Molecular Properties of Rous Chicken Sarcoma
Hyaluronic Acid-Protein Complex
A. CAPUTO AND M. L. MARCANTE
("Regina Elena" Institt, te for Cancer Research, Rome, Italy)
H y a l u r o n i c acid (HA), first isolated b y M e y e r
(1r is a c o m p o u n d of considerable biological interest. T h i s p o l y s a c c h a r i d e has been f o u n d in
m a n y a n i m a l tissues, especially those of mesenc h y m a l origin, and in t h e capsules of some t y p e s of
bacteria.
F u r t h e r m o r e , a m u c i n o u s p o l y s a c c h a r i d e of t h e
H A t y p e was d e t e c t e d a n d isolated from fowl sarc o m a by K a b a t (10) a n d by C l a u d e (5). T h e s e
findings h a v e been confirmed r e c e n t l y by W a r r e n
a n d co-workers (17), b y C a p u t o (5), and by H a r ris, M a l m g r e n , and Sylv6n (9).
I n t h e course of some research related to t h e
i m p o r t a n c e of m u c o p r o t e i n s in t u m o r growth, we
b e c a m e i n t e r e s t e d in t h e isolation a n d c h a r a c t e r ization of Rous c h i c k e n s a r c o m a HA.
I n this p a p e r will be described t h e results of a
s t u d y o n t h e p h y s i c o c h e m i c a l properties of H A
isolated f r o m Rous chicken sarcoma, c o m p a r i n g it
w i t h t h e same c o m p o u n d p r e p a r e d f r o m n o r m a l
sources, such as cock's comb, ox s y n o v i a l fluid, and
h u m a n umbilical cord.
MATERIALS
AND METHODS
T u m o r s were collected f r o m ~ - 3 - m o n t h - o l d
chickens. T h e m a t e r i a l , freed of necrotic areas a n d
reduced to small pieces, was d e f a t t e d with cold
a c e t o n e c o n t a i n i n g a few drops of acetic acid; t h e
acid acetone was twice replaced with cold acetone.
After 48 hours t h e pieces were dried and t h e n
homogenized in a W a r i n g B l e n d o r with 10 volumes of distilled water. T h e p H was t h e n b r o u g h t
to 9 b y a d d i n g a few drops of 0.1 N N a O H .
Extraction was carried out for 48 hours with occasional
stirring. The mixture was filtered through cheesecloth, and the
centrifugally cleared extract was acidified to pH 4.0 with 0.1 N
HC1. The salt concentration of the extract was brought to
0.`2 M by adding NaC1, and the mucopolysaccharide was then
precipitated by addition of e volumes of acetone. The precipitate, collected with a stirring rod, was redissolved with distilled water, dialyzed against distilled water until salt-free, and
then lyophilized. All manipulations were performed in the cold
room at +2 ~ C.
The same procedure has been employed to isolate HA from
other sources, such as cock's comb and human umbilical cord.
Received for publication December 8, 1958.
In the case of ox synovial fluid after defatting, the pH was
brought to 4.0, and then precipitation of HA complex was obtained, as described above, by adding NaC1 and `2 volumes of
cold acetone.
From chemical analysis it is apparent that HA prepared by
this method must be considered as an HA protein complex.
Quantitative estimations of protein by means of the biuret reaction (8) have shown that the HA from Rous chicken sarcoma
contains about 15 per cent of protein and the HA's from umbilical cord, ox synovial fluid, and cock's comb contain, respectively, 10, 13, and `20 per cent of protein.
None of the preparations contained heparin, as ascertained
by the clot-forming method. The absence of phosphorus and of
a specific absorption in the U.V. region both indicate that the
HA-protein complex does not contain nucleic acids.
Electrophoretic experiments were performed with moving
boundary Perkin-Elmer apparatus, and mobilities (~ X 10-5
sq. era. sec-t volt-I) were calculated as means of the values of
the ascending and descending limbs. All the runs were made
with buffer systems according to Miller and Golder (18).
The sedimentation studies were made in the Spinco electrically driven ultracentrifuge at 59780 r.p.m. The values of the
sedimentation constant, corrected for viscosity and density of
water to '20~ C., are given in Svedberg units (S~.~0 )< 10-ta).
Diffusion measurements were made at + 1~ C. in the electrophoresis separation cell according to the height-area method.
For sedimentation and diffusion constant measurements, the
media viscosities were determined on a Ostwald capillary viscosimeter with a water flowtime of 4`24see. The densities of the
media were calculated in a specific gravity pipette.
RESULTS
T h e electrophoretic b e h a v i o r of Rous s a r c o m a
H A - p r o t e i n complex is c h a r a c t e r i z e d b y t h e presence of one very sharp single b o u n d a r y , which
migrates r a p i d l y with a m o b i l i t y , at p H 7.0, of
# = 14.5 )< 10 -5. F o r H A - p r o t e i n complex isol a t e d from sources different from R o u s sarcoma,
such as cock's comb, h u m a n umbilical cord, a n d ox
s y n o v i a l fluid, we h a v e o b t a i n e d t h e same electrophoretic pattern.
I n t h e u l t r a c e n t r i f u g e , the Rous s a r c o m a HAp r o t e i n complex sediments as a monodisperse system, as i n d i c a t e d b y t h e presence of one single
very sharp b o u n d a r y at b o t h higher a n d lower conc e n t r a t i o n s . T h e same characteristics h a v e b e e n
f o u n d for t h e c o m p o u n d s p r e p a r e d f r o m o t h e r
sources; t h e s e d i m e n t a t i o n d i a g r a m s of Rous sarc o m a a n d cock's comb H A - p r o t e i n complex are
shown in C h a r t 1.
1010
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CAPUTO AND IM~ARCANTE--ROU8 Sarcoma Hyaluronic Acid-Protein Complex
The sedimentation constant of HA-protein complex depends to a considerable extent upon its
viscosity, and therefore the values are closely related to the concentrations, as is shown in Chart 2,
where the sedimentation constant, corrected for
temperature and viscosity of medium, is extrapolated to 0 concentration.
We have studied the stability of our preparations in the ultracentrifuge as a function of pH:
as shown in Chart 3 the behavior of tumoral HAprotein complex closely resembles that of HA from
different sources. The pH stability region is confined to the zone pH 5.0 and 8.0, above and below
which the sedimentation constants at infinite dilution decrease. HA precipitates from solution below
pH 8.0.
In order to ascertain the influence of the medium on the stability of HA-protein complex a few
1011
lZ.
12
\
Sw, 20
10
8
I
I
.2
!
.Z,
I
.6
I
.8
1.0 1.2
gm.~/o
CHANT 2.--Relation between sedimentation constant
values and concentration of hyaluronic acid prepared from
different sources.
Rous chicken sarcoma ( O ~ O ) .
Cock's comb ( O ~ O ) .
Human umbilical cord ( +
.+).
Ox synovial fluid ( ~
~).
pH 7.0 phosphate buffer with ionic strength, 0.1.
CHART 1.--Sedimentation diagrams of Rous sarcoma
hyaluronic acid (left) and cock's comb hyaluronic acid (right),
pH 7.0; phosphate buffer with ionic strength, 0.1; HA concentration, 1 per cent. Photographs taken 5400 seconds after
reaching full speed (59780 r.p.m.).
15
SOw,20
14
experiments were performed with high salt concentrations. Changes were observed in the ultracentrifuge at the highest salt concentrations (Chart
4).
In the diffusion experiments each run was performed in duplicate, and each half of the cell was
filled with a different sample of HA. The photographs were taken at intervals of between 70 and
120 hours, and the values averaged. In Table 1 are
reported the values obtained for HA-protein complex of different sources.
The partial specific volume has been measured
in 5-ml. pyknometers by plotting the weights of
the contents of the pyknometers for several concentrations of HA-protein complex against the
concentration expressed as a weight fraction. The
same mean value of V = 0.703 has been obtained,
without appreciable differences, for HA-protein
complex of each source. The molecular weight has
been calculated using the usual formula of Svedberg, where M = Rts/D(1 - fr), and substitut-
13
0
J
12
10
'
3
l
5
|
I
7
I
I
I
9
1
CHART 8.--Sedimentation constants at infinite dilution of
Rous sarcoma HA (O
~) and cock's comb HA (O
O)
as a function of pH. Acetate, phosphate and veronalate buffer
according to Miller and Golder. Ionic strength, 0.1.
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Research.
Cancer Research
101~
ing the values found for s, D and f'. The data are
reported in Table 2.
DISCUSSION
Evidence has been obtained to show that HA is
present in appreciable quantities in Rous chicken
sarcoma, thus confirming the reports of previous
findings (Kabat [10], Claude [5], Warren and coworkers [17], Caputo [3], Harris and co-workers
[9]). HA has been isolated by a method which rep13
12
S ,2o
Vol. 19, November, 1959
In a comparison of our results with those reported by previous workers (1, 4, 6, 11, 14-16), it
will be noted that the discrepancies are not significant when the molecular weight data were obtained with the same procedure. The only difference found was in the partial specific volume,
which was slightly different from those reported by
Ogston and Startler (14) and by Varga (16). The
value we have found falls within the range of many
other polysaccharides, and the discrepancies could
be attributed to the different percentages of protein present in our preparations.
To ascertain whether the difference in molecular
weight is due to the degree of polymerization or to
a peculiar arrangement of the molecule, we have
studied some other physicochemical properties,
TABLE 1
10
DIFFUSION CONSTANT OF HYALURONIC
ACID-PROTEIN COMPLEX
Source
Concentration
(gin. %)
Dw. 2oXlO -7
Rous sarcoma (chicken)
Comb (cock)
Umbilical cord (human)
Umbilical cord (human)
Synovial fluid (ox)
0.5
0.5
0.4
0.3
0.3
1.0
1.0
0.9
1.1
1.0
TABLE
i
l
I
1
2
3
NaCI molarily
CHART 4.--Sedimentation constants at infinite dilution of
Rous sarcoma HA (O
0) and cock's comb HA ( 0 ~ 0 )
as function of NaC1 molarity. Miller and Golder, pH 7.0
phosphate buffer with increasing quantities of NaCI.
resents a slight modification of the old procedure
of acetone precipitation.
The mucopolysaccharide obtained from Rous
sarcoma and other sources by this procedure must
be considered as HA-protein complex and is a relatively pure substance as judged from some of its
physicochemical properties. Thus, it behaves in
the electrophoresis as a monodisperse compound
exhibiting one single, sharp boundary with a high
mobility. The homogeneity of HA-protein complex has been confirmed by the ultracentrifuge experiments, in which in the sedimentation diagram
is present one single, very sharp boundary.
A variation in molecular weight has been found
between HA-protein complex from the tumor and
that from normal sources: for the first a value of
760,000 has been found, while for the others the
molecular weight is of the order of 1 million.
MOLECULAR WEIGHT AND FRICTIONAL RATIO OF
HYALURONIC ACID-PROTEIN COMPLEX
Source
Rous sarcoma (chicken)
Comb (cock)
Umbilical cord (human)
Synovial fluid (ox)
Mw X l0 s
f/f~
0.76
1.01
1.03
~.75
~.65
~. 65
2.60
1.07
such as the pH stability in the ultracentrifuge and
the salt effect. With regard to the stability of HAprotein complex at different pH values, both the
material prepared from tumor and that isolated
from a normal tissue of the same species (cock's
comb) behaved similarly. The two compounds are
stable only between pH 5.0 and 8.0; below pH 5.0
and above pH 8.0 they begin to dissociate into low
molecular weight decomposition products. These
findings are in good agreement with the data previously obtained by Laurent and Gergely (l l) and
by Blix and Snellman (1).
The appearance of the sedimentation diagrams
of tumor or comb HA-protein complex is not modified by the addition of salt. However, the salt ef-
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Research.
CAPUTO AND
MARCANTE--Rou8 Sarcoma Hyaluronic Acid-Protein Complex
feet is evident from the computed sedimentation
constants.
Evidence has been presented in this paper that
HA occurs in Rous sarcoma in a molecular state
slightly different from that isolated from normal
sources. If we consider that the tumor HA-protein
complex has physicochemical properties similar to
the HA isolated from normal sources, with the exception of a lower molecular weight, it might be
concluded that the difference is due to a smaller
degree of polymerization.
Now the following question arises: is the tumor
HA synthesized in the same way as in a normal
tissue, or is a depolymerizing system present in the
tumor which is able to act on the usually formed
HA? It seems unlikely to admit that the synthesis
of HA in tumor might follow steps different from
those normally observed.
For this purpose we have performed a few experiments, in which it has been observed that the
incubation of synovial fluid HA with the tumor
extract, deprived of its HA, was able to reduce the
molecular weight of synovial fluid HA from 1.07 to
0.80 • 106. It seems, therefore, according to Gersh
and Catchpole (7), that the tumor contains an enzymatic system which depolymerizes HA. Preliminary observations on the mechanism of action
of this depolymerizing system have shown that it
is almost different from the well known hyaluronidase, a n d p e r h a p s s o m e e n z y m e of t h e p r o t e o l y t i c
type could p l a y a g r e a t role i n t h e d e g r a d a t i o n reaction. Studies on the isolation and characterizat i o n of t h e H A d e p o l y m e r i z i n g s y s t e m p r e s e n t i n
t h e R o u s s a r c o m a are i n progress, a n d t h e r e s u l t s
will b e r e p o r t e d i n a f o r t h c o m i n g p a p e r .
SUMMARY
The molecular properties of HA-protein complex isolated from Rous chicken sarcoma have
been studied. I t has been shown that tumoral HAprotein complex behaves in electrophoresis and in
the ultracentrifuge as a monodisperse system.
The molecular weight of tumor HA-protein
complex is 760,000, while that isolated from nor-
1013
mal sources, such as cock's comb, ox synovial fluid,
and human umbilical cord, has a molecular weight
of the order of 1 million.
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Research.
Molecular Properties of Rous Chicken Sarcoma Hyaluronic
Acid-Protein Complex
A. Caputo and M. L. Marcante
Cancer Res 1959;19:1010-1013.
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