Journal o f Protein Chemistry, Vol. 8, No. 2, 1989
Circular Dichroic Study of Conformational Changes
in Ovalbumin
Prem P. Batra, 1 Katsushi Sasa, 2 Takuya Ueki, 2 and
Kunio Takeda 2
Received August 19, 1988
By simulation of the circular dichroic spectra (Greenfield and Fasman (1969)) and using
reference spectra of Chen et al. (1974), native ovalbumin was estimated to contain 33%
a-helix, 5% fl-structure, and 62% random coil. Ovalbumin resisted conformational changes
in solutions of urea and o f SDS. However, guanidine induced transition, starting at about
2 M and completing at about 4.5 M. A t concentrations exceeding 4.5 M guanidine, ovalbumin
existed as 6-7% a-helical, 12-13% fl-structure, and 80-81% random coil. Ovalbumin after
denaturation in 6 M guanidine or in 8 M urea (incubated at 4°(? for 24 hr) did not recover
the native conformation but acquired a new conformation in each case, with a somewhat
destabilized helical structure:
ovalbumin; CD studies; guanidine; urea; sodium dodecyl sulfate;
conformation; secondary structure; denaturation-renaturation.
KEY WORDS:
INTRODUCTION
Ovalbumin was obtained in crystalline form some 100 years ago; since then, the
purified protein has been available in gram quantities. It is partly for this reason
that it has been used extensively for biochemical studies (Taborsky, 1974). Ovalbumin is a glycoprotein consisting of a single polypeptide chain of 385 amino acids;
the oligosaccharide moiety is linked to an asparagine residue by an N-glycosidic
bond. The protein is crosslinked by one disulfide bond and has four sulfhydryl
groups (Fothergill et al., 1970). Ovalbumin messenger R N A (mRNA) has been
sequenced (McReynolds et al., 1978), and the complete amino acid sequence of
ovalbumin was reported in 1981 (Nisbet et al., 1981).
Several studies on the noncovalent structure of ovalbumin have appeared
(Simpson and Kauzmann, 1953; Schellman et al., 1953; Imahori, 1960; Holt and
1Department of Biochemistry, Wright State University, Dayton, Ohio 45435.
2 Department of Applied Chemistry, Okayama University of Science, Okayama 700, Japan.
3 Abbreviations used: CD, circular dichroism; SDS, sodium dodecyl sulfate.
221
0277-8033/89/0400-0221506.00/0
~) 1989 Plenum Publishing Corporation
222
Batra, Sasa, Ueki, and Takeda
Creeth, 1972; Ahmad and Salahuddin, 1976; Klausner et al., 1983; Egelandsdal,
1986; Timasheff and Gorbunoff, 1967; Gorbunoff, 1969; Jirgensons, 1966). However,
only a gross description is available concerning the secondary structure of the
protein. Thus, it has been reported that the native protein exists in a compact
and globular conformation containing 25-30% a-helix and "some" /3-structure
(Timasheff and Gorbunoff, 1967; Gorbunoff, 1969). Similarly, no quantitative studies
have been conducted on the effects of guanidine, urea, and surfactants such as
sodium dodecyl sulfate (SDS) on the secondary structures of ovalbumin, although
it has been reported that ovalbumin occurs as a crosslinked random coil in 6 M
guanidine (Ahmad and Salahuddin, 1976). There has also been controversy concerning the ability to renature ovalbumin after chemical denaturation. Some investigators
have reported recovering the native conformation after removal of the denaturant
(Ahmad and Salahuddin, 1976), while others have reported the inability to do so
(Simpson and Kauzmann, 1953; Schellman et al., 1953; Imahori, 1960; Holt and
Creeth, 1972). Klausner et al. (1983) reported that after denaturation, ovalbumin
does not return to its native conformation but assumes a new stable conformation
with an exposed hydrophobic domain.
In this circular dichroic (CD) study, we have determined the relative contents
of a-helix,/3-structure, and random coil of native ovalbumin and in solutions of
guanidine, urea, and SDS. This was done by using the circular dichroic reference
spectra of the corresponding structures determined by Chen et al. (1974) from
proteins of known structures. We have also found that ovalbumin after denaturation
from 6 M guanidine or from 8 M urea acquires a new conformation in each case
and different from the native conformation.
1. MATERIALS AND METHODS
The sources of guanidine, urea, and SDS have been described (Takeda et al.,
1981, 1987). Ovalbumin (lot number 76F-8045) was obtained from Sigma Chemical
Co. The extinction coefficient e of ovalbumin in phosphate buffer of pH 7.0 and
ionic strength of 0.014 (buffer A) was found to be 2.69 × 1 0 4 M - 1 • c m - 1 at 280 nm;
this value was used subsequently to determine the concentration of ovalbumin.
Buffer A was used throughout these studies.
CD measurements were made with a JASCO J-500A spectropolarimeter
equipped with a DP-501 data processor at 25°C. The concentration of ovalbumin
was maintained at around 10/zM. Details of CD measurements were previously
described (Takeda et al., 1981). In experiments involving conformational transitions,
the denaturing agents (guanidine, urea, and SDS) were mixed with the protein
solution for 5 min at 25°C before the CD spectra were measured. The relative
proportions of the secondary structures were estimated according to the CD curvefitting method (Greenfield and Fasman, 1969). The CD spectrum was simulated
using the reference spectra for the a-helix,/3-structure, and random coil determined
by Chen et al. (1974) from five proteins of known structures. The simulation was
done in the wavelength range 200-240 nm at 1-nm intervals. Since urea and guanidine
Secondary Structure of Ovalbumin
223
at higher concentrations interfered in CD measurements below 210 nm, the simulated
wavelength range was raised to 210-240 nm at those concentrations.
2. RESULTS AND D I S C U S S I O N
2.1. CD Spectrum of Ovalbumin
An example of a CD spectrum of ovalbumin determined in 6.4 M guanidine
in the wavelength range of 200-240 nm is shown in Fig. 1. Also shown is the simulated
CD spectrum based upon the reference spectra for the a-helix, /3-structure, and
random coil reported by Chen et al. (1974). The simulation was done in the
wavelength region 200-240 nm. The spectrum was also calculated in the wavelength
ranges 190-200 and 240-250 nm using the relative proportions of the three structures
determined in the 200- to 240-nm range. It is obvious that there is good agreement
between the experimentally determined and the simulated CD spectra of ovalbumin.
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guanidine. Ovalbumin (10 p M ) was mixed for 5 min
at 25°C before C D measurements. Computation was
done in the 200- to 240-nm range at 1-nm intervals.
The formula at the top indicates that the computed
spectrum consists of the three structural elements at
the given fractions. The spectrum was also calculated
in the 190- to 200- and 240- to 250-nm ranges using
the relative proportions of the three structures determined at 200-240 nm.
224
Batra, Sasa, Ueki, and Takeda
Note that the CD spectrum of ovalbumin in 6.4 M guanidine lacks the doublenegative maxima typical of the a-helix; indeed, the simulation indicates that the
contents of a-helix, fl-structure and random coil in this solution are 6%, 13%, and
81%, respectively.
2.2. Effects of Guanidine, Urea, and S D S on the Secondary Structure of Ovalbumin
The effects of guanidine, urea, and SDS on the secondary structures of ovaibumin are shown in Figs. 2-4. In the absence of these compounds, ovalbumin exists
as 33% a-helical, 5% /3-structure, and 62% random coil. Around 2.0 M guanidine
(Fig. 2), the protein starts to undergo transition, which is seen to be completed at
around 4.5 M guanidine. At this point, only 6-7% a-helix remains, while the
/3-structure and the random coil contents have increased to 12-13% and 80-81%,
respectively. Thus, we conclude that in solutions of guanidine above 4.5 M, most
of the helical structure is converted into the fl-structure and random coil, the latter
being present in excess of 80%. These results are in general agreement with those
reported by Ahmad and Salahuddin (1976), who studied changes in the secondary
structures of ovalbumin by measuring changes in the intrinsic viscosity as well as
changes in the absorption spectra at 288 nm and 293 nm. They also reported the
transition starting above 1 M guanidine and completing at about 4.5 M guanidine.
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Fig. 2. Changes in the relative proportions of a-helix (a),/3-structure
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concentration. Guanidine was mixed with the protein solution 5 rain
prior to the CD measurements at 25°C. The helical content as well as
the sum of the a-helical and /3-structure contents at each guanidine
concentration was plotted. Thus, each of the labeled areas gives the
fraction of the correspondingstructure. The same procedure was used
also to depict the data in Figs. 3-6.
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226
Batra, Sasa, Ueki, and Takeda
From the viscosity measurements, these workers concluded that in 6 M guanidine,
ovalbumin existed in a crosslinked random-coil conformation.
In contrast to guanidine, urea did not induce a conformational change in
ovalbumin even at a concentration of 9 M, when it was mixed with the protein
solution for 5 rain prior to the CD measurements (Fig. 3). However, if the mixing
time exceeded 4 hr, a limited transition was observed in an 8 M urea solution (data
not shown). Thus, it seems that denaturation by urea is somewhat time dependent.
Guanidine and urea are believed to induce transition by interfering with the
hydrophobic regions in the interior as well as in the hydrogen-bonding pattern
involved in the polar regions of the peptide chain in a protein molecule. By contrast,
surfactants such as SDS cause unfolding of proteins by their ability to weaken or
disrupt the hydrophobic interactions (Nozaki and Tanford, 1963, 1970; Kauzmann,
1959). The effect of SDS on the secondary structure of ovalbumin is shown in Fig.
4. As can be seen, SDS even at a concentration of 10 mM has almost no effect on
the conformation of the protein. The inability of SDS to unfold the protein molecule
is perhaps attributable to the fact that ovalbumin contains only a small amount of
the a-helical structure to begin with. Supporting this conclusion is the finding that
SDS does not alter the secondary structure of ribonuclease (a-helical content 19%)
(unpublished data) but has a very significant effect on the conformation of bovine
serum albumin (a-helix decreases from 66% to 52% in 10 mM SDS) (Takeda et
al., 1987), and of myoglobin (a-helix decreases from 82% to 58% in 0.6 mM SDS)
(Takeda et al., 1988).
2.3. Renaturation Study of Chemically Denatured Ovalbumin
Attempts were made to determine whether ovalbumin could be renatured after
denaturation with 6 M guanidine or 8 M urea (following incubation at 4°C for 24 hr).
After exhaustive dialysis against buffer A at 4°C (48 hr with repeated buffer changes),
conformational transitions induced by guanidine and urea were reinvestigated.
Figure 5 shows the effects of guanidine and urea on ovalbumin dialyzed from 6 M
guanidine. Figure 6 shows the effects of the same denaturants on the protein dialyzed
from 8 M urea. Two points need to be emphasized:
1. Ovalbumin dialyzed from 6 M guanidine or from 8 M urea has a new
conformation in each case which, in turn, differs from the native conformation.
Thus, ovalbumin dialyzed from 6 M guanidine (Fig. 5) contains 24% a-helix, 29%
/3-structure, and 47% random coil, while ovalbumin dialyzed from 8 M urea (Fig.
6) contains 25% a-helix, 24% /3-structure, and 51% random coil. But native
ovalbumin consists of 33% a-helix, 5% /3-structure, and 62% random coil. It is
obvious that after denaturation ovalbumin does not return to its native conformation
but assumes a new conformation. These results are in agreement with those of
Klausner et al. (1983), who also reported that ovalbumin after denaturation in 6 M
guanidine or 8 M urea acquires a new conformation. However, our results have
further extended this observation to suggest that the newly acquired conformation
depends upon the denaturant from which renaturation is attempted. It is also
interesting to note that compared with the native conformation, the dialyzed
Secondary Structure of Ovalbumin
227
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6 M guanidine. (a) As a function of guanidine concentration, (b) As a
function of urea concentration. For further details, see the legend to
Fig. 2.
o v a l b u m i n ( w h e t h e r f r o m 6 M g u a n i d i n e o r from 8 M urea) has a strikingly h i g h e r
c o n t e n t o f fl-structure, b u t a l o w e r c o n t e n t o f b o t h the a - h e l i x a n d the r a n d o m coil.
2. N o t o n l y does o v a l b u m i n acquire a new c o n f o r m a t i o n after d e n a t u r a t i o n
a n d dialysis, the a - h e l i c a l structure also exists in a d e s t a b i l i z e d f o r m in the r e n a t u r e d
o v a l b u m i n . This is p a r t i c u l a r l y true w h e n r e n a t u r a t i o n is a t t e m p t e d from 6 M
g u a n i d i n e , as can be d e m o n s t r a t e d w h e n we s t u d y the effects o f g u a n i d i n e a n d u r e a
on the s e c o n d a r y structures. The d a t a in Fig. 5a i n d i c a t e that g u a n i d i n e starts to
Batra, Sasa, Ueki, and Takeda
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(fl), and random coil (C) in renatured ovalbumin after dialysis from
8 M urea (24-hr incubation at 4°C). (a) As a function of guanidine
concentration, (b) As a function of urea concentration. For further
details, see the legend to Fig. 2.
induce transition at a much lower concentration (<1 M) in ovalbumin renatured
from 6 M guanidine than is the case with native ovalbumin ( > 2 M guanidine) (Fig.
2). Furthermore, urea also appears to induce transition to some extent in renatured
ovalbumin (Fig. 5b), but not so in native ovalbumin (Fig. 3). Guanidine and urea
also induce transition in ovalbumin renatured from 8 M urea in a similar fashion
(Fig. 6), although the transition is not as clear cut as in the case of ovalbumin
Secondary Structure of Ovalbumin
229
renatured from 6 M guanidine (Fig. 5). However, in this case as well, guanidine
and urea appear to induce transition at somewhat lower concentrations compared
with native ovalbumin, but exceeding those required to induce transition in ovalbumin renatured from 6 M guanidine. Thus, it appears that the a-helical structure
of ovalbumin renatured from 8 M urea is somewhat more stable than that of
ovalbumin renatured from 6 M guanidine, but less so than that of native ovalbumin.
REFERENCES
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