Download Protein Molecules in Solution

Protein Molecules in Solution
By IRVING M. KLOTZ, PH.D.
A protein imolecule in solution miay undergo a host of interactions: with solvent, with
small molecules and ions, with itself, with other macromolecules. In a physiologic milieu,
all of these possibilities are pre-sent. A variety of interactions will occur. On the basis
of the behavior of these substances in simiiple svstemiis, reasonable guesses caan often be
mlade as to which interactions will predomiiinate in a biologic environmilent.
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IN THIS paper, we shall examinie soime of
the properties, and the behavior, of proteins in solution. Our view will be a maolecular
one and we shall interpret the behavior of
proteins in terms of the structure of these
milacromolecules anid their interactions with
their enviroiinment. It is appropriate, therefore, to summarize briefly at the outset, some
of the salient features of the structure of
proteini molecules.
+ H
HN H
C-OH
O
Brief Review of Protein Structure
3.4A
CH-CH
--
H~~
H~~
H% /
Proteinis are supermolecules made up of
small amiino acid building blocks. The chains
of amino acids are held together by peptide
linkages, each residue contributing about
3.5 A to the length of the chain (fig. 1). As
has long been recognized, specificity of proteins must reside ultimately in the particular
sequenee of amlino acids in the chains of the
supermnolecule. As a result of the magnificent
skills of several individuals, sequences (sometimes called the primary structutre) are Inow
known completely for 2 proteins, insulin1 and
ribonuclease,' 3 and almost completely for several others. The arrangement in insulin is
shown in figure 2.
Overwhelmiing evidenlee exists that withini
a protein molecule the polypeptide chain does
not occur as a stretched-out string of residues.
While the basic configurations assumed by the
chains are not yet established unequivocally,
and certainly must differ among different
classes of protein, the best model so far is the
a-helix coil suggested by Pauling, Corey, and
Branson4 (fig. 3). With such a coiling, it takes
approximately 7 amino acids to traverse 10 A
From the Departinent of
CH=CH
1
HC-CH
H 0-1
0O
Figure 1
Peptide bond (in broken-line circle) bet ween ,9
amino acids, tyrosine (top) (and alaiiine (bottom).
Scale in aniigstromi tunits is shoucn at left.
in the direction of the axis of the helix. In a
helix, furthermore, a given amino acid has
as neighbors n-ot onlly the 2 amino acids to
which it is attached bv peptide linkages, but
also those residues 1 turn in the helix above
or below. If these steric relationships were
knowl, and as yet they are completely obscure, we would say that the secondary structure of the protein miolecule had been established.
If the basic configuration of the polypeptide
is a helical coil, then it is clear from the known
dimensions of globular proteins that these
helices must be folded over in various configurations so as to be contained, in imost cases,
in a volume which is very roughly spherical
in shape. Sueh conformations have usually
Chemistry, -Northwestern
University, Evanston, Ill.
828
Circulation, Volume XXI, May 1960
PROTEIN MOLECULES IN SOLUTION
829
Ala Lys Pro *Thr Tyr Phe
-
Figure 2
Sequence of amino acids in insulin. There are 2 peptide chains linked together by disulfide bonds represented by heavily blackened parts in the diagram. Gly = glycine;
Phe = phenylalanine; etc. (Republished by permission of Naturwissenschaften.37)
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been called the tertiary structure of the protein molecule. Despite heroic efforts over an
extended period, information concerning 3dimensional configuration is available for only
1 protein, myoglobin,5 (fig. 4) which shows a
very complicated pattern of foldings, the biologic significance of which is not comprehended at all.
Nevert-heless, even in the absence of detailed
structural models, we do have extensive
knowledge of the over-all sizes and gross
shapes of mnany protein macromolecules. A
few of these are shown in figure 5. It is only
*
I
from these very gross models that we can begin our considerations of the properties of
protein molecules.
Interactions of Protein Molecules with Solvent
In any discussion of the behavior of proteins in solution, i.e., of the interactions of
these macromolecules with their environment,
we recognize that the surfaces shown in figure
5 cannot actually be as smooth as the drawings might imply. If the figure were expanded
sufficiently we would be able to visualize the
specific side-chains of the individual amino
acid residues. A few polar side-chains are
shown schematically in figure 6. Such polar
and nonpolar substituents play a vital role in
the inlteraction of the macromolecule with its
environment.
That the environment of the protein surface
is very different from that in the bulk of the
solution is evident from the marked differences in the chemical behavior of functional
Circulation, Volume XXI, May 1 960
Figure 3
Schematic diagram
z
*n
of helical configuration of a
polypeptide chain (solid line) with hydrogen bonds
represented by broken lines.
groups which are attached to protein sidechains. For example, sulfhydryl groups in
simple compounds, such as glutathione, react
rapidly and com-pletely with m-aniy substances,
particularly heavy metals:
R -SH +Ag = R -SAg±+H+. (1)
The same -SH group attached to a protein
(for example, f3-lactoglobulin) acts as if it
were completely hiddeni fromi any attacking
reag,ent, such as Ag+ (fig. 7). The diffusion
current of Ag+ added to protein in buffer rises
just as it would if protein- were absent. Only
if the protein is denat-ured, for example with
KLOTZ
830
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voA4
Figure 4
Configuration of mYoglobin. The gray disk is the hemne group, the little bla1ck ball attached
to it, an atrificially introduced group requiredl for the x-ray analysis of this macrotm-olecule. Thl e white parts are te polypeptide confliguration. ([eUpublished b?y permission
of Endeavour. 38)
Table 1
Some l7minsn(l
P2 's in Pr?oteins
PK
Protein
Group
Expected
Fotund
TRibonuelease
OH
10
9.9 (3 groups)
>11.5 (.3 groiips)
Ovalbuiyiii
OH
10
>1-
I{eiiioglobixi
HN NH+
6.5
<
5
Circutation, Volume XXI. May 1960
PROTEIN MOLECULES IN SOLUTION
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urea, does the -SH group become visible to
the Ag+. Denaturation is said to "unmask"
this mereaptan in the protein molecule.
Similarly, if we examine the titration characteristics of acid-base groups of a protein,
we often find peculiarities (table 1). For example, the phenolic -OH groups of tyrosine
residues often will not dissociate H+ ions at
pH values much higher than are characteristic for this substituent.i 7 Also, basic residues,
such as histidine, may not accept a hydrogen
ion until the pH is lowered much below that
at which an imidazolium ion is normally
formed.8 A more detailed description of such
unusual acid-base behavior is shown in figure
8 for an -N(CH3)2 group which has been
artificially introduced into a protein; hydrogen ions are taken up only at much stronger
acidities than are necessary when the same
group is attached to a simple amino acid.9
There is thus no question that a protein environment may modify the properties of a
substituent. However, the molecular basis of
the changed behavior is less certain. I should
like to present primarily our views'0 on this
subject.
As we have suggested recently, many aspects of the behavior of proteins in solutioni
can be interpreted advantageously if we assign some special properties to the water in
the immediate neighborhood of the macromolecule. There are good reasons, aside from the
properties of proteins as such, for assuming
a special ice-like character of regions of the
hydrate water of these macromolecules. As
was mentioned previously, proteins contain a
large number and variety of nonpolar sidechains (table 2); in simple molecules these
side-chains possess the remarkable property
of forming crystalline hydrates consisting
predominantly of molecules of water in an
ice-like lattice with occasional holes into which
a nonpolar molecule may fit.1" 12 Such nonpolar hydrates are stable at temperatures well
above the melting point of ordinary pure ice;
it is the preseniee of the nonpolar molecules
in the holes of the polyhedrons (figs. 9 and
10) formed by the H20 molecules which stabilizes the ice-like lattice.
Circulation, Volume XXI, May 1960
831
SIZES AND SHAPES OF
SOME PROTEINS
IOOA
H20
e
CrGlucos.
No+ Cl- Glucose
N
Chymotrypsinogen
Myoglobin
,B-Lactoglobulin
Hemoglobin
SFibrinogen
Hemocyanin
Turnip Yellow
Virus
Figure 5
Sizes and shapes of some protein molecules. For
comparison, some small molecules and a scale are
shown at the top.
In a protein molecule many nonpolar sidechains exist in juxtaposition. Thus, there
could be a "coupling" of the ice-like lattices
which are formed around each residue with a
consequent strengthening of the resultant hydrate structure. The structure of the lattice
which is formed by the coupling of water of
hydration of protein side-chains may not be
832
KLOTZ
>. :-..Xu
.::PRO T EIN.
-:S
.
:(~~~~'4H
±
H'
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+
+
Figure 6
Schematic diagram of some ionic side-cha-ins in
protein
molecules.
Table 2
:TA-LACTOGLOBULIN
CComparison of Amino Acid Side-Cha ins with
IMolecutles TWhich Form Hydrates
0/
Some molecules
which form
crystalline
hydrates
Some amino acid side-chains
/
/
(Ala)
-CH3
GH s
(Val)
--CHz--CH<XCHs
(Leu)
-C
CR4
CHs
CH3
2CH<
Buffer
Ur / /
A
cf
_
AgNO3
_,
Figure 7
Detection of -SH groups by amperometric Ag+
titration of protein P-lactoglobulin. In buffer, current due to free Aig+ goes up immediately upon
addition of AgNO3. ,i.e., -SH grounps on protein
±SH urea,scurt sotays
are not detectable. In buffer
ait zero for substantiia addition of
of
due to removal of {A+
by -SH
--.1
L-'y
Ag,crra behantor
protein.
X-
-1'11
-CHR--SH
(Cys)
-CHz--CHR--S CIC3
(Met)
-CR2-
(Phe)
/
CHI
C
CR3CH
XCTI,CH
CHSH
CH3 S-CR3
/9
the same as that of nonpolar hydrates of small
molecules, such as those listed in table 2, but
the possibilities for cooperative interaction in
the macromolecule should be eveu greater,
since the residues are even initially relatively
fixed in position by the framiework of the protein molecule. A very schematic and exaggerated representationa of such an ice-like lattice is shown in figure 11.
The preseniee of patches or ribbonis of icelike coveriugs around various regions of a protein nmoleeule would affect both the kinetics of
Circulation, Volume XXI, May 1960
833
PROTEIN MOLECULES IN SOLUTION
N (CH5)2
TITRATION OF
s02- R
Fraction Acid Form
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pH
Figure 8
Uptake of H+ as a function of pH by dimethylamino group (of molecule shown) when
attached to glycine and bovine serum albumin (BSA), respectively. A much lower pH.
i.e., stronger acidity, is required to
place H+ on conjugate to the protein.
its reactions and its state of equilibrium. Rates
of diffusion of essentially all substances to a
reactive site under an ice layer would be
greatly reduced. With Li', for example, it is
known'3 that the rate of diffusion in iee is at
least 10,000 times slower than in liquid water.
On this basis, one can readily understand why
Ag' titrates very slowly with -SH residues
in many proteins, e.g., fl-lactoglobulin (fig. 7).
Judging from the observation that mereaptans
form erystalline hydrates, we might conclude
reasonably that -SH groups in proteins could
participate cooperatively with neighboring
side-chains in the formation of an ice patch.
Diffusion of Ag+ through this ice patch
would be greatly hindered as compared to the
movement of this ion in ordinary water. Collsequently, the protein mereaptan acts as if it
were masked.
Similarly, the shift in the titration curve
of protein-bound groups such as -N(CH3)2
toward a lower pH (fig. 8) can be readily inCirculation, Volume XXI, May
1960
terpreted. Such a shift means, in essence,
that the environmient is more favorable to
(CH3 ) 21\N than to (CH3) 2NH'. Sueh a bias
seems very reasonable, for the creation of a
charged group in place of the uncharged one
would require some breakdown of the ice lattice of the protein.
Interactions of Protein Molecules with Small
Molecules and Ions
In addition to interacting with solvent, proteins may also interact with all types of small
ions and molecules in the solution. The stability of the complexes which are formed
depends largely upon the character of the
macromolecule and the small ligand, but it is
also affected by the nature of the environment
in the solution. An exhaustive survey of these
factors would be inappropriate for this symposium, but a few representative examples of
some dominant factors in these interactions
will be described.
834
KLOTZ
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Figure 9
JLIodel of arrangement of water mnolecules in crystalline hydrates of nonipola,r taial(cules.
Each baill represents 1 water molecule. The molecules are arranged in ptntagoinal planes,
12 of wbhich enclose a dodeaahedral space. The hole insidle this polyhedron is about 3 A
in diameter. (Republishedc by permission of Zcitschritff fir Elelctroclh'n?ic.1)
Metal Ions and Hydrogen Ionis
Figure 6 was iutl oduced inito this paper
largely to slhow sonic J)olar side-chains wlhicl
occur ini practically all proteinis and which
are capable of forminog complexes witlh maimv
metal ions. Practically all metal ions forml
complexes wvith some protein side-chainis.
Neverthcl4ss, there are differenlees in specificity amiong the rm-etals; soIIme examples of these
differeinees are summarized in table 3. In
(gencral, tlhe alkali iietlals (e.g., Nat) do not
form coimiplexes, althi.oug,h with special proteinis (e.g., myosin14) bind(iing is observed,
probably due to a clustering of appropriate
resid-ues of the macromolecule. The alkalilneeartlh m1-etals (e.g., Cat+) formn complexes
largely with car boxylate or' phiosphate sidechainis. HeaIvier metals, particularly of the
transition series, are bound to practically all
groups of the protein which are capable of
Circulation, Volutme XXI, May 1960
835
PROTEIN MOLECULES IN SOLUTION
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Figure 10
JPolyhedlrous of type of figure 9 arranged to form a large lattice, as is found in crystalline
nonpolar hydrates. Compartments of 5, 6 and 7 A are available for enclosure of nonipolar
molecules wlithin icater structure. (Republished by permission of Oxford University
Press.39)
Table 3
Bindiny of Metal lons by tSome Sbubstitnents inl
ProteinsHG=CHO-P-O
Na
(."a+ +
Fe+++
Zni
Cu++
C
/ t0
I
HN
N
W
\C/
0
0-
H
0
+
0
+
+
+
0
0
+
± -+
+
A
+
HNH
S-
0
0
0
0
+
+
+
+[+
0
0
+
+
+
+ +±+
Hg++
++ +±+
+
+
*TEor furtiher qiuantitative estinates of affinities, see
(Trd an-id Wileox.13a
+
+
+
+
+
+
interacting with II+. Nevertheless, there are
differeciees ii:i specificity. Beyond the very
qualitative iniformatioln of table 3, one can
point to the very high affilnity of Fe-much
grieater thani that of Cu-for pheinolic oxygens
Circulation, Volumte XXI, May 1960
Figure 11
Schematic diagramii of ice-like character of hydration sheath airounzd nonpolar groups in protein
molecules. Small circ-les represent waiter molecules.
KLOTZ
836
MOLES BOUND METAL
MOLES TOTAL PROTEIN
1O0
5
pH
Zn++
pH
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8 .8
+~~~+
6.80
6.08
-
7.6
-I 0.0
I
-3.0
LOG
-2.0
[Me+
] FREE
Figure 12
Uptake of Zn++, and of Ca+, respectively, by serum albumin at different pH's. Ordinate
represents number of Zn++ (or Ca±±) ions bound by 1 molecule of protein; abscissa
gives the logarithm of the concentration of free metal ion (in qvater) in equilibrium with
the bound metal ion on the protein.
in compounds such as tyrosine. On the other
hand, Cu is bound more strongly to amine
nitrogens or to mereaptan groups. An interesting case not shown in table 3 is Pb ion
which, like Zn or Cu, is bound strongly to
mereaptan groups. On the other hand, Pb
shows a much greater affinity for carboxyl
than for amine groups; the opposite preference is characteristic of Zn and Cu.
There are also specificities among protein
molecules, although these are not too well understood in molecular terms, except perhaps
for the heme pigments in which a definite
prosthetic group can be isolated. Thus, conalbumin or transferrin shows a remarkable affinity for Fe, the interaction occurring with
tyrosine residues;15 yet, many other proteins
have a substantial complement of tyrosines,
but no unusual avidity for Fe.
The ligand groups shown in table 3 are all
capable of binding H+ ions. It is not surpris-
ing, therefore, that H+ ions should compete
with metal ions and, hence,
that the binding
of metals is dependent on the pH. For example, Fe(III)-conalbumin is stable above pH 7
but loses Fe(III) below pH 7 (if citrate is
present'5). More detailed results for albumiin
complexes with Zn'6 and with Ca'1 are illustrated in figure 12. In all cases, lower pH, i.e.,
increasing acidity, lowers the amount of
bound metal ion.
The extent of binding of metal ions by proteins may also be controlled in part by "metal
buffers, i.e., complexing and chelating agents
with a strong affinity for the cation. These
agents thus compete with the protein for the
ietal. For example, the binding of zinc ions
by serum albumin at pH 6.4 can be reduced
by addition of glycine,'8 which forms complexes with the metal. Similarly, acetate competes with protein for lead ions, iodide for
mereury,'8 citrate for copper.'9 These "metal
Circulation, Volume XXI, May 1960
837
PROTEIN MOLECULES IN SOLUTION
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-4
log [Am. Acifree
Figure 13
Comparison of the extent of binding of L-tryptophan with that of D-tryptophan by
serum albumin. Ordinate gives number of bound amino acid molecules on each protein
molecule, and abscissa shows concentration of free tryptophan in equilibrium ivith that
bound to protein.
buffers" can be used to control the extent of
binding of cations and, hence, the physical or
biologic consequences of such binding.
Organic Molecules
Proteins also form complexes with organic
ions and molecules. For purposes of illustration, we shall consider just a few complexes
of serum albumin. In a sense, this protein is
not representative because it binds a great
variety of molecules and ions whereas one
usually emphasizes the high specificity of the
interactions of proteins, e.g., enzymes or antibodies. We shall not discuss the molecular
interpretations of such specificity. It should
be pointed out, however, that serum albumin
also shows remarkable discriminatory properties. A most striking illustration occurs with
tryptophan (fig. 13), the L-isomer being
Circulation, Volume XXI, May 1960
bound substantially, the D-isomer markedly
less so.20 Preferences also are shown for posi-
tional isomers. For example, albumin distinguishes between o-, m-, and p-methyl red (fig.
14), not only with respect to affinity but also,
as judged from spectroscopic manifestations,
with respect to the nature of the interaction.
As with metal ions, the binding of organic
ions by proteins may be decreased by competition of other anions. Thus, the addition of
chloride, acetate or phthalate ions, reduces
the binding of methyl orange by serum albumin (fig. 15). Obviously, the type of buffer
solution used may influence substantially the
interaction between macromolecule and small
ion. Nonionic organic molecules may also compete with ionic. Thus, decanol reduces the
binding of tryptophan,20 and alcohols, from
838
KI,OTZ
3\N
N=N
w3
C
Go O2
z
0
a.
PROTEIN
Li
0
z
-1I
D 0
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H3C\
e0 O
co
J
0 0
/=N
N
H *-
0
12-13A
/
CO- H3
PROTE IN
PROTEIN
Figure 14
Structural formulas of some positional isomers
between which serum albumin can discriminate
either in extent of binding or in effect of protein
on color of small molecule. Also shown are distances between groups on protein inrolved in
binding of these small molecules, for comparison
with distances between (CHS)2N- and -COO- substituents on each small molecule. Protein sidechains are too far apart (12 to 13 A) to link to
both (CH,3)2N- and -COO- of top molecule; hence
linkage is to -COO- only, and binding and spectroscopic effects differ in this case from results with
lower 2 molecules.
-5.0
LOG I DY EIFREE
Figure 15
Difference in extent of binding of methyl orange
by serum albumin in presence of different buffTers:
*, acetate buffer, 0, phthalate buffer. Lower binding curve in presence of latter buffer showvs that
phthalate ion interferes more thacn does acetate.
methanol to decanol, reduce the binding of
methyl orange by serum albumin.
It is also possible to increase the uptake of
organic molecules and ions by a variety of
stratagems, 2 of which will be mentioned here.
A molecule such as pyridine-2-azo-p-dimethylaniline (I) is not, by itself, bound appreciably by proteins. However, if very small
amounts of metal, e.g., Zn++ are added to the
solution, very substantial binding of (I) follows (fig. 16). As we now kniow, the metal
N-N-=N
-
-N(CH3)2
(I)
ion acts as a bridge between protein and organic molecule (fig. 17). The net result is a
strong cooperative interaction. Zinc ion and
(I) have a small affinity for each other (k
0.2 x 103) even in the absence of protein;
attached to a protein, the affinity of Zn++ for
(I) rises almost 50-fold (k = 9 X 103).
Equally striking increases in the binding
of anions by protein molecules are observed
in the presence of glycine (fig. 18), but here
Circulation, Volume XXI, May 1960
PROTEIN MOLECULIES IN SOLUTION
839
2
z
WZn+i
li
C 0
a
z
cn
D 00
ED
.1
Uf)
Uf)
LJ
0 0
2 ri
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No Zn
0
fv
I
*-
-5
LOG
-4
[DYE]
FREE
Figure 16
Increase in degree of binding of a dye (pyridine2-azo-p-dimethylaniline) when Zn++ is added to
the protein solution. Each curve shows number of
bound dye molecules on each protein molecule, as
a function of free dye concentration in the solution.
PROTEIN
.Zn
ON =
N
o
(C H3
Figure 17
a bridge
Schematic diagram of Zn++ ion acting
between protein and small molecule. This mediating effect of Zn++ increases number of dye molecules bound to protein molecule.
as
the mechanism of the effect must be markedly
different, for glycine itself is not bound by
serum albumin. Furthermore, the concentrations of this amino acid which are required to
increase the binding of protein22 are very
high (0.5 to 2 M) compared to those at which
metals manifest their effects. At such high
Circulation, Volume XXI, May 1960
log
(A)
Figure 18
Increased binding of methyl orange by serum
albumin when glycine is added to the solution:
0, no glycine; 0, 2.5 M glycine. Each curve shows
number of bound dye molecules per protein molecule as a function of the concentration (A) of free
dye in solution.
concentrations of amino acid, the dielectric
constant of the solution is altered appreciably
and, hence, interactions between charged
species could be changed. Nevertheless, as we
have shown, this electrostatic effect is not the
dominant one since even the binding of uncharged molecules by proteins is increased. Instead, it is our feeling, for reasons described
elsewhere,22 that the influence of glycine is to
be understood in terms of what this amino
acid can do to the solvent rather than to the
protein. Zwitterionic glycine perturbs the
water in its neighborhood and, hence, also the
hydration of the organic molecule in the
aqueous solution. Consequently, the organic
moleeule turns more readily to the more hospitable environment of the protein envelope
where, with the assistance of nonpolar side-
840
KLOTZ
As has been mentioned earlier, mercury
combines with -SH groups of proteins. An
analogous reaction occurs with organic mercurials, e.g., between (II),
(CH8)2N-Q-
p
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Figure 19
Top: protein molecule, P, with some water of
hydration (small circles) around nonpolar regions.
Bottom: protein complex with an anionic hydrocarbon molecule, A, showing increased hydration
in the complex due to cooperative effect betwveen
P and A.
chains of the macromolecule, a cooperative
stabilization of the water lattice may ensue
(fig. 19). On this basis, one can see that
glycine should affect the binding of uncharged
organic molecules as well as of organic ions.
So far, we have considered examples of protein complexes which are essentially reversible
(in the thermodynamic sense), that is, which
can be dissociated by lowering the concentrations of the reacting species. It should be
pointed out that covalently bound complexes
may also be formed at physiologic temperatures and pH's. Among the most interesting
of these from a physiologic viewpoint are
those involving the mercaptan or disulfide
substituents of proteins.
N = N-D-
Hg-OOCCH8
(II)
and almost any protein with mereaptan
groups:
P - SHl+RHgX--.P-- S--HgR +HX. (2)
The mereaptide bonds formed are very stable;
although the rate of the reaction is not always
high, the stoichiometry23 is very sharp (fig.
20). Similarly, sharp stoichiometry is observed in the reaction of disulfide compounds
with protein mereaptan groups :24
P-SH + R-S-S-R --- P-S-S-R. (3)
Intramacromolecular Interactions of Proteins
It has been known for some time that for
many proteins, the kinetic-molecular unit
which we see at physiologic pH's and temperatures is not necessarily the smallest molecular species. Under other conditions, the
macromolecule may dissociate (or associate)
reversibly into smaller (or larger) units. Some
of the earliest work in this field is that of
Eriksson-Quensel and Svedberg,25 who demonstrated a number of disaggregation reactions in hemocyanins. A more recent study
with ft-lactoglobulin,2628 is shown in figure 21.
The normal molecular weight for this protein is around 35,000. From the shape shown
in figure 5, which is based on x-ray investigations, one might not be too surprised to see
that this molecule dissociates into 2 equal fragments at low pH. Furthermore, as figure 21
shows, aggregation into units which have
molecular weights of 70,000 may also occur at
low temperatures, although this is not as complete as the reaction of disaggregation.
In general, disaggregation occurs at pH's
far from the isoelectric point, i.e., where the
protein acquires a substantial charge. Sueh
behavior is characteristic of many other proteins, for example, chymotrypsin29 and insulin,30 as well as of hemocyanin and
B&-lactoglobulin. WVith insulin the electrostatic
Circulation, Volume XXI, May
90an
841
PROTEIN MOLECULES IN SOLUTION
1.0k-
r
A 0-a
o
o0
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0.5
r)
0
8 °
C
0
0
0
l
p~~~~
O
P-S-Hg-R
-SH+R-Hg-X
1-
15
30
45
[dye]free aqueous
M
60
X
Figure 20
Extent of binding of an azomercurial by serum albumin. The binding rises very sharply
even at exceedingly small concentrations of mercurial, and levels off very sharply when
combination with all available -SH groups has occurred.
basis of the dissociation has been indicated by
further experiments.30 In acid solution the
unit of molecular weight near 36,000 dissociates into particles which have a molecular
weight of 12,000 when the charge reaches
about +20 to +30 per trimer. In the presence
of SCN- ions, known to be bound to insulin in
acid solution, lower pH's must be attained to
dissociate the trimer. Obviously, the bound
anions reduce the internal electrostatic repulsion in the insulin molecule and hence stabilize the trimer. These observations indicate
that many protein molecules are composed of
smaller units linked together by relatively
weak bonds. The nature of these bonds has
still not been established unequivocally.
Intermacromolecular Interactions
In addition to interacting with themselves,
protein molecules of a given kind may form
complexes with other types of proteins or with
other types of macromolecules, including polyCirculation, Volume XXI, May 1960
saccharides and nueleic acids. One can distinguish in these combinations 2 general categories of complexes: (1) nonspecific, in which
electrostatic factors usually predominate; (2)
specific, in which distinctive structural factors
are of major significance. As an example of
each category, we shall cite protein-nucleic
acid interactions.
Interactions of proteins with nucleic acids
are excellent examples of cases in which opposite electrostatic charge is the primary condition for reaction. Precipitates are formed,
for example, between nucleic acid and lysozyme, ovalbumin, serum albumin, fibrinogen, casein, and aetin, respectively ;31-33 in all
cases the protein is cationic, the nucleic acid,
anionic. As we would expect for a complex
held together primarily by electrostatic forces,
small quantities of salt usually redissolve and
dissociate the combination. Soluble complexes
may also be formed.32 34 In all these cases, the
KLOTZ
842
Molecular Weight
-
60,000)
0
~~~~~~~~~20C.
IO---0 0'
'IO
0
50,000o
1
I~~~~~~~~~~~~~~~~~~~~~~
O
//
0
40,0ooo
0~
-0-0-0-
30,0001
25° C.
20o,0o0
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
6-v
10,000
2.0
3.0
4.0
5.0
pH
Figure 21
Variations in molecular weight of /3-lactoglobulini. Solid line correlates results at 25 C.,
broken line at 2 C. (Method of presentation taken from Tanford.28) The "normal" moleculaxr weight of /3-lactoglobulin at its isoelectric point (pH 5) and higher is 35,000. At lowtt
temperature and pH's of 4 to 5, the molecules aggregate to dimers. At very low pH, the
molecule breaks up into 2 subutitts; compare with diagram in figure 5 which is based on
x-ray data.
wide spectrum of proteius with which nucleic
acid complexes are formed shows the absence
of any specificity in these combinations. Theoretical calculations35 also lead to the conclusion that electrostatic attraction can account
quantitatively for the strength of association.
In contrast to these nonspecific complexes,
one may cite the highly specific complexes between nucleic acid and protein which are exemplified in the viruses. These combinations
lead to special supermolecular structures,
with characteristic arrangements of constituent macromolecules. A model for tobacco
mosaic virus36' 36a, 36b iS showin in figure 22.
This supermolecule, with a nlolecular weight
of about 40,000,000, has an over-all shape of
a long thin rod, 3,000 A in length and 170 A
in width. It is composed of about 5 per cent
ribonucleic acid and 95 per cent protein. The
association, however, is hardly the random
type found in the complexes described in the
preceding paragraph. The nucleic acid seemiis
to be a single strand winding its wvay through
the rod in a helical fashion. In contrast, the
protein componeiit seemlls to be present in subunits which have a molecular weight of about
17,000. These subunits envelop the coiled
strand of nlueleic acid by winding around it,
also in a helical array.
There are other rod-shaped viruses with
structures similar to that of tobacco mosaic
virus. Many small viruses, however, are quite
different in structure, being spherical in shape
(for example, turnip yellow virus, fig. S).
Again, interactioni between niucleic acid and
protein leads to a very specific structure, but
with the nucleic acid in the core of the spherical supermolecule anil protein subunits arranged symmetrieally in an outer tbick shell.
Circulation, Volume XXI, May 1960
-PROTEIN MOLECULES INT SOLUTTION
843
Conclusion
see that a protein molecule in
solution may undergo( a hlost of intteractionis:
witli solvent, withl smnall moleeules land ioiis,
wvitlh itself, witlh other mnaeromolecules. In a
jthy-So1dogie miliell all of these possibilities
resciLt tIheniselv es a.d(1 wvill ronipete amiong
thlemselves. Reasonable guesses (as t-o whieb
xwill be the doiminaiit inter-et 10118 (1ii often
be Ina(le Lfioini lelia-vioi observe(l in somie of'
thie simple sy slens iwllie we hiave (lescribed.
It seem11S reaW-sonlable to expert tait further
studies of s11(c1 int ew-rartions in arntifieial simple
vitviroiiinieiits 1iiayf iltiiately provide! a finin
basis f1 relaftilln) thle strlneture, of ninae romoleruldes to liciir biologir art ivity.
'Thus,iwe
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References
N
1. lh ri^ A.
\ P., S ANOVR, F., SLITII, L A.ANT)
:Errf u,N IL.: l)isulphide 1)0(ods of insilin. BioCeieii. .J. 60: 541, 1955.
2. lJiTs;, C 11. \V., STEN, W. If., A \N- tOOR, S.:
S1(tuies oil the
11 sittitre of rihoimleclr':ise. 11(
Svmposlum (o11 Piroteini Struiceture. Neuberger,
A., Eld. Liondon, Methlinll & C1o. Lttl., 195S,
pt. 211.
ii, A. P., AsN ANFINseN, C. IL: Stlldies oll
3. As
thle (lisultide bridIg-es ill rihollnuelease. B3iochimii. e(t bId)hYs. acta 24: 633, 19;57.
4. L'ING, 1.., (CoRY\, . I'., \NND IBRANSON, It. I.:
p
St ructu e of r-ote
ns: Two xv l rogell-bolnded
lelic:il eolfiguratiills of tlle polYp)eptile ch"lil.
Prloc. NlA-l. Acuol. Sci., U.S. 37: 205, 1951.
sw
IKv\w
1
C., D1hno, G., 1)NTrZIS, I. AM., P.A1SI 1, II
.,NDWCO\FVFi , IJ.: A. three dinieii,8io01,1t modlel of the myoglobin mIIolecule ohtamdni(l iv X royt! nalysis. Na(lture 181: 662,
1958.
XXI) \
6. (nsnsmniu, .1 . A.,
A.: State of
tmr)osinie in egg all)iil 1d1(1 in insilulin as deby spectropliotol3metric titration. Bioterf'ini
chemo. J. 37: 302, 1943.
7. T NNFORDJ, C. Ti XSTEiX, .7 . S., -.-ND P DS,
P. G.: Phlenloli,
lc1droxyl ionizaition in protr IsS. I i-.
Hibolclese. .1. Ani. Chewm. Soc.
77: 6409, 1955.
S. 1IEYCEIOKR, S., XI) SinsARiIT, 3.: IncreTF(ase in
aci(l-dbilnding sites oil denatrlluation of boIse
ferrihemnoglobin ait
,O. .1. Am. Clew,l. Soc. 81:
10.
nucleic acid hell.. 7'he
p)rotneif
siibltoits also uoint
Kliol'z, 1M.AL, .o) LPu:ss, I-. A.: Ionie eq(uilibria
aroutndl n a hclical a-rr'a q. Tile cueter of the r od
is h0ollo0w (butt filled with water mooculies) for a
distance of aboutl; &0 - arocl)d the aris. (RepvblishedI byJ Permission O(f J<ndea(o.301
im a pIroteill conjug.ate, of a sulfonamide type.
Bliochim. et biophys. acta 38: 57, 196(.
Protein hydration and(] behavior. Science
128: 81.5, 19-58.
11. ST'WKRELERG, M. V., AND ML:LLEiR, IH. R.: Feste
Gashydrate. IT. Strullktur uiil Ralumehemie.
Ztschr. Elektrocbeiu. 58: 25, 1954.
5679, 1959.
9.
Figure 22
X-4 model of tobat co mosaic virusE. The ithite ellip.soids rej)present hr proteiu Subunits (each, abodt
18',000 molecular weight); tile block coil, the ribo-
Circulation, Volume XAI, May 1960
844
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
12. -, AND MEUTHEN, B.: Feste Gashydrate. VII.
Hydrate Wasserlislicher Xther. Ztschr. Elektrochem. 62: 130, 1958.
13. EIGEN, M., AND DEMAEYER, L.: Self-dissociation and protonic charge transport in water
and ice. Proe. Roy. Soc. London s.A:247:
505, 1958.
13a. GURD, F. R. N., AND WILCOX, P. E.: Complex
formation between metallic cations and proteins, peptides, and amino acids. Advane.
Protein Chem. 11: 311, 1956.
14. LEWIS, M. S., AND SAROFF, H. A.: Binding of
ions to muscle proteins: Measurements on the
binding of potassium and sodium ions to myosin A, myosin B and actin. J. Am. Chem. Soc.
79: 2112, 1957.
15. WARNER, R. C., AND WEBER, I.: Metal combining
properties of conalbumin. J. Am. Chem. Soc.
75: 5094, 1953.
16. GURD, F. R. N., AND GOODMAN, D. S.: Preparation and properties of serum and plasma proteins. XXXII. The interaction of human
serum albumin with zinc ions. J. Am. Chem.
Soc. 74: 670, 1952.
17. KATZ, S., AND KLOTZ, I. M.: Interactions of calcium with serum albumin. Arch. Biochem.
44: 351, 1953.
18. GURD, F. R. N.: Specificity of metal-protein interactions. In Ion Transport Across Membranes.
Clarke, H. T., Ed. New York, Academic Press,
1954, p. 264.
19. KLOTZ, I. M., AND FIESS, H. A.: Thermodynamics
of metalloprotein combinations: Buffer effects in copper-albumin complexes. J. Phys.
and Colloid Chem. 55: 101, 1951.
20. MCCMENAMY, R. H., AND ONCLEY, J. L.: Specific
binding of L-tryptophan to serum albumin.
J. Biol. Chem. 233: 1436, 1958.
21. KLOTZ, I. M., BURKHARD, R. K., AND URQUHART.
J. M.: Structural specificities in the interactions of some organic ions with serum albumin.
J. Am. Chem. Soc. 74: 202, 1952.
22. -, AND LUBORSKY, S. W.: Binding of organic
ions by proteins: Effects of changes in solTent and their implications as to the nature
of the complexes. J. Am. Chem. Soc. 81:
5119, 1959.
23. HOROWITZ, M. G., AND KLOTZ, I. M.: Interactions
of an azomercurial with proteins. Arch. Biochem. 63: 77, 1956.
94. KLOTZ, I. M., AYERS, J., Ho, J. Y. C., HOROWITZ, M. G., AND HEINEY, R. E.: Interactionns
of proteins with disulfide compounds: Some
implications for electron transport in proteins.
J. Am. Chem. Soc. 80: 2132, 1958.
25. ERIKSSON-QUENSEL, I. B., AND SVEDBERG, T.:
Molecular weights and pH-stability regions of
the hemocyanins. Biol. Bull. 71: 498, 1936.
26. TOWNEND, R., AND TIMASHEFF, S. N.: The pH
KLOTZ
dependence of the association of 8-lactoglobulin. Arch. Biochem. 63: 482, 1956.
27. -: Molecular weight of P-lactoglobulin. J. Am.
Chem. Soc. 79: 3613, 1957.
28. TANFORD, C.: Configuration of globular proteins
in aqueous solution and its dependence on pH.
In Symposiumi on Protein Structure. Neuberger, A., Ed. London, Methuen & Co. Ltd.,
1958, p. 61.
29. SCHWERT, G., AND KAUFM AN, S.: Molecular size
and shape of the pancreatic proteases. III.
a-Chymotrypsin. J. Biol. Chem. 190: 807, 1951.
30. FREDERICQ E., AND NEURATH, H.: Interaction of
insulin with thiocyanate and other anions: The
minimum niolecular weight of insulin. J. Am.
Chem. Soc. 72: 2684, 1950.
31. CHEPINOGA, 0. P., AND GROSBLAT, R. SH.: Characteristics of salt-like compounds of deoxyribonucleic acid with proteins. Ukrain. Biokhim.
Zhur. 21: 121, 1949. Chem. Abstr. 48: 4014,
1954.
32. GEIDUSCHEK, E. P., AND DOTY, P.: A light scattering investigatioii of the interaction of sodium desoxyribonucleate with bovine serum albumin. Biochim. et biophys. acta 9: 609, 1952.
33. TONGUR, V. S., DISKINA, B. S., AND SPITKOVSKII,
D. M.: Production and properties of artificial
complexes of desoxyribonucleic acid with serum
albumin. Biokhimiya 22: 879, 1957.
34. HAM,ER, D.: Interaction of acids of high molecular weight with albumin. Biochem. J. 56:
610, 1954.
35. STEINER, R. F.: Reversible association processes
of globular proteins. II. Electrostatic complexes of plasma albumin and lyzozyme. Arch.
Biochem. 47: 56, 1953.
36. FRANKLIN, R. E., KLUG, A., AND HOLMES, K. C.:
X-ray diffraction studies of the structure and
morphology of tobacco mosaic virus. In Ciba
Foundation Symposium on the Nature of
Viruses. Wolstenholme, G. E. W., and Miller,
E. C. P., Eds. London, Churchill Ltd., 1957,
p. 39.
36a. CRICK, F. H. C., AND WATSON, J. D.: Virus
structure: General principles. In Ciba Foundation Symposium on the Nature of Viruses.
Wolstenholme, G. E. W., and Miller. E. C. P.,
Eds. London, Churchill Ltd., 1957, p. 5.
36b. CASPAR, D.: Discussion. In Ciba Foundation
Symposium on the Nature of Viruses. Wolstenholme, G. E. W., and Miller, E. C. P., Eds.
London, Churchill Ltd., 1957, p. 14.
37. Tuppy, H.: Aminosiure Sequenzen in Proteinen. Naturwissensehaften 46: 35, 1959.
38. PERUTZ, M. F.: Some recent advances in molecular biology. Endeavour 17: 190, 1958.
39. WELLS, A. F.: The Third Dimension in Chemistry. New York, Oxford University Press, 1956,
plate X.
Circulation, Volume XXI, May 1960
Protein Molecules in Solution
IRVING M. KLOTZ
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Circulation. 1960;21:828-844
doi: 10.1161/01.CIR.21.5.828
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