibrills stretch within the fibril-
through the whole length of the allotropic
Q discs, their ends being fixed at their en-
trance into the isotropic I disc. In this ot~e,
if water would accumulate in the intermicellary
spaces the whole structure would show in itg
cross section an onionlike structure which is
~p~sented very ~o~ematic~ly in fig. 1.

We could, however, equally well suppose
that the speces between the miaell-bundles do
not stretch from 1 to 1 dim but are much shore
ter. The geometrical principle could be applied
to this structure also, Very s~#m8ti~lly this
is shown in fig. 2.

Finally, the same principle could be ap-
plied also to the single n&ells, as shown in
plate III, fig. 1. In this case the rigid fibrillary
structure corresponds to the C-backbone of the
single myosine molecules.                      a    lsg.1. b

a

Extending our geomet~~a~ consideration we could also think of the same
principle as being applied repeatedly in different dimensions. I& us take the
structure represented in fig. 2. This structure could 8nanBwer for a contraction
of 1/S. If within this st,r&ure the single micells contract t&o, the system
could show a further &or&.&g of l/3. But if the fibril takes up wster, this way
than the single primitive fibfill8e till not t&r&& any more in 8 sfxsight line from
I disc to I disc,, but the whole Q segment swells up and acquires an onionlike
st~~ture (as in fig, s), which alao means a further possibility of shortening.
                 Speculating for one more minute

we can also imagine a water uptake
that Ieads to a 8tretehing of the fibrill,
Let us glance once more st fig. 2 b. If
in this struct~e water uptake boomed
excessive and breaks the lateral oonnec-
tions of the mice11 bundles the singIe
small intermicsllar spaces unite into long
splite, which means lengthening. Also,
if we suppose water aooumulat~g in plate
III, fig. 1 between the n&ells snd preb
sing the mice& t~~tber, this w8ter 8ooa-
mu&ion will also lead to a lengthening.

Thus geometrical oonsidsrations allow
US to picture a water uptake that lemds
to ~orte~g and a water uptake that
Ieetda to len~hen~g, so it will not only
be the quantity but also the location of
water which matters and we could picture
a shift of water oven within the fibril that
&jg from contraction to reb%xation.

  I am aware that ai1 this is only pure
theoretical speculation at the moment.


104                   A. SHUNT-QY&tt%YI

But I will tell you presently about observations on myosine threads which in one case
contra&, when they take up water, then again relax, while still taking up water: observations,
which make ones thoughts drift this way and seek for geometrical explanations.

H. H. WEBIJR l") has shown that a strong solution of myosino, if squirted in a thin
stream into water, solidifies to a thread. WEBPR, in his careful work, showed that many
of the properties of myosine can be studied with great advantage in these threads. If
the thread is stretohed the myosine mice& arrange themselves len~h~e and parallel
to eaohother and a+xmme in this way a structure, in many ways analogous to the contracltile
Q discs of the muscle fibriIl.

Such an orientated thread can be stretched elastioally and if let loose contracts to a great
extent again. If the thread is dried in the stretched oondition and then wetted with an alkaline
salt solution it shortens and one would be inclined believe that this shor~n~g is simply due to
its elasticity reap. previous stretching and has further no interest. This is, however, not the case,
since the thread, if transferred from the alkaline salt solution into water relaxes spontaneously,
reaching or even exceeding its original length; if retransferred into the salt solution it contracts
again, if put into water it relaxes and so the play goes on indefinitely. But you must not
picture these changes as slight variations of length. The ohangesinlength are as much as 20-250/,,
thus of about the same ma~tude as normal contractions of muscle. The changes are quite
fast too. Within two minutes the thread mostly reaches its maximum length or shortness and
the most of this is done within the first minute. The rate is dependent on the diameter of the
thread. The times given relate to a thread of 0,l mm diameter. Threads of half this diameter
will move about 4-P times as fast whioh means that in fibrillary dimensions the whole change
would take place within u-s. The rate of motion depends also on the nature of the alkali. NH&OH
acts faster than Na&Os or NaOH.

Now this, I think, is most remarkable. You take muscle, dissolve its contractile element,
rearrange this in a way that comes the closest to its ~rangement in muscle and add salts and
alkali to it and it contracts, You take these elements away (i. e. put the thread into water) and
it relaxes again and we know that salts and NH,OH are formed during contra&ion. Naturally,
I do not mean to say that by this the rn~h~rn of muscular contraction is explained. Far from it
the problem just begins. What I mean to say is that the phenomenon is most remarkable and
deserves careful study and it is not only remarkable, it is amusing too.

It would be a premature attempt to try to give a physi~o-~he~oal explanation of these
changes in length and at the moment I oan do more than to mention a few observations.

If the thread is dried in an unstretched condition, a salt solution, instead of causing a con-
traction, only causes a slight lengthening. But from the observations just refered it is evident
that the stretching does not condition the subsequent oontraction as such, by elasticity, since
the thread is able to relax and contract again. Evidently the strete~g conditions this ability of
eontraction and relaxation by the coaxial arrangement of the myosine n&ells.

The changes of length are also connectted with oonsiderable changes in diameter, i. e. ohan-
ges in swelling. Older physiolo~~l literature conta~s many attempts to explain muscular
oontraction by the swelling of colloids. One would thus feel tempted to explain the contraction
and relaxation of the thread simply by swelling and dehydration. But the first experiment
shows that things are not so simple. If a thread is put into the alkaline salt solution it swells
etrongly and contracts, but when it is now put into water it goes on swelling while it relaxes.
The next contraction in the salt solution goes hand in hand with a strong decrease of swelling.
Thus we can produce at will a contrition with simultaneous swelling or dehydration, and can
produce swelling with contraction or relaxation.

But however this may be, the fact remains, that contraction and relaxation of the threads


ON PROTOPLASM10 lJTRUOTURE AND FUNUTIONB           105

is connected with strong changes in swelling and shifts of water and thus water must play an
important r6le in the meohanism of these changes in length but evidently it is not the quantity
only but also the speoifio looalisation of water which matters. Nsturally, the migration of water
is, at least to a great extent, only secundary to the action of our aiodine salt solution on myosine.

It is possible to connect the single phases of the described contraction-relaxation cycle
with the different ions of our salt solution. KC1 in itself gives only contraction, while OH ions
are necessary for the relaxation.

The action of phosphate has hitherto been studied only in relation to swelling. In this
respect the action of ph~phate on myosine is quite specific. While neutral salts like KC1 make the
myosine swell considerably only in higher concentrations (0,l m and above) phosphate gives a
strong swelling in small concentrations (0,Ol m) which can actually be formed during con-
traction. While, within certain limits, the a&ion of KC1 is dependent on its concentration,
phosph&te reaches its maximal action already at 0,Ol mol. and a further increase of the phos-
phate concentration does not entail a stronger action. Only at high concentrations (above 0,l
mol.), where the action of the kation seems to oome into play, can the swelling further be in-
oreased,

VI. The double refra&ion of flow. (DRF).

As globular and rod shaped proteins have such widely different physical properties
it is of the greatest importance to know whioh group the single proteins belong to.

  The classical method for establishing the dimensions of a molecule is its X-ray examination.
Unfortunately this method will give results only if the particles are arranged with regularity,
as in a crystal, and can not be applied to solutions.

  A very simple method which allows to demonstrate the rod-shape of suspended particles
is the DRF. This has the following foundation: if rod-shaped objects are floating in a streaming
fluid the rods will tend to arrange themselves with their long axis parallel to the direction of
flow. This is nicely demonstrated by the apparatus of M. GEREND~S (plate III, fig. Sl), a circular
channel in which the water is kept in motion by a paddle wheel. If rod-shaped objects like wax
threads, are floated in the moving fluid, they will arrmnge themselves parallel to the direction of
flow in the long narrow part (plate III, fig. S) where the velocity-gradient is the highest, the
veloaity of the fluid being the greatest in the middle and the smallest along the walls.

  Rod shaped molecules mostly conduct light at a different velocity along their small and
long axis; thus if they are arranged parallel to each other their solution will be doubly
refrauting. Even if the single molecules are not doubly refracting but there is a difference in the
refraction index of the solvent and the solute, rod shaped particles will be doubly refracting
(rod-double refraction) once arranged coaxially.

  Thus, if we have a solution containing rod-shaped particles and press it through a narrow
split or squirt it in a thin jet into a stagnant fluid the particles will arrange themselves coaxially
and will give a double refraction. If we observe this moving fluid between crossed Nicols in the
polar&&ion microscope the fluid will appear luminous.

All this can very conve~ently be demonstrated in the chamber construoted by M. GE-
REND&t (plate III, fig. 4). This chamber, made of oelluloid is 6 mm deep and is covered on
both sides by glass plates. The fluid is pressed, by means of a syringe, through a narrow
(1 mm) split and enters a little basin. If the chamber is filled with 8 fluid, containing rod-
sh8ped particles, like myosine, and is observed under the polarisation microscope, 811 is dark
while the fluid is sta~ant. As soon as the fluid is put into motion a strong lum~osity appears
(plate III, fig. S), which, in the narrow split is especially strong along the wall where the
velooity gradient is the biggest. The middle of the split remains dark. We can also observe
the fluid entering the basin as a luminous stream.

  The average position of the p8rticles in the streamiug fluid will be the resultant of different
forces. The velocity gradient tends to arrange the molecules parallel to the direction of the flow
while the thermio agitation tends to disarrange them. The longer the particles relatively, the


106                    A. SZENT-ClYktC+YI

easier will they be orientated, and the better will they keep this orientation. Under
the conditions of our experiment, and I want to emphasis0 this, only relatively big and
very long particles give a strong DRF. The method gives no answer to the question
whether the particles are single macro-molecules or molecule aggregates, n&ells.

Another factor which influences the orientation of floaCng particles is hydrodynamic
pressure which tends to turn the particles at 45' to the direction of the flow. The relatively
longer the particle the easier it will remain parallel to the direction of the flow. The average
position of the particles can be found in the polarisation microscope by measuring the plains of
the maximum luminosity or maximum darkness of the streaming fluid, by the measurement of
the so-called angle of isocline which also gives an indication of the relative length of the particles.

VII. Structure-proteins.

  Aooording to the theory presented in the previous chapters muscular contraction
is due to the interaction of water ions and rod-molecules, and is a form of water transport.
This takes me back to the beginning of this lecture, where I said that the most varied
cellular functions might be the different applications of the selfsame simple prinoiple.
Water transport is certainly one of the most basic and primitive functions of any living
organism. But, if this is so a protein fraction, analogous to myosine, should be found in
any cell. Naturally we need not expect that the mioells of this protein should be arranged
coaxially and reveal themselves thus in the intact cell by a double refraction or X-ray
spectrum as they do in muscle where this arrangement is conditioned by the meohanioal
function.

But even if we drop all this speculation about water transport and simply ask ourselves
what we have to expect about the shape of proteins if we take into account solely the known facts
about the physical properties of rod-shaped and globular molecules, we will come to the con-
clusion that wherever the organism wants to build up a solid structure it will resort to the rod-
shape and wherever it needs a certain mobility it will apply the globular form. To the former group
will belong the proteins making up the solide edifice, the morphological structure of the
cell; into the latter group will belong proteins of set retions (proteins of milk, different
enzymes and hormones) proteins, of t r a n s p o r t a t i o n (serum proteins), reserve proteins
(ovalbumin) and certain mobile intracellular enzymes (e. g. lacticodehydrogenase).

  This postulate seems, however, to be in contradiction with the experience of protein che-
mistry. The shape of, the molecules of a very great number of different animals and vegetable
proteins has been studied and most of them have been found to be globular. According to this
general experience we only find long, fibrous molecules in special cases only where there is a
mechanical function, like in muscle (myosine), hair (keratine), collagene, fibrine, vegetable fibres
etc. Rod-shaped molecules are thus looked upon at present as specific and rare exceptions
conditioned by some mechanical function.

  Thus the general experience of protein chemistry pleads against the correctness of our ideas.
But let us see, whether research has not been misled unconsciously by some secondary factor.
Let US see what proteins the physioo-chemist will take if he wants to study the shape of molecules.
Naturally he will select the easily accessible or extractible ones, which must be, because of their
extractability, mobile, thus of globular shape. He will unconsciously leave aside the rod-shaped
proteins which, by their physical nature, tend to give solid textures and are thus less readily
accessible. These proteins, forming the major part of the bulk of the cell might actually be fi-
brous and it seems possible that fibrous molecules have been found in tissue only, having
mechanical function, not because the fibrous form is limited to these tissues but because the
mechanical function aonditions a coaxial arrangement which makes the fibrous nature of these
molecules easily recognisable by their double refraction and X-ray spectrum.

Our rough, all-day experience is in agreement with these considerations. If we mince 8


ON PROTOPLASYIG STRUOTURE AND FUN'CITIONS          107

tissue, let us say k&my, and extract it with water, about l/6 of the tot81 protein goes into solu-
tion. These easily extractible proteins are, as shown by the lack of DRF and other physical
properties, globular. But in spite of the considerable loss of protein it will be impossible to
find anything missing if we examine the tissue under the microscope. The whole morphologic81
structure is still there; the globular proteins disaolved had no considerable part in the building
of the morphological structure.

How can we hope now to de&&grate these structure-proteins into single micells or mole-
cules? In analogy to myosine we can expect that these proteins will need a higher salt concen-
tration and a certain alkalinity for their dissolution. But at the same time, knowing that the
micells of these proteins, if rod-shaped, are less regularly arranged than myosine and are not
separated by intermicellar spaces, we can expect that a higher salt concentration and alkalinity
will be insufficient to desintegrate the structure. Since the forces, holding protein molecules
together, are often ,,?t&ogen bcntds" we can hope to gain our end by adding a substance which
is known to split such bonds. Such a substance is urea in high concentration, which accor-
ding to WEBER and ST&ER 17) also desintegrates myosine mice& into molecules.

If the kidney is minced and suspended and stirred in 8 salt solution containing 60% urea,
as done by I. BANU and myself, the tissue particles will soon swell and the fluid itself will
assume 8 very sticky consistence which is already an indication of the presence of rod-
shaped molecules. If the tissue particles 8re centrifuged off and the supernatant fluid diluted
with the same urea-salt solution it will be found to give an intense DRF. The angle of
i so oline as shown by M. GEREND&I, also indicates very long molecules. Plate III, fig. 5 is actually
the photograph of the DRF of such a solution from kidney. It is not the best picture we have
obtained but was the first DRF observed. It was indeed a great excitement to see this splendid
DRF in our tissue extrsct the first time after all these long trams of thought.

The substance, giving the DRF is a protein and behaves similary to myosine. It can be
precipitated by diluting its solution with five times its own volume of water and neutr8lising it.
It can be redissolved in the same urea-salt solution and will give the same DRF again. It can
also be pulled into threads and examined r~ntgenoscopically. The rontgenoscopio measurement
kindly made by Prof. ST. NARAY SZAB~, has given a periodicity in distances of 9,5,4,6 and 2,75 A".
These data are in agreement with the data obtained on other fibrous proteins, like myosine and
keratine, so that these first measurements indicate that this substance is built on the same
pattern with other known fibrous proteins. In analogy with myosine I will call this protein
F%IlOSblO or in general structure protein 1. This substance makes up about one third of the total
protein of the kidney.

There are two striking differences between renosine end myosine, While myosine is positi-
veIy double refracting renosine shows a negative double refraction. In the former the index
ellipsoid lies lengthwise, in the latter crosswise to the axis of the molecule; in other words the
plane of the greater refraction lies in the long axis bf the myosine molecule while in renosine the
greater refraction index is found in the cross section. This is the first known example of a negs-
tively double r&acting protein.*)

But there is also another, still more striking difference between renosine and myosine.
While myosine contains no phosphorus, renosine contains phosphorus in a non-extraotible
form and seems to be thus' a nuoleoproteid. Since the cellular nucleus is microscopically
still intact after the urea-salt extraction and could hardly make l/S of the total protein, renosine
can not be of nuclear origin.

The sticky appearance of the extract is due to the thixotropy of renosine. If the thixotropy
is broken up the sticky appearance vanishes giving place to a true and high viscosity, evidence
of long, rod-shaped particles.

Renosine is a representative of a specific proteinfraction apparently present in all

  *) Muscle seems to contain, side by side with myosine, also 8 fibrous protein, auelogous to
renosine: a protein soluble in urea-salt only and having a negative DRF. See BANGA'S subsequent
paper.


108                   A. SBENT-aYi&GYI

tissues. If the same procedure of extraction is ~ppIied to liver, brain, nerves, rn&~~y
glands, p&rot&, lymphglands, whole embryos, EHRLIU~ or Rous sarooma, EHRLI~R mr-
&noms, the same result is obtained. Only pancreas behaves differently.

After the tissue has exhaustively been extracted with the urea salt solution it is still there
with one third of its total protein. Even examination under microscope does not reveal
anything missing: the rno~holo~i~~l structure is still present.

If this extracted tissue is suspended in a BOY0 urea-solution ~ont&~g 2% NrtOH (15 ml.
to 1 g of tissue) and stirred for a few minutes and then heated for 5 minutes under continuous
stirring to 60' 0, practically the whole tissue dissolves. Sf cooled down again we are left with 8
very sticky fluid, the sticky appe&~nce of which is again due to t~xotropy. After the thixo-
tropy is disturbed we are left with a fluid of high and true viscosity with a strong DRF.

If the solution is diluted by 5-10 times its own volume of water and then slightly acidified,
the material giving the DRF precipitates and can be r~issolved in alkeline uree and shows the
same DRF again. I will cdl this fraction structure-protein 11 without any claim of homogenity.
~~tur&lly, the solution must contain different substances, including the nuclear material. What
is important at the moment, is that the DRF undoubtedly shows that also this most insoluble
pert of a;nimal cellular m&e&l is built of rod-shaped molecules.

An objection aould be raised against the content of this chapter. One could ~fly that the
molecules examined are not realty rod-shaped, but they might be globular and be unfoIded into
rods only under action of our solvents. For this reason & series of globular proteins, like ~&se&
lectalbumin, serum-albumin and globuline, ovalbumin, edestine, gel&tine have been ex8-
mined in our chamber also under action of our solvents. They gave no DRF oomparsble to the
RRF of the desoribed structure proteins.

            VIII..  On the ~at~~~ of eert& viruses.
When hearing about rod-shaped molecules many of you will think st once of ~&GYI
vegetable viruses, like the tobacco mosa;ic virns, isolated by STANLEY, which owing to
their rod-shepe also show a DRF.

It is now generally believed that these viruses sre of exogenous origin, This belief is based
chiefly on three arguments.

Fin&y, it has been shown, that the tobacco mosaic virusis rod-shaped. Since it w&s thought
that rod shaped molecules are not found in the protoplasm this was taken as evidence of their
exogenous nature. But we hgve seen that the structural part of protoplasm is built up of rod-
shaped molecules. As BANGA has shown this is also true for vegetable cells &. ohloroplssts.
With this observation the problem &sea whether these viruses are not m feet protopl&sm-
molecules of the plant itself: molecules, which for some reason or other attained s suspension
stability and started herewith an independent existence. So it becomes necessary to review also
the other arguments brought forward in favour of the exogenous nature.

The seoond strong argument for the exogenous nature of these viruses is the discovery of
BAWDIBN and PIXIE that the virus contsins phosphors, and is a nucleoproteid, thus an
independent gUld complete organism. Rut I have mentioned before that the protein fmotion I
does contain phosphorus, s;nd contains this element in quantities comparable to the
quantity of P found in viruses. I will give, side by side, the% P oontent of viruses, taken
from an article of STANLEY 18) and the P oontent of struoture protein I, t&ken from the
pper of hNffA.

As the tsbla f shows the ~~~rnent is olose.

The third evidence for the exogenous nature of a virus, is its immunolo~~al behaviour.
The tobacoo mosaic virus behaves immunoIogically in a different way than the protein of the
tobaooo letat. At the moment I oannot disprove this %rg~ment, I can only show its weakness.
For if we say: tobacco-protein, what protein do we mean? After all probability the soluble,
stumble gfobulsr proteins, while the insomble fibrous proteins p~babIy do not come into


ON PROTOPLASMIC STRUUTURE AND FUNCTIONS

109

Tobacco mosaic . . . . . . . . . . . . . .

Aruba mosaic  ..............
Cucumber mosaio  ............
Latent mosaic of potato  ......

Tobaoco ring spot ............
Tobacco bushy stunt   ........
Chicken tumor. ...............

TABLE I.

0,43
0,39
0,60
0,45-0,55
0p1
0,55-0,60
0,51-05s
o&-o,5
334
&S-1.5
0.7

Kidney . . . . . . . . . . . . . . . . . .
Liver . . . . . . . . . . . . . . . . . . . .

Brain   . , . , . . . . . . . . . . . . . .

Parotis . . . . . . . . , . . . . . . . . .

iif9
0:53
1,OS
A19
0153

Mammary gland . . . . . . . . . .

2,58
1,94

Lung . . . . . . . . . . . . . . . . . 1. .     0,60
                      0.67

EHRLICH Samome . . . . . . . . I   lj43
                     1.3

Chicken tumor (Rous) . . . . I   0;55
                  0,56

the picture at all. If they had been brought into solution and tested they might have given
entirely different immunological reactions as compared to globular proteins.

I2L Summary and cone~u~ion.

To sum up, I have given you a brief survey of the ex~r~entai work done in my
laboratory during the last year and have shown you that the volume-contraction
of muscle is prolonged under action of veratrine (E. ERNB'I), which is new evidence
in favour of the assumption that the production of ions has an irn~rt~t r61e in musoular
oontraction. I have also shown that under action of veratrine more potassium is libe-
rated and given off by the contractting muscle (E. ERNST and E. M~Rocz),). I have pre-
sented geometrical considerations and models which show how a shift of water might
lead to contraction. I have shown, how myosine threads are capable of contraction and
relaxat,ion under influence of ions. I have also shown that the bulk including the
greatest part of the morphological structure of animal cells is made up of fibrous
molecules (BANGA) and I have discussed the bearings of this finding on the virus theory.

All this work has been done with the ultimate aim to arrive at an understanding
of the muscle as a machine and correlate struoture, contraction, oxidation and fermen-
tation. With this aim in sight also our earlier work on oxidation and fermentation has
been continued and we tried to go on pulling the machine to bits. Thus E. and K.
LAKI isolated the fumarase in crystals and identified its coenzyme. F. B. $~RAUB orys-
tallised the lactioo-dehydrogenase *) (Biochem Jl., in print) and studied its kinetics.
E. ANNAU made some hopeful beginnings in fat oxidation describ~g a new leoithine-
dehydrogenase and its coenzyme.

All these experiments which I have put before you are but first steps towards our

  *) The lactic0 dehydrogenase, as shown by the lack of DRF, is globular (1,6% solution of the
crystalline enzyme). This enzyme is readily extracted from muscle by water or weak saline, which
solubility in itself is an indication of a globular nature. Fumarase and the yellow enzyme of the muscle
are not extracted by water but all the same they are globular because they show no DF&F (1,3% SO-
lution of the crystalline fumarase and 0,5x solution of the pure yellow enzyme, isolated by F. B,
STRAUB). These two enzymes, in isolated condition, are readily soluble in water. This indicates that
in the tissue they are bound to insoluble, thus fibrous proteins. Our first experiments indicate that
the situation is probably similar in the case of the suoomodehydrogenase and the indophenoloxydase.


110                    A. BZENT-QY6RCiYI

distant goal, the understanding of the cell ae a whole. Preeenting my results has taken you
to a sowing rather than to a harvesting. My one reason for taking to you about this in-
complete work was to invite you to join in and share the pleasure we are having our-
selves at my laboratory.

  1) Z. M. Bacq, M. Goffart, A. Szent-Gyijrgyi, Nature 148, 522 (1939). -2) Z. M. Baoq, Arch.
intemat. Pharm., 68,59 (1939). - 3) Z. M. Bacq, M. Goffart, Aroh. internat. Phys. 99,189 (1939). -
4) E. Ernst, Frioker, Arch. ges. Phys. (PFL~~GER) 284, 360 (1934). - 5) P. Ernst, Ibid. 209, 613,
(1925). - 6) E. Ernst, J. Koozkhs, Ibid. 239,691(1938). - 7) J. T. Edsall, Jl. of Biol. Chem. 89,
289 (1930). - 8) J. T. Edsall, A. L. v. Muralt, Ibid. 89, 315, 351 (1930). - 9) H. Staudinger: Die
hochmolekularen organisohen Verbindungen, Kautschuk und Cellulose, J. Springer, (1932). - 10)
H. H. Weber, Naturwiss. 27, 33 (1939); Erg. Physiol. 86, 107 (1934). - 11) E. Ernst, Sz. Preisz,
Z. f. Biol. 96,185 (1935). - 12) McDougall, Jl. of Anat. and Physiol. 81,410 (1897); 82,187 (1898). -
13) E. B. Meigs, Amer. Jl. Phys. 14,138 (1905). - 14) 0. v. Fiirth, Erg. Physiol. 17,363 (1919). -
16) J. Bernstein, Arch. ges. Phys. (PFL~~GER) 138, 136 (1909). - 16) H. H. Weber, Arch. ges. Phys.
(PFL~~oER)~~~, 205(1934). - 17) H. H. Weber, R. StSver, Biooh. Zs. 259, 269 (1933). - 18) W. M.
Stanley, Science in Progress, Yale University press 1939.