[CANCER RESEARCH 37, 1356-1359, May 1977]
Interactions
between
“Fever―
Proteins
and Normal
Serum
Proteins in Febrile Cancer Patient&
Charles W. Young, Wilner Dessources,
and Sadie Hodas
The Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
(7). By comparison with purified bovine serum albumin,
ovalbumin, ANase, chymotrypsin, and cytochrome C, these
When analyzed by cationic discontinuous electrophoresis
proteins behave in the polyacrylamide gel electrophoretic
in urea-containing polyacrylamide gels, plasma or serum
systems as if they have molecular weights of 30,000 dal
from febrile individuals contains trace quantities of five tons and isoelectric points between 5 and 9. Comparison of
protein bands that are not recognizable in the blood of
the content of these trace proteins in serum and CSF2
normal individuals. These proteins appear and disappear in suggests the existence in vivo of extensive interaction be
parallel in sequential samples. Cerebrospinal fluid from
tween the trace proteins and the abundant larger protein
febrile and nonfebrile individuals contains a protein band
species in plasma. This paper reports on studies which have
that is electrophoretically identical with only one of these
provided in vitro evidence for such interactions. They ap
proteins.
Sincethetraceproteins
migrate,inurea-contain pear to be based principally upon hydrogen bonding and
ing polyacrylamide gel electrophoresis, as if they had a electrostatic attraction ; however, hydrophobic interactions
molecular size of 30,000 daltons, their absence from cere
with lipoproteins and chybomicrons may also occur.
brospinal fluid implies the existence, in vivo, of interactions
between them and other serum proteins. Under nondisso
ciating conditions, four of the bands appear to circulate in MATERIALS AND METHODS
physical interaction with one another. In molecular sieve
A detailed summary has been published previously on the
chromatography at neutral pH in lipid-free sera, the trace
reagents
and methods employed in cationic polyacrylamide
proteins have an approximate molecular size of 165,000
gel
electrophoresis
in urea-containing gels (6). Anionic
daltons; in lipemic sera they have a molecular weight of
polyacrylamide
gel
electrophoresis
was carried out in 15- x
200,000 daitons. Their behavior in gel filtration and in ion
1.5-cm tubes according to Green's modification (2) of the
exchange chromatography excludes extensive interaction
method of Davis (I). Molecular sieve chromatography with
with any of the following: immunogbobulin M, immunogbo
Sephadex
G-75 and G-200 (Pharmacia Fine Chemicals, Pis
bulin G, a2-macrogbobulin, haptoglobin, and albumin. In
cataway,
N.
J.) was carried out in the conventional manner.
teractions between these and other serum proteins are re
duced by high concentrations of urea and by low pH. The Ion-exchange chromatography performed on QAE-Sepha
dex A-50 was modified from the method of Joustra and
mechanisms responsible for the observed protein-protein
Lundgren (4).
associations would appear to include electrostatic attrac
tion, hydrogen bonding, and weak hydrophobic interaction.
RESULTS
SUMMARY
INTRODUCTION
By means of cationic disc electrophoresis in urea-con
taming polyacrylamide gels, Young et a!. (6, 7) have de
scribed 5 distinct protein bands in serum or plasma of
febrile patients which are not found in nonfebrile individ
uals. Sequential analysis of serum from patients with a fever
of limited duration (e.g., Bacillus Ca!mette-Guërin immuno
therapy or postsurgical wound infection) disclosed parallel
ism in the concentration, appearance, and disappearance
of these “fever―
proteins (6). Although the identity of these
proteins is not established, they have been differentiated
from the following acute phase proteins: C-reactive protein,
haptogbobin , a,-acid glycoprotein , and a2-macroglobulin
I This
work
was
supported
in part
by Grants
from
the
Bodman
Foundation
and DeDombrowski Fund and by Grants CA 08748 and CA 15928 from the
National Cancer Institute.
Received November 24, 1976; accepted February 3, 1977.
1356
Comparison of Trace Protein Content of Sera from Fe
bribe and Afebrile Individuals with That of CSF. Sera ob
tamed from febrile individuals with a wide variety of disor
ders contain trace quantities of 5 distinct protein bands.
These overlap when observed in urea-containing cationic
polyacrylamide gel electrophoresis at pH 3.8, giving a dou
blet appearance (Fig. 1). When these sera are analyzed by
cationic polyacrylamide gel electrophoresis in urea-con
taming gels near neutral pH, the DP remain in the sample
gel (6); 3 protein bands that were previously obscured
within the doublet now become evident (Fig. 1). lrom the
most cathodal they are presently referred to as a , b , and c
bands. When studied by polyacrylamide gel electrophoresis
at pH 3.8, CSF contains a band coincident with the distal
2 The
abbreviations
used
are:
CSF,
cerebrospinal
fluid;
QAE,
quaternary
aminoethyl; DP, doublet proteins.
CANCER RESEARCH VOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
Serum Protein Interactions in Febrile Patients
component of the serum doublet. In polyacrylamide gel
electrophoresis at pH 6.0, CSF displays only a single com
ponent with approximately the same mobility as the plasma
a band (Fig. 1). The apparent electrophoretic overlapping
between the cationic band in CSF and the a band in serum
from febrile individuals is supported by coelectrophoretic
studies recorded in densitometric form in Chart 1.
At no time
have the b or c bands
or the doublet
proteins
present in febrile sera been observed in CSF. However, the
a band is consistently found in CSF, both in febrile and
afebrile individuals. Although we have not yet examined in
detail the relationship between plasma and CSF concentra
tions of the a band, this protein may filter to some degree
through the blood brain barrier while the other trace pro
teins in question appear to be excluded. This physiological
observation implies that the DP, b, and c proteins have a
higher molecular weight in vivo than the @30,000daltons
suggested by their migration in urea-containing gels. This
could be produced by self-aggregation or by their interac
tion with the abundant high-molecular-weight plasma pro
teins.
over, the DP, b , and c proteins appear to interact with each
other or with a common binding protein. Although urea is
primarily used to disrupt hydrogen bond interactions, it
produces some alteration of hydrophobic forces as well (3).
However, since the small-molecular-weight hydrophobic re
agent (CH.@CH2)4NCl
had no effect upon the elution volume
of the trace proteins from Sephadex G-200 in defatted sam
pies, it seems unlikely that hydrophobic binding of DP, b,
and c is significant in nonlipemic serum.
Studieson the SpecIficItyofTrace ProteinBInding.Inan
effort to identify specific binding proteins, febrile serum
was chromatographed and rechromatographed on Sepha
dex G-200, removing thereby most of the proteins of very
high
molecular
weight,
i.e.,
a2-macroglobulin,
haptoglo
bins of genotype 2-2, etc., as well as most of the serum
albumin. This partially purified fraction was then applied to
a QAE-Sephadex A-SO column (Tris-HC1, pH 7; I = 0.1) to
permit elution of the lgG fraction. The column was then
developed with a linear sodium chloride gradient, 0.0 to 0.4
M. The bulk of the remaining
plasma
proteins
and the trace
DP, b, and c proteins exited together in a single peak (Chart
Behaviorof Trace ProteinsIn NondissociatlngMedia. In 4). This post-ion exchange fraction was subjected to pre
contrast to their apparent low molecular weight in poly
parative anionic polyacrylamide gel electrophoresis (1, 2) in
acrylamide gel electrophoresis with urea, the trace proteins a 5.5% polyacrylamide gel; the proteins were eluted from
generally behaved as if they had a molecular size slightly in sliced gels and rerun in the pH 3.8 cationic-urea polyacryl
excess of 150,000 when passed through Sephadex G-200 amide gel electrophoresis system. This produced evidence
under near physiological buffer conditions (0.1 M NaCI-0.05 of multiple sites of protein interaction , the most extensive of
which was coincident with the transferrin band (Fig. 2).
M phosphate,
pH 7.0) (Chart 2). Four of the 5 protein bands,
i.e., b, c, and the DP, could be clearlydifferentiatedin
chromatographic fractions; their elution volumes were con
sistently superimposed. Significant departure from this
conventional Sephadex G-200 elution pattern of trace pro
teins was observed with lipemic sera (Chart 3). In the Ii
pemic samples, both the DP and the b and C elutions were
shifted toward void volume. Following ultracentrifugation
which removed 98% of the lipid but extracted only 30% of
the fever proteins, the latter exited in the conventional spot,
just prior to and coincident with the immunogbobulin peak.
This suggests the existence of loose hydrophobic interac
tions between these trace proteins and serum lipids; more
6v:
—.
:i
v:D
c@
0
t
Volume(ml)
Chart 2. Elution pattern from a Sephadex G-200 column (2.5 x 40 cm) of
ultracentrifugally defatted serum from a febrile individual with monocytic
leukemia. Buffer, 0.05 M phosphate, pH 7.0-0.1 M NaCl.
5
,@
Chart 1. Densitometric analysis at 540 nm of cationic polyacrylamide gel
electrophoresis gels at pH 6.0 of febrile plasma (—), CSF (—--),and a
combination of two (- - -) that had been subjected to electrophoresis within
the same gel. The areas beneath the curve in arbitrary units were as follows:
Serum:a,22;b,99;c,152.CSF:A,27.SerumandCSF:a',50;b',100;andc',
166. The units are the weight in mg of the “cut
outs―of the densitometric
tracings.
200
Volume (ml)
Chart 3. Elution pattern of lipemic serum from a Sephadex G-200 column
from a febrile individual with renal cell cancer. Buffer and column dimen
sions as in Chart 2.
MAY 1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
13S7
C. W. Young et a!.
Studies on the Nature of the NonhydrophobicInterac shape and size; net charge also influences a protein's mi
tionsof the Fever ProteinswithOtherSerumProteins.The gration in polyacrylamide gel electrophoresis. Urea and low
@
@
relations between hydrogen bonding and the elution vol
ume of the trace proteins were explored by molecular sieve
chromatographic experiments in urea-containing buffers.
Studies with G-200 Sephadex in urea-containing buffers
were technically unsatisfactory. However, when applied to a
urea-containing (6 M) Sephadex G-75 column, the com
bined elution of b plus c and DP overlapped and followed
that of serum albumin. This indicates that urea had pro
duced an appreciable reduction in the apparent molecular
weight of DP, b, and c (Chart 5). When urea (6 M) was
combined with acidification by formic acid to eliminate fur
ther the charge-based interactions, the apparent reduction
in molecular size was enhanced (Chart 6). All of the trace
febrile proteins now eluted as a group in the interval be
tween albumin and muramidase.
Acidification alone (HCI to pH 2.0), influencing both
charge-charge and hyd rogen-bond-based interactions, re
duced the apparent size of the trace proteins, but the result
ing elution pattern was heterogeneous; the b and c bands
eluted coincident with a residual albumin component while
the DP exited after the albumin peak (Chart 7).
pH undoubtedly alter the shape of the proteins under study;
however, the magnitude of the observed changes seems
bestexplainedby reductioninprotein-protein
interactions
in the presence of these dissociating agents. These interac
tions do not appear to be random; some of the most promi
I
a
g @-11OO@
@.l
Li0
100
50
ElutionVolume (ml)
Chart 5. Elution pattern from a Sephadex G-75 column (2.5 x 34 cm; 0.1 M
NaCI-0.05 N phosphate, pH 7.0-6 M urea) of serum from a febrile patient with
advanced Hodgkin's disease. The elution of b and c (not shown) overlapped
DP.
DISCUSSION
6 0[
@
The identity and physiological role of these proteins are at
present unknown. The parallelism of concentration, ap
pearance, and disappearance of “DP―,
b, and c in sera (6)
as well as their shared segregation from a variety of serum
proteins in gel filtration and ion-exchange chromatography
implies both a physical and physiological interrelationship.
Although we have not as yet identified conditions other than
fever that are associated with significant serum levels of
“DP―,
b, and c, direct involvement of these proteins in the
production of fever seems unlikely because of their evident
exclusion from the central nervous system.
The present studies offer a reasonable explanation for the
failure of these proteins to enter the CSF. Comparison of
the chromatographic and electrophoretic behavior of these
proteins in the presence and absence of dissociating agents
has demonstrated major changes. The molecular behavior
of proteins in gel filtration is a function of both molecular
Chart 4. Gradient elution pattern from a QAE-Sephadex A-SOcolumn (2.5x 40-cm
Tris-HCI,
pH 7.0;
I = 0.1)
of the
DP-,
a-,
b-,
and
c-rich
fraction
obtained post-Sephadex G-200 filtration of serum of a febrile individual with
monocytic leukemia.
13@8
C
S
a.
0
25
Elution volume ml
Chart 6. Elution pattern from a Sephadex G-75 column (2.5 x 34 cm: 10%
(v/v) formic acid-6 M urea) of serum from a febrile patient with advanced
Hodgkin's disease. The elution of b and c (not shown) overlapped DP.
200
Volume (ml)
3.0
‘a
Elution volume ml)
Chart 7. Elution pattern from a 2.5- x 70-cm Sephadex G-200 column of
serum from a febrile
patient with advanced
Hodgkin's
disease.
0.1 M NaCI
0.01 N HCI, pH 2; 0, DP; A, b and c. Arrow, point of peak elution residual
albumin; over 70% of the albumin had precipitated during acidification.
CANCER RESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
@@@.0
Serum Protein Interactions in Febrile Patients
nent plasma proteins, i.e., albumin, lgG, and haptogbobin,
did not participate in any significant degree. Neither are the
interactions remarkably specific; they can be altered by the
presence of lipoproteins and chybomicrons; multiple pro
teins appear to be involved at high pH.
The cationic polyacrylamide gel electrophoresis assay
techniques in this study employ urea to reduce hydrogen
bond interactions and the electrophoretic voltage to coun
teract protein-protein associations based on charge. The
pH 3.8 polyacrylamide gel electrophoresis assay further
reduces electrostatic interaction by its low pH. In the pH 6.0
polyacrylamide gel electrophoresis system, the trailing ion,
picoline,
might alsodecreaseproteininteractions
by pro
viding a positively charged aromatic ring nitrogen which
could lessen protein-protein binding between positively
charged histidyl groups and electron-rich sequences. Used
in conjunction in gel filtration, urea and low pH apparently
eliminated these interactions; however, when either was
used alone, the separation of trace proteins from most of
the serum components was incomplete. Therefore, no sin
gle interaction mechanism appears to be responsible for the
protein-protein associations under consideration. Since re
ducing agents are not employed in the polyacrylamide gel
electrophoresis assay, disulfide linkages between proteins,
e.g., between the K chain of lgG and a-antitrypsin (5), are
not involved.
Our present studies are directed toward the purification
of these proteins. After the elution of igG from a QAE
Sephadex column (Chart 4), the trace proteins can be
eluted and considerable purification of the column is devel
oped by the initial buffer made 6 M with respect to urea (6).
Under these conditions, the more cathodal a, b, and c
bands elute within a single-bed volume while the relatively
neutral doublet proteins come off in the 2nd- and 3rd-bed
volumes. Subsequent purification to homogenicity by
large-scale cationic polyacrylamide gel electrophoresis in
urea-containing gels appears possible. These observations
will be described in later communications.
REFERENCES
1. Davis, B. J. Disc Electrophoresis. II: Method and Application to Human
Serum Proteins. Ann. N. V. Acad. Sd., 121: 404-427, 1964.
2. Green, S., Dobrjansky, A., Carswell, E., Kassel, R. L., Old, L. J., Fiore,
N., and Schwartz, M. K. Partial Purification of a Serum Factor that
Causes Necrosis of Tumors. Proc. NatI. Acad. Sci. U. S., 73: 381-385,
1976.
3. Jencks, W. P. Hydrophobic Forces. In: Catalysis in Chemistry and Enzy
mology, pp. 393-436. New York: McGraw Hill, Inc., 1969.
4. Joustra, M. , and Lundgren, H. Preparation of a Freeze Dried Monomeric
and Immunochemically Pure IgG by a Rapid and Reproducible Chro
matographic Technique. In: H. Pesters (ed), Protides of the Biological
Fluids, pp. 511-515. New York: Pergamon Press, Inc., 1970.
5. Laurell, C-B., and Thulin, E. Thioldisulfide Interchange in the Binding of
Bence Jones Proteins in a,-Antitrypsin, Prealbumin and Albumin. J.
Exptl. Med., 141: 453-465, 1975.
6. Young, C. w., Dessources, W., Hodas, S., and Bittar, E. S. Use of
Cationic Disc Electrophoresis Neutral pH in the Evaluation of Trace
Proteins in Human Plasma. Cancer Res., 35: 1991-1995, 1975.
7. Young, C. W., Hodas, S., Dessources, w., and Korngold, L. Observa
tions on Trace Proteins in Plasma of Febnle Patients by Cationic Disc
Electrophoresis in Acrylamide Gel at pH 3.8. Cancer Res., 35: 1985-1990,
1975.
_@-0@
/
,
U,
C
(I,
C
a)
ci
a
b
Cd
e
f
g
Gel Cut Number
h
Fig. 1. Gel electrophoretograms of: normal serum. a, e; febrile serum, b,
Fig. 2. Electrophoretic migration of the DP ( 0) in anionic polyacrylamide
gel electrophoresis in relationship to the migration of other serum proteins
as indicated by the photograph of the Coomassie blue-stained gel. The
f; partially purified fever proteins, c, g; CSF, d, h. Analytical conditions:
serumfractionsubjectedto electrophoresiswasobtainedbygradientelution
cationic polyacrylamide gel electrophoresis in urea-containing gels at pH 3.8
(a to d) and pH 6.0 (e to h). The sample quantities were: Serum, 5 @.tI
in pH 3.8
assay and 20 g@Iin pH 6.0 assay; CSF, 150 Ml. Stain, Coomassie blue. —.,
position of the a, b, and c bands.
from a quaternary aminoethyl Sephadex A-50 column similar to that illus
trated in Chart 4. Minimal residual albumin was present to which the trace
proteins did not complex. In the illustrated run, the electrophoresis was
carried long enough for the albumin to run off the edge of the gel.
MAY 1977
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.
13@9
Interactions between ''Fever'' Proteins and Normal Serum
Proteins in Febrile Cancer Patients
Charles W. Young, Wilner Dessources and Sadie Hodas
Cancer Res 1977;37:1356-1359.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/37/5/1356
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1977 American Association for Cancer Research.