Reprinted from the Proceedings of the NATIONAL ACADEMY OB SCIENCES,
         Vol. 22, No. 7, pp. 439447.  July, 1936.

ON THE STRUCTURE OF NATIVE, DENATURED, AND
          COAGULATED PROTEINS

       BY A. E. MIRSKY* AND LINW PAULING

GATES CHEMICAL LABORATORY, CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA,
                      CALIFORNIA

Communicated June 1, 1936

In this paper a structural theory of protein denaturation and coagulation
is presented. Since denaturation is a fundamental property of a large group
of proteins, a theory of denaturation is essentially a general theory of the
structure of native and denatured proteins. In its present form our theory
is definite and detailed in some respects and vague in others; refinement in
regard to the latter could be achieved on the basis of the results of experi-
ments which the theory suggests.  The theory (some features of which
have been proposed by other investigators) provides a simple structural
interpretation not only of the phenomena connected with denaturation and
coagulation which are usually discussed (specificity, solubility, etc.) but
also of others, such as the availability of groups, the entropy of denatura-
tion, the effect of ultra-violet light, the heat of activation and its depen-
dence on pH, coagulation through dehydration, etc.

I.  The experimental basis upon which the present theory rests will be
briefly described.
1.  The most significant change that occurs in denaturation is the loss of
certain highly specific properties by the native protein. Specific differences
between members of a series of related native proteins and specific enzy-
matic activities of native proteins disappear on denaturation, as the fol-
lowing observations demonstrate :
(a)  Many native proteins can be crystallized and the crystal form is
characteristic of each protein.  No denatured protein has been crys-
tallized.


440         CHEMISTRY: MIRSK Y A ND PA ULING    PROC. N. A. S.

(b) Most native proteins manifest in their immunological properties a
high degree of specificity, which is diminished by denaturation.'
(c) The native hemoglobins of closely related animal species can be
distinguished from each other by differences in crystal form, solubility,2
gas aflinities, positions of absorption bands, and other properties.3  On the
other hand, in the denatured hemoglobins some of these properties, the posi-
tions of the absorption bands, for example, can be subjected to precise
measurement, and it is found that differences between the various hemo-
globins have disappeared.4
(d) A number of enzymes have recently been isolated as crystalline
proteins. When these proteins are denatured their enzymatic activity
vanishes. In pepsin and trypsin, where an especially careful study has
been made of this phenomenon, there is a close correlation between loss of
activity and formation of denatured protein.6
2. Striking changes in the physical properties of a protein take place
during denaturation. At its isoelectric point a denatured protein 4s in-
soluble, although the corresponding native protein may be quite soluble.
It was the loss of solubility that first drew attention to the phenomenon of
denaturation, and denaturation is now usually defined by the change in
solubility. The denatured protein after precipitation has taken place is
called a coagulated protein, the process of coagulation being considered
to include both denaturation and aggregation of denatured protein in the
form of a coagulum.6 If the denatured protein is dissolved, by acid, alkali,
or urea, the solution is found to be far more viscous than a solution of native
protein of the same concentration.7
3. Changes in the availability of sulfhydryl, disulfide, and phenol
groups appear as a consequence of denaturation.  All of the SH and S-S
groups found in a protein after hydrolysis can be detected in a denatured
protein even before hydrolysis, while in the corresponding native protein
only a fraction of these groups is detectable. In native egg albumin no SH
or phenol groups are detectable. ,In other native proteins (hemoglobin,
myosin, proteins of the crystalline lens, for example) some groups can be
detected; new groups appear when the protein is made more alkaline,
although not alkaline enough to cause denaturation, and then disappear
when the original pH is restored. In all the different ways of coagulating
a protein a close correlation between appearance of groups and loss of
solubility has until recently been observed.* Now, however, it has been
found that when myosin is rendered insoluble by drying (or when water
is removed by freezing) there is no change in availability of its SH groups.g
Furthermore, if the insoluble myosin is treated with a typical denaturing
agent, such as heat or acid, all of the SH groups in the protein become
available, although no change in solubility is observed.  Hitherto protein
coagulation due to dehydration has not been distinguished from coagulation


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caused by other agents, but the tests for availability show that in coagula-
tion by dehydration the change in protein constitution is distinctly different
from that caused by any of the known protein coagulating agents.  In our
theory of coagulation both types of change will be considered.  Probably
both types occur biologically.  It has been suggested that when light con-
verts visual purple, a conjugated protein, into visual yellow the former is
denatured in much the same way as it is by heat, alcohol, or acid. lo  And it
has been shown that in the course of fertilization and in the rigor of muscle
a coagulation of protein similar to that caused by dehydration takes
place.11n12

4.  In a list of the large number of different agents, with apparently
little in common, that cause denaturation are heat, acid, alkali, alcohol,
urea, salicylate, surface action, ultra-violet light, high pressure.  The
temperature coefficient of heat denaturation of many proteins (egg albumin,
ferrihemoglobin, trypsin, etc.) is about 600 for a rise in temperature of ten
degrees, and from this the energy of activation can be calculated. On
either side of a point between the isoelectric point and neutrality the
temperature coefficient of denaturation by heat is diminished.13 A dry
preparation of egg albumin is not readily denatured by heat.6

5. The denaturation of certain proteins, notably hemoglobin, serum
albumin, and trypsin, is reversible.`4  From the effect of temperature on
the equilibrium constant, the heat of reaction and the entropy change can
be calculated.  In the denaturation of hemoglobin by salicylate and in the
denaturation of trypsin by heat or acid, the equilibrium between native
and denatured protein is not affected by changes in the total concentration
of protein present, l5 from which it can be inferred that in the denaturation
of hemoglobin and trypsin by these agents no change in molecular weight
takes place.  Since typical denaturation can occur without change in the
molecular weight, it is important to distinguish between denaturation and
the changes in particle size observed by Svedberg. Although denatura-
tion can occur without change in molecular weight, under certain conditions,
as in the denaturation of myosin by urea, denaturation may be accom-
panied by depolymerization. Weber found the molecular weight of myosin
to be of the order of a million and that of myosin in urea to be thirty-five
thousand.`6 On the other hand, it is unlikely that depolymerization is
always accompanied by denaturation, for under some of the conditions of
depolymerization described by Svedberg it is improbable that denaturation
(loss of specificity, loss of solubility, or appearance of previously inaccessible
groups) takes place. In the case of hemocyanin, for example, decomposition
into products l/z and l/i6 of the size of the original molecule readily occurs,
and these smaller particles seem to have the properties of native hemo-
cyaninn

6.  The shape of the native protein molecule appears to have little sig-


442

CHEMISTR Y: MIRSK Y A ND PA ULING    PROC. N. A. S.

nificance for an understanding of denaturation.  Denaturation occurs in the
spherical molecules of egg albumin and hemoglobin, in the elongated par-
ticles of soluble myosini8 and (as indicated by SH groups becoming detect-
able) even in myosin that has been formed into insoluble fibres by drying.g

7.  In certain conjugated proteins stability of the protein and presence
of the prosthetic group are related.  In hemoglobin, the yellow oxidizing
ferment of Warburg, and visual purple, it is necessary to denature the
protein in order to detach the prosthetic group with the use of present
methods.10s19  After removal of the prosthetic group it is possible to reverse
the denaturation of globin and the protein of the oxidizing ferment, but
these native proteins are more unstable (with respect to denaturation)
than they are when conjugated with their prosthetic groups.  In hemoglo-
bin the ease of denaturation depends upon the state of the prosthetic group.
Carbon monoxide hemoglobin, for example, is less readily denatured by
heat, acid, or alkali than are oxyhemoglobin and ferrihemoglobin.20 In
visual purple presence of the prosthetic group causes the protein to be de-
natured by visible light.  Denaturation in this case appears to be revers-
ible.lO

II. Our' conception of a native protein molecule (showing specific
properties) is the following.  The molecule consists of one polypeptide
chain which continues without interruption throughout the molecule (or,
Jn certain cases, of two or more such chains); this chain is folded into a
uniquely defined configuration, in which it is held by hydrogen bondP
between the peptide nitrogen and oxygen atoms and also between the free
amino and carboxyl groups of the diamino and dicarboxyl amino acid
residues.

We shall not enter into a long discussion of the precise configurations of native proteins,
about which, indeed, little reliable information is available.  From the x-ray investiga-
tions of Astbury** and his collaborators it seems probable that in most native proteins
the polypeptide chain, with the extended or one of the contracted configurations dis-
cussed by Astbury, folds back on itself in such a way as to form a layer in which peptide
nitrogen and oxygen atoms of adjacent chains are held together by hydrogen bonds;
several of these layers are then superposed to form the complete molecule, the bonds
between layers (aside from the continuation of the polypeptide chain from one layer to
the next) being hydrogen bonds between side-chain amino and carboxyl groups.  In
general not all of the side chain groups will be used in forming bonds within the mole-
cule; some will be free on the surface of the molecule.

The importance of the hydrogen bond in protein structure can hardly be overempha-
sized. No complete review of the large amount of recent work on this bond is available;
we shall mention only the most striking of its properties.*r The hydrogen bond consists
of a hydrogen atom which bonds two electronegative atoms together (F, 0, N), the hy-
drogen atom lying between the two bonded atoms. The bond is essentially electrostatic
in nature. The bonded atoms are held more closely together than non-bonded atoms,
the N-H-O distance being about 2.8 A. The energy of a strong hydrogen bond is 5000 to
8000 cal. per mole, the lower value being approximately correct for an N-H-O bond as


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in proteins.  Side-chain bonds in proteins we consider to involve usually an amino and a
carboxyl group, the nitrogen atom forming a hydrogen bond with each of two oxygen
atoms and holding also one unshared hydrogen atom.  In acid solutions hydrogen
bonds may be formed between two carboxyl groups, as in the double molecules of formic
acid."3

The characteristic specific properties of native proteins we attribute to
their uniquely defmed configurations.

The denatured protein molecule we consider to be characterized by the ab-
sence of a uniquely defined con$guration.  As the result of increase in tem-
perature or of attack by reagents (as discussed below) the side-chain
hydrogen bonds are broken, leaving the molecule free to assume any one
of a very large number of configurations.  It is evident that with loss of the
uniquely defined configuration there would be loss of the specific properties
of the native protein; it would not be possible to grow crystals from mole-
,cules of varying shapes, for example, nor to distinguish between closely
related proteins when the molecules of each protein show a variability
in configuration large compared with the differences in configuration of the
different proteins.

Strong support of this view of the phenomenon of denaturation is pro-
vided by the known difference in entropy of native and denatured proteins,
which is about 100 E. U. for trypsin, the entropy of the denatured form
being the greater.5 This very large entropy difference cannot be ascribed
to a difference in the translational, vibrational, or rotational motion,
but must be due to a difference in the number of accessible configurations.
It corresponds to about 10zo accessible configurations for a denatured
protein molecule: The large entropy of denaturation thus shows clearly
that the phenomenon of denaturation consists in the change of the mole-
cules of the native protein to a much less completely specified state.

The magnitude of the heat change, entropy change, and activation energy
of denaturation (about 30,000 cal./mole, 100 E. U., and 150,000 cal./mole,
respectively, for trypsin) can be interpreted in terms of our hydrogen-bond
picture of the protein molecule.  We consider the native protein molecule
to be held in its definite configuration by side-chain hydrogen bonds, about
fifty in number for this protein (corresponding to about twenty-five amino
and twenty-five carboxyl side chains), each with a bond energy of about
5000 cal./mole, as in simpler systems.  The activation energy of 150,000
cal./mole shows that in order for the molecule to lose its native configura-
tion about thirty of the bonds must be broken.  Some of the side-chain
groups then again form hydrogen bonds ;  the heat of denaturation shows
that on the average there are about six fewer such bonds in the denatured
molecule than in the native molecule. (The activation energy for transition
of a denatured protein molecule from one configuration to another is
without doubt smaller than the activation energy of denaturation, as it


444           CHEMISTRY: MIRSK Y AND PA ULING    PROC. N. A. S.

involves breaking a smaller number of hydrogen bonds, so that at a tempera-
ture at which the rate of denaturation is appreciable the denatured mole-
cule would run rapidly through its various configurations.) The magnitude
of the entropy of denaturation also fits into our picture; it corresponds to
the number of configurations obtained by forming hydrogen bonds at
random between about twenty amino side chains and twenty carboxyl
side chains.

The reagents which cause denaturation are all substances which affect
hydrogen-bond formation. Alcohol, urea, and salicylate are well-known
hydrogen-bond-forming substances; they form hydrogen bonds with the
protein side chains, which are thus prevented from combining with each
other and holding the protein in its native configuration.  Acids act by
supplying protons individually to the electronegative atoms which would
otherp share protons, and bases by removing from the molecule the
protons needed for hydrogen-bond formation. This conception provides
an explanation of the facts that the isoelectric point of a protein shifts
toward the neutral point on denaturationz4 and that the pH at which the
activation energy for denaturation has its maximum value is in general
not at the isoelectric point of the native protein, but between this point
and the neutral point.25 In the native protein molecule of the usual type
some amino and carboxyl side-chain groups are paired together by forming
hydrogen bonds. The acid-base properties of the molecule are in the main
determined by the groups which are left free.  On denaturation some of
the paired groups are freed, amino and carboxyl in equal numbers, and in
consequence the isoelectric point of the denatured protein is shifted toward
neutrality. We have pictured the process of denaturation as involving
the rupture of a large number of hydrogen bonds, to form a labile acti-
vated molecule. This labile molecule is stabilized by action of base on its
free carboxyl groups or of acid on its free amino groups, the activation
energy thus having a maximum at a pH value between the neutral point
and the isoelectric point of the native protein.

The action of ultra-violet light must be different.  It is not possible to
formulate a reasonable mechanism whereby a quantum of light can break
twenty or thirty hydrogen bonds.  Instead the light must attack the mole-
cule in a different place, probably breaking the main polypeptide chain
after absorption in a tyrosine or other phenolic residue, as suggested by
Mitchell.26  That this occurs is indicated by the observationz7 that after
illumination with ultra-violet light in the cold denaturation occurs only
on warming, though then at a lower temperature than without illumina-
tion; it is clear that illumination in the cold causes a break in the mole-
cule, which, however, is restrained to configurations near to its native con-
figuration by the side-chain hydrogen bonds.  (That some loosening of the
molecule occurs is shown by the observation of an increase in the available


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groups in egg albumin after illumination in the cold.)  On warming, these
bonds are broken; because of the break in the molecule, however, it can
be denatured "in parts," and hence at a lower temperature than before
illumination.  We predict that it will be found that denaturation by il-
lumination with ultra-violet light is in general not reversible.

In a conjugated protein the prosthetic group plays a part in holding
the molecule in the native configuration (hemoglobin, yellow oxidizing
ferment, visual purple).  It is possible in such a protein for reversible
denaturation to take place after absorption of light by the prosthetic group,
no permanent damage being done to the molecule.

In a protein coagulum side-chain hydrogen bonds hold adjacent mole-
cules together. Native proteins do not coagulate because most of the side
chains are in protected positions inside of the molecule; denatured proteins
(at the isolectric point) do coagulate because they have a larger number
of free side chains and because in the course of time, as the molecule
assumes various configurations, all of the side chains become free. The
increase of viscosity of protein solutions on denaturation we attribute to
the change from the compact configuration of the native protein molecules
to mote extended configurations.

A native protein molecule of small molecular weight may have free side
chains so arranged as to permit it to combine with similar molecules to form
a polymer with properties differing little from those of the small molecules.
The observations of Svedberg and his collaborators indicate that this is the
case for hemocyanin, casein, and certain other proteins.

Our theory of denaturation leads to definite predictions regarding the
availability of groups to attack by reagents.  In the large compact mole-
cule of a native protein, of the order of magnitude of 50 L% in diameter and
having the same structure as every other molecule of the protein, all
groups would be protected from attack by reagents except those on the sur-
face or near the surface. After denaturation of the protein, however, the
molecule (in solution) would in the course of time assume various configura-
tions, and every group in the molecule would become available to attack.
These statements are in complete agreement with the experimental results
regarding sulfhydryl, &sulfide, and phenol groups mentioned above.  The
observation that the number of available grOups is increased when the
solution is made alkaline, though not alkaline enough to cause denaturation,
shows that under these conditions some of the hydrogen bonds in the protein
molecule are broken, causing it to assume a configuration somewhat more
open than its original configuration.
Although a denatured protein molecule in a coagulum is not free to as-

sume all configurations, being restrained by bonds to its neighbors, in
general its configuration will be so open as to make all groups accessible to
attack.  However, as mentioned above, there is no change in the avail-


446         CHEMISTRY: MIRSK Y A ND PA ULING

PROC. N. A. S.

ability of groups in myosin when it is rendered insoluble by drying or when
water is removed by freezing. We interpret this as showing that coagula-
tion of this type is not accompanied by denaturation; that is, the molecules
do not lose their uniquely dejined conjigurations. On dehydration of the
native protein the surface side chains of adjacent molecules form bonds
sufficiently strong to produce a coagulum of native protein molecules.  It
would be expected that the mechanical bolstering effect of adjacent mole-
cules in this coagulum would aid the molecules to retain their native
configurations; this is observed to be the case for egg albumin and some
other proteins, which in this state are denatured by heat only at a tempera-
ture considerably higher than before dehydration.6

Some features of the theory of protein structure discussed above have
been suggested before. Many investigators have correlated the specific
properties of native proteins with definitely specified molecular configura-
tions and the loss of specific properties on denaturation with change in con-
figuration. Astbury and his collaborators in particular, in discussing their
x-ray investigations, which have provided so much valuable information on
protein structure, have stated 22 that dehydration and temperature de-
naturation lead to destruction of the original special configuration of the
native protein, giving a debris consisting simply of peptide chains. Our
picture agrees with theirs except in regard to the effect of dehydration,
which we believe to consist primarily in the coagulation of molecules with
essentially unchanged structure. The hydrogen bonds which we postulate
to exist between side-chain carboxyl and amino groups (each nitrogen
atom attached by hydrogen to two oxygen atoms, with the distances
N-H-O equal to 2.8 A) are a refinement of the side-chain salt-like linkages
discussed by Astbury. Our picture of the spreading of protein films on
water, involving the unfolding of compact molecules to layers one amino-
acid-residue thick, is not essentially different from that of Gorter and
Neurath, who have suggested unfolding in connection with film formation,
though not with denaturation in general.28

In this paper we have discussed in some detail a very drastic change in
configuration of protein molecules, that connected with denaturation.  We
have pointed out that the large entropy of denaturation provides strong
support for our suggested structures of native and denatured protein mole-
cules, and that many other phenomena can also be interpreted in a simple
way from this point of view. There also has been put forth recently some
evidence that small changes in configuration of proteins occur, which play
an important part in protein behavior. Thus Northrop and his collabora-
tors have prepared an enzymatically inactive protein which on slight
hydrolysis by trypsin is transformed into native trypsin,2g and another
which is similarly transformed by pepsin into native pepsin30 and Gorter31
has shown that myosin spreads on water only after slight hydrolysis.  The


VOL. 22, 1936     CHEMISTRY: MIRSK Y AND PA ULING            447

structural interpretation of these phenomena can be made only after
further experimental information is available.
* On leave of absence from the Hospital of the Rockefeller Institute.
1 Zinsser, H., and Ostenberg, Z., Proc. New York Path. Sot.. 14,78 (1914).
* Landsteiner, R., and Heidelberger, M., Jour. Gem Physiol., 6, 131 (1933).
3 Barcroft, J., The Respiralory Function of the Blood, Part II, Cambridge Univ. Press,
1928.
4 Anson. M. L., and Mirsky, A. E., Jour. Physiol., 60,50 (1925).
6 Northrop, J. H., Jour. Gen. Physiol., 13, 756 (1930); 16, 33, 323 (1932).
6 Chick, H., and Martin, C. J., Jour. Physiol., 40, 404 (1910); 43, 1 (1911-12); 45,
61, 261 (1912-13).
7 Anson, M. L., and Mirsky, A. E., Jour. Gen. Physiol., 15,341 (1932).
8 Heffter, A., Media. nat. Arch., 1, 81 (1907);  Arnold, V., Zeit. Physiol. Chew, 70,
300 (1911); Mirsky, A. E., and Anson, M. L., Jour. Gen. Physiol., 18, 307 (1934-35);
19, 427, 439 (1936).
0 Mirsky, A. E., unpublished experiments.
10 Wald, G., Jour. Gen. Physiol., 19, 351 (1935); Mirsky, A. E., Proc. Nat. Acad. Sci.,
22, 147 (1936).
I1 Mirsky, A. E., Jour. Gen. Physiol., 19 (1936), in press.
I2 Mirsky, A. E., unpublished experiments.
12 Lewis, P. S., Biochem. Jour., 20, 978, 984 (1927); Loughlin, W. J., Biockem. Jour.
27, 1779 (1933).
11 Anson, M. L., and Mirsky, A. E., Jour. Gen. Physiol., 9, 169 (1925); Jour. Phys.
Chem., 35, 185 (1931).
l6 Anson, M. L., and Mirsky, A. E., Jour. Gen. Physiol., 17, 393, 399 (1934).
I6 Weber, H. H., Ergeb. d. Physiol., 36, 109 (1934).
I7 Svedberg, T., Science, 79, 327 (1934).
I* von Muralt, A., and Ed&l; J. T., Jour. Biol. Chew., 89,315 (1930).
19 Theorell, H., Biochem. Zeitschr., 278, 263 (1935).
*a Hartridge, H., Jour. Physiol., 44, 34 (1912).
*I Latimer, W. M., and Rodebush, W. H., Jour. Am. Chew. Sot., 42, 1419 (1920);
Pauling, L., Proc. Nat. Acad. Sci., 14,359 (1928) ; Sidgwick, N. V., The Electronic Theory
of Valqncy, Oxford University Press, 1929; Bernal, J. D., and Megaw, H. D., Proc.
Roy. Sot., A151, 384 (1935).
22 Astbury, W. T., and Street, A., Phil. Trans. Roy. SOC., A230, 75 (1931); Astbury
and Woods, H. J., Ibid., A232, 333 (1933); Astbury and Sisson, W. A., Proc. Roy. SOG..
AlSO, 533 (1935); Astbury and Lomax, R., Jour. Chem. Sot., 1935,846; Astbury, Dick-
inson, S., and Bailey, K., Biochem. Jour. 29, 2351 (1935);  see also Meyer, K. H., and
Mark, H., Der Aufbau der Hochpolymeren Organischen Naturstoffe, Akademische Verlags-
gesellschaft, M. B. H., Leipzig, 1930.
23 Pauling, L., and Brockway, L. O., Proc. Nat. Acad. Sci., 20, 336 (1934).
*4 Heidelberger, M., and Pederson, K. O., Jour. Gen. Physiol., 19,95 (1935); Pederson,
K. O., Nature, 128, 150 (1931).
s Laughlin, W. J., Biochem. Jour., 27, 1779 (1933).
26 Mitchell, J. S., Nature, 137,509 (1936).
27 Bovie, W. T., Science, 37, 373 (1913).
28 Gorter, E., and Grendel, F., Proc. Acad. Sci. Amsterdam, 29,371 (1926); Neurath,
H., J. Phys. Chem., 40, 361 (1936).
29 Kunitz, M., and Northrop, J. H., Science, 80,505 (1934).
3o Herriott, R. M., and Northrop, J. H., Ibid., 83,469 (1936).
al Gorter, E., Nature. 137, 502 (1936).