J. gen. Virol. 0977), 35, 45-52
45
Printed in Great Britain
Effect of Chaotropic Salts and Protein Denaturants on the
Thermal Stability of Mouse Fibroblast Interferon
By R. J. J A R I W A L L A ,
S. E. G R O S S B E R G
AND J. J. S E D M A K
Department of Microbiology, The Medical College of Wisconsin,
Milwaukee, Wisconsin 53233, U.S.A.
(Accepted 3 November I976)
SUMMARY
Altering the aqueous environment, especially with agents that affect hydrogen
bonds, markedly affects the stability of mouse L cell interferon. Low p H stabilizes
interferon whereas high p H labilizes it; heavy water further enhances interferon
thermostability at p H 2 but not at p H 9. Exposure to the protein denaturants,
4 M-guanidine hydrochloride and 6 M-urea, significantly decreases the activity of
interferon at p H 2 and pH 9; however, the residual interferon activity is relatively
thermostable. Certain chaotropic salts protect interferon against thermal destruction, and in terms of effectiveness, their sequence is in the order SCN- > I - i> Cl= CIO4- = Br- > NO3-. interferon becomes more stable to heat as the NaSCN
concentration is increased from o.25 M to z'o M. Molecular sieve chromatography
of interferon in the presence of I'5 M-NaSCN at p H 7 shows a shift in its apparent
mol. wt. from 2500o to 42000. Unlike most proteins, the unfolded conformation
of interferon appears to be more stable to heat than the molecule with a smaller
Stokes' radius.
INTRODUCTION
Interferons are considered to be glycoproteins that are stable to low pH or heat. Since
heating has frequently been used to probe the structure and behaviour of proteins under
various other environmental conditions (Tanford, I968), we have analysed several physicochemical factors affecting the thermal inactivation ofmurine interferon (Jariwalla, Grossberg
& Sedmak, I975)- The idea that at least a portion of the molecule may interact with the
aqueous environment in such a way as to diminish the rate of heat inactivation led us to
study low tool. wt. compounds affecting the solvation of proteins, such as protein denaturants and chaotropic salts, i.e., salts whose anions increase the solubility of the non-polar
regions of proteins (Hatefi & Hanstein, I969).
Protein denaturants, like guanidine hydrochloride (GuHC1) and urea, have been reported
to have various effects on the biological activity of interferons. Thus, 8 M-urea destroyed the
activity of mouse interferon, but neither chicken (Merigan, Winget & Dixon, I965) nor
human leukocyte interferons (Mogensen & Cantell, I973) were adversely affected. In the
latter report, it was shown that 4 to 8 M-GuHC1 protected the interferon from thermal
inactivation. Urea or GuHCl (Kadri & Kohlhage, i969) completely destroyed virusinduced high tool. wt. interferon in the serum of rabbits, but only partly inactivated the
smaller tool. wt. species also present. The present study has attempted to determine if
reagents that affect the hydrogen-bonded structure of proteins or alter their hydrophobic
interactions change the resistance of mouse L cell interferon to thermal inactivation.
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46
R.J.
JARIWALLA,
S. E. G R O S S B E R G A N D J. J. S E D M A K
METHODS
Cell cultures. The cultural conditions for the L929mouse fibroblast line have been described
(Jariwalla et al. 1975).
Preparation, purification and assay of L cell interferon. The preparation of interferon in
L cells with Newcastle disease virus and its partial purification using zeolite elution have
been described (Jariwalla et aL I975). Interferon activity was measured by haemagglutinin
(HA) yield-reduction bioassay (Oie et al. 1972) with GD-VII virus as the challenge, the
endpoint being a o'5 log (7° ~o) reduction in virus HA yield. When samples of the National
Institutes of Health mouse reference standard preparation Goo2-9o4-511 (Grossberg,
Jameson & Sedmak, I974), with an assigned potency of I2ooo units/container, was assayed
by our method, the mean titre obtained was 12ooo, with a standard deviation of ± 6i %.
Zeolite-purified interferon preparations had asp. act. of 5"9 to 7"7 × I o5 units/mg protein.
Heat-inactivation tests. Interferon samples containing the different reagents were stored
at 4 °C for 24 h and then subjected to heat-inactivation in a linear non-isothermal (LNS)
test (Zoglio et aL 1968; Greiff & Greiff, 1972) and also a static temperature test conducted at
68 °C. For such tests I ml quantities of interferon distributed in I5 × 48 mm screw-cap glass
vials were submerged in water baths. In the LNS test the bath temperature was continuously
raised at a rate of 4 °C/h between 4o and 9o °C over a period of 13 h by a motor-driven
attachment on the magnet of the thermoregulator (Constant Temperature Circulator,
Model ED 'Unithermal'). Samples were taken when the bath reached 4o, 50, 6o, 70, 80 and
9o °C; at each temperature it was checked with a thermometer that the temperature inside
a vial was the same as that of the water bath.
Samples were cooled by placing them at room temperature for 3o rain, and then stored
at - 7o °C until assayed. The time of cooling from 68 °C (the static temperature test usually
employed) to room temperature was approx. I5 rain (Jariwalla et al. I975).
Buffer solutions. Interferon samples at pH 2 were prepared in o'I5 M-KC1-HC1 buffer
solutions, and at pH values 7 and 9 in o.I M-Na~HPO4-NaH2PO4.
Reagents. The following chemicals, of reagent grade or higher purity, were dissolved
directly in, or added to, the interferon preparation to give the desired final concentration:
NaSCN, NaC10~, NaNO3, NaBr, NaI, NaCl, urea-free of any contaminating cyanate
(Mallinckrodt Chemical Works, St Louis, Missouri), GuHC1 (Eastman Organic Chemicals,
Rochester, New York) and heavy water (deuterium oxide D20 from Matheson, Coleman
& Bell, Cincinnati, Ohio). The reagents were removed by dialysis at 4 °C before the assay
of interferon activity.
Molecular weight estimation. The mol. wt. of mouse interferon were estimated by molecular
sieve chromatography using Sephadex G-IOO columns at pH 7 in the presence or absence
of 1"5 M-NaSCN. Columns were exhaustively washed and equilibrated with o.t M-Na2HPO~NaH2PO4 (pH 7) at 4 °C or in o.I M-tris-HC1, I'5 M-NaSCN (pH 7) at room temperature.
The void volume was determined with blue dextran 2000. The columns were calibrated with
markers of known mol. wt., namely, ovalbumin, soybean trypsin inhibitor and lysozyme or
cytochrome c, selected since they resist conformational changes under denaturing conditions
such as exposure to pH 2 (Tanford, I968; R. J. Jariwalla et al. unpublished observations).
Zeolite-purified mouse interferon was dialysed against o.r M-sodium phosphate (pH 7) or
o-I M-tris-HC1, I'5 M-NaSCN (pH 7) at 4 °C before being applied to each column. Fractions
(2.0 ml) were diluted and assayed for interferon activity without dialysis; preliminary
experiments showed that concentrations up to o'05 M-NaSCN had no deleterious effect on
either cell cultures or virus HA yield.
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Thermal stability o f mouse interferon
47
Table I. The effects of some additives on the stability at 68 °C
of partially purified L cell interferon
Interferon activity (units/ml)
after heating (rain)
Additive
Expt. I
None
Urea, 6 M
None
Urea, 6 M
Expt 2
None
NaSCN, 2 M
NaI, 2 M
NaC1, 2 M
NaC104, 2 M
NaBr, 2 M
NaNO3, 2 M
pH
o*
60
2
2
9
9
7600
2160
76o0
5O0
(Ioo)]"
(28"4)
(too)
(6.6)
7
7
7
7
7
7
7
7000
659 °
7tlO
6600
7200
896O
II750
(1oo)t
(94q)
(~oI"6)
(94"3)
(Io2"9)
028"0)
067"9)
150
3000 (39"5)
I350 (i7"8)
6 0 ( < 0"8)
< 50 ( < 0"7)
--------
---
480
5980
3720
336o
I820
4o30
3390
240
750
135o
--
<
--
<
(6"9)
(85"4)
(53'I)
(48'0)
(26"0)
(57'6)
(48"4)
(9'9)
(I7"8)
50 (< 0 - 7 )
5 ° (< o'7)
40
3660
I49O
IOOO
91o
IO7O
520
(0.6)
(52"3)
(2I'3)
(14"3)
(I3"O)
(I5"3)
(7"4)
* Sample held for 24 h at 4 °C before heating.
t Figures in parentheses s h o w the residual activity as ~ o f u n h e a t e d control without additive.
RESULTS
Effects of protein denaturants and chaotropic salts on stability
The stability of murine interferon was tested in the presence of urea, GuHC1 and chaotropic salts - agents that break the hydrogen-bonded structure of proteins (Table 1). After 24 h
exposure to urea at 4 °C, interferon lost 70 % to 93 % of its original activity. The interferon
activity remaining after heating at 68 °C for 240 min at pH 2 was twofold greater in the
presence of urea than in its absence. Thus, although urea decreased the original activity of
interferon at p H 2, it enhanced the stability of the residual interferon during subsequent
heating at this pH. However, urea did not protect interferon during heating at pH 9,
possibly due to the decomposition of urea to cyanate upon heating at high p H (Tanford,
1968). Some chaotropic ions had greater protective effects on the heat stability of interferon
than others. Treatment of interferon with these salts for z4 h at 4 °C and pH 2 or pH 7
or p H 9 did not significantly alter activity. When subsequently heated at 68 °C for 24o min,
the control interferon sample at pH 7 retained < 1% of its original activity as compared
to 52% for NaSCN, 2I ~o for NaI, 14% for NaCt, I 3 % for NaC104, I 5 ~ o for NaBr and
7 % for NaNOz. Thus, in terms of their effectiveness in stabilizing interferon against heat
inactivation, these chaotropic ions ranked in the order: S C N - > I - / > C I - = C104= Br- > NOa-.
The stabilizing effect of some chaotropic salts at pH values 2 and 9 is seen in Fig. I.
Samples of interferon were first treated for 24 h at pH 2 or pH 9 and 4 °C, and this did not
alter their activity. At pH 2 the residual interferon activity after 24o min at 68 °C was Io %
for the control, 25 % for NaSCN and 5 % for NaI, but less than 1% for NaNOa (Fig. I a).
At pH 9, the residual interferon activity after 240 min at 68 °C was < 1% for the
control, 4 % for NaSCN, 2.2% for NaI, and < 1% for NaNOa (Fig. l b). NaSCN
increased the stability of interferon at pH 9 but not at p H 2. At pH 7, the stability of heated
interferon increased as the NaSCN concentration was increased from o'25 M to 2"0 M
(Fig. 2).
4
VIR 35
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48
R.J.
JARIWALLA~
(~0
I
S. E. G R O S S B E R G
I
AND
[
(h)
I
J. J. S E D M A K
t
I
100
50
'\\\"",,\
10
~;~
"~
5
--
A
•
N
1
N
\
\
--
\
\
\
\
\
N
N
~
-
0'5
\
I
60
0'1
\
m
m
\
Z
\,
i
150
\
m
I
60
I
240
I
150
I
240
Time (rain)
Fig. I. Stability at 68 °C o f partially purified interferon in 2 M-chaotropic salts at (a) p H 2 a n d
(b) p H 9. © - - ©, Control; • - - 0 ,
NaSCN; &--&, NaI; A -- ~, NaNOs.
100
z
50
i;_ lO
=
5
)
o
1
0-5-B
B
0.1
I
60
I
150
Time (min)
I
240
1
Fig. 2. Stability at 68 °C o f partially purified interferon as a function of N a S C N concentration.
Salt molarity: [] - - K], 2 M; I - - ' ,
I'5 M; A ---- ~ , t M; & - - A , O'5 M; • ------ • , O'25 M;
O - - © , control.
In addition to stabilizing the L cell interferon in liquid preparations, NaSCN also enhanced
the stability of freeze-dried interferon. Interferon prepared in o'5 M-NaSCN at pH 7 was
freeze-dried and heated in the LNS test at a rate of r'5 °C/h, followed by cooling to room
temperature. After progressive heating to 85 °C, the sample in NaSCN retained 27.2 % of
its original activity as compared to 2"2 % for the control.
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Thermal stability o f mouse interferon
49
Table 2. The effect of GuHCl and heavy water (D20) on the heat stability of
partially purified L cell interferon in the linear non-isothermal stability test
Interferon activity (units/ml)
after heating to
r
Additive
None
GuHC1, 4 M
Heavy water (D20), 50 ~
None
GuHCI, 4 M
Heavy water (D20), 50 ~
pH
Before heating*
60 °C
80 °C
2
2
2
9
9
9
49o0 0oo)~
640
(13"I)
3900
(79"6)
38oo (1oo)
90O (23"O)
345
(9"1)
195o (39"8)
225
(4"6)
3900 (79'6)
95
(2"5)
225
(5"9)
< 50 ( < 1"3)
23o
(4'7)
90
(1"8)
740 (I5"I)
< 5 o ( < 1"3)
9O (2"4)
< 50 ( < I'3)
* Sample held for 24 h at 4 °C.
t Figures in parentheses show the residual activity as ~ of unheated control with no additive.
Another protein denaturant, GuHC1, was effective in stabilizing residual interferon activity
against heat inactivation in the LNS test, but the large loss of activity, before heating, 77 to
87 % after 24 h at 4 °C (Table 2), limits its usefulness as a stabilizing agent. However, the
residual activity appeared more resistant to heating in the presence of GuHC1 than in its
absence. For example, after being heated progressively to 60 °C, interferon in GuHC1 at
pH 9 maintained 25 % of the residual activity present before heating as compared to 2"5 %
in the absence of GuHC1.
The effect of heavy water
Heavy water (deuterium oxide, D20) can strengthen hydrogen bonds of proteins or alter
their hydrophobic interactions (Wiberg, 5955; Aleksandrov et al. ~966). Interferon prepared
in 5o % D20 and kept at 4 °C for 24 h, showed no significant change in activity at pH 2
before heating (Table z), whereas 9 ~% of the original activity of interferon was lost at pH 9.
When interferon in D20 at pH z was progressively heated to 6o °C in the LNS test, it lost
virtually none of its activity, but it lost 6o % of its activity in the absence of D20. At pH 9,
D20 did not stabilize interferon during heating.
Effect of NaSCN on Stokes' radius and/or mol. wt. of interferon
When L cell interferon was chromatographed on a Sephadex G-Ioo column at pH 7, its
activity was recovered in a single peak corresponding to an apparent tool. wt. of 25000.
However, on a pH 7 column containing I-5 M-NaSCN, 90 % of its activity was recovered on
a single peak in the region of mol. wt. 4zooo (Fig. 3). Thus, the apparent tool. wt. and/or
Stokes' radius of mouse fibroblast interferon increased in the presence of NaSCN.
DISCUSSION
An analysis of environmental factors affecting the thermal inactivation of mouse fibroblast
interferon showed that acid conditions, i.e. pH 2 to pH 4 (which labilize most proteins)
stabilized interferon, apparently by unfolding it and increasing its mol. wt. or Stokes' radius
(Jariwalla et al. r975). The present study further substantiates this finding by demonstrating
that interferon is protected against inactivation by heat in the presence of reagents, such as
GuHC1 or urea, which can unfold globular proteins. Low pH can deform the higher
(secondary, tertiary or quaternary) orders of protein structure, primarily by affecting
4-2
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50
R . J . J A R I W A L L A , S. E. GROSSBERG AND J. J. SEDMAK
10
9
7
x
-
(a)
87 - O
6
5
4
(b)
~
~
umin
~ S o y b e a n
~ trypsin
32
b
] I ~ N ~ hibit°r
l/
~
- 1200
1100
1000
- 900
800
-
C
-
700
600 -E
=
400 ~~=
ytochro
y4,1~l=l~J. ~
I
a.a" I I [ I ~.J-,,,J.,,J.I
28 32 36 40 44 48 5)_7g~ 6]) 64 68
26-'28 30 32 34 36 38 4-0-4-2-4=4
V
V0
Fraction number
200
100
Fig. 3. Molecular sieve chromatography of partialIy purified L cell interferon through a column
of Sephadex G-Ioo. (a) Interferon (I x io ~ units in I ml) was applied to a 7z x I'5 cm column in
o. I M-sodium phosphate, pH 7, at 4 °C. Recovery of interferon activity was about 60 ~. (b) Interferon
(4"5 x lO3 units in 0"5 ml) was applied to a 53 x I cm column in o'I M-tris-HC1, I '5 M-NaSCN, pH 7,
at room temperature. Recovery was about 90 ~ of interferon activity.
electrostaticinteractions. Thus, the observations that interferon was also stable to heating in
solutions of other deforming agents, such as NaSCN, urea or GuHCI, suggested that
conformational state(s) of interferon resistant to thermal inactivation may be generated.
Alternatively, since denatured proteins are commonly aggregated (Joly, I965), these agents,
which also act to dissociate proteins (Tanford, I968; yon Hippel & Schleich, I969), may
stabilize interferon by preventing aggregation during heating.
Data obtained from molecular sieve chromatography show that mouse L cell interferon
in neutral I'5 M-NaSCN has an apparent mol. wt. of 42ooo, an increase from the 25ooo
measured in neutral, low molarity solutions. A similar shift in apparent mol. wt. occurs at
pH 2 (Jariwalla et al. I975). Since acid pH and molar NaSCN act as strong dissociating
agents, it is unlikely that the 42 ooo mol. wt. species of interferon resulted from aggregation.
Certain globular proteins containing disulphide bonds, such as lysozyme, ribonuclease,
and soybean trypsin inhibitor, are resistant to denaturation at acid pH as measured by
optical rotation (Tanford, I968; Wang & Kassell, I974). In addition to disrupting the
hydrogen-bonded structure of these proteins (Lumry & Eyring, I954), urea and GuHC1
may also weaken their hydrophobic interactions, either directly, resulting in the subsequent
hydration of the protein molecule (Haschemeyer & Haschemeyer, I97a), or indirectly, by
exerting an effect on the hydrogen-bonded structure of water, similar to the so-called
lyotropic or Hofmeister-type effect produced by high concentrations of chaotropic salts
(Hammes & Schwann, I967; von Hippel & Schleich, t969). Since chaotropic salts also affect
hydrophobic interactions, proteins in solutions of these salts acquire a cross-linked, partially
unfolded conformation which is not a firmly cross-linked random coil (Tanford, ~968; yon
Hippel & Schleich, I969). Thus, the degree of solvation and unfolding may affect the
thermal stability of the interferon.
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T h e r m a l stability o f mouse interferon
5I
Very few studies have attempted to relate the physical properties of proteins undergoing
denaturation and renaturation to their biological activities. Teipel & Koshland (I97Ia, b)
studied the activity of enzymes undergoing denaturation and renaturation during treatment
with GuHC1. Regaining of enzymatic activity correlated with the regain of native structure.
Some polypeptide chains refolded within I rain to an inactive structure similar, but not identical, to the conformation of the native enzyme, after which biological activity was slowly
regained. Since the resulting amount of enzyme activity related to the proportion of polypeptide chains initially renatured correctly, the energy states in the environment during the
cooling phase after thermal denaturation of interferon may determine the residual biological
activity. Normal (i.e. relatively slow) cooling may allow time for the protein to refold during
renaturation in its most stable, or at least its most biologically active, conformation
(Jariwalla et al. 1975).
How D20 enhances the stability of interferon at pH 2 is not clear, it is known that the
exchangeable H atoms in proteins are rapidly replaced by deuterium, which forms bonds
having greater strength than the hydrogen bond (Wiberg, ~955; Aleksandrov et al. I966).
Protonation of interferon at pH 2 may favour greater substitution of deuterium for hydrogen
and/or a lower energy state, requiring the addition of more calories to alter or break the
hydrogen bonds. [n addition, replacing H20 with D20 alters several properties of the solvent,
including the structure of water and the dissociation constants of substances dissolved in it,
thereby influencing bonds maintaining the higher order structures of protein molecules.
A model which accounts for various observations during thermal inactivation of mouse
fibroblast interferon postulated that the molecule may unfold on prolonged heating and
refold to an inactive conformation on cooling (Jariwalla et al. I975), and that during the
cooling at pH 2, a significant fraction of molecules having the unfolded conformation may
refold correctly, accounting for their thermal stability. We suggest that this same model may
be applied to the observations reported here. Thus, a large fraction of those molecules
having extended conformations in a chaotropic salt may refold during cooling and removal or
dilution of the salt to the conformation active at pH 7; only a small fraction of those having
an unfolded conformation in GuHCI or urea may refold similarly, accounting for the lower
residual activities after treatment with these protein denaturants. The direct evidence for
such conformational changes could come from measurements by circular dichroism, which
will require large amounts of interferon purified to homogeneity.
The studies reported herein demonstrate for the first time that chaotropic salts can
stabilize a protein against thermal inactivation. Since these salts can weaken intermolecular
or intramolecular hydrophobic interactions of macromolecules (Hatefi & Hamstein, i969) ,
they may be of use in the elution of interferon bound to immobilized ligands. We have
recently applied this principle to the elution of human fibroblast interferon linked noncovalently to concanavalin A-Sepharose (Jariwalla, Sedmak & Grossberg, I976).
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