Tohoku
J. exp.,
Med.
1975,
116, 179-182
Conformational Studies of Hepatitis B Surface Antigen (HBsAg) by Amino Acid Analysis NORIYOSHI ISHIDA*
SUKENO, HIROYUKI
SHIRAISHI and
NAKAO
Department of Bacteriology,*Tohoku University School of Medicine and Miyagi Prefectural Institute of Public Health, Sendai
SUKENO,N., SHIRAISHI,H. and ISHIDA, N. Conformational Studies of Hepatitis B Surface Antigen (HBsAg) by Amino Acid Analysis. Tohoku J. exp. Med., 1975, 116 (2), 179-182 The amino acid composition of hepatitis B surface antigen (HBsAg) purified from plasma of asymptomatic carriers was examined for further structural analyses. The protein of HBsAg spherical particles was characterized by the high contents of cystine, proline and tryptophan residues. A high a-helical content of HBsAg was discussed on amino acid composition. HBsAg; hepatitis; amino acid composition
Hepatitis B surface antigen (HBsAg), the lipoprotein surface component of hepatitis B virus (HBV) can be found in the sera of patients and asymptomatic carriers as numerous 22-nm spherical structures. These particles may be produced by infected hepatocytes together with tubular forms and Dane particles and these three particles share the same antigenicity coded by viral genome. Papers from this laboratory disclosed the fact that the reduction of disulfide bonds may lead to the disappearance of the HBs antigenicity (Sukeno et al. 1972b) without influencing the a-helical structure of the spherical particles (Sukeno et al. 1972a). For further conformational discussion, the amino acid analyses of HBsAg were conducted in this work. Vyas et al. (1972) and Dressman et al. (1972), once worked along the same line, but they did not put emphasis on the high content of cystine, proline and tryptophan in this particular protein. MATERIALS AND METHODS Highly nitrogen method by
at
suspension rate
hr
in
contain
248,000 •~ a
preformed
any
antihuman
g.
human serum
HBsAg
Received
at Final linear
serum antibody,
was
a
complement
liter
of
plasma
(Sukeno
layerd
separation
with 1
68,000 •~
was
zonal at
ration
purified
from described
centrifugation
the
HBsAg
obtained
previously
treated
32
purified was
g
for
on
et 16
hr,
fixation of
al.
1972b).
followed
a discontinuous
68,000 •~
g
purification sucrose
16
hr,
gradient as was
March
was
(5-30%). revealed
used
for
adw.
for publication,
the
22, 1975. 179
of by
amino
4096
HBsAg
was
(40
isopycnic
analyses.
mg
to
the
the
enzyme.
60%
banding by
obtained
0.1
pelleted
The and
rate
W/V) in
for
sepa did
with The
for
CsCI
zonal
preparation
immunoelectrophoresis acid
per
according
digestion.
sucrose
accomplished The
by
1:
the
pronase
followed
of HBsAg
components and
by
of carriers
Briefly,
gradient
for
titer
asymptomatic
subtype
not
rabbit of
180
N. Sukeno
et al.
The amino acid analysis was performed after hydrolysis in 6 N hydrochloric acid at 110° for 24 hr in vacuo (500 jig Hg) on a Hitachi model KLA-5 automatic amino acid analyzer with standard long and short columns. The content of half-cystine was determined as S-earboxymethylcysteine (S-CM cysteine). For this, purified HBsAg was reduced in 0.01 M Tris (hydroxymethyl-aminomethane)-HC1 at pH 8.0 containing 0.1 M dithiothreitol (DTT) and 8 M urea, and alkylated with 0.5 M iodoacetamide.
RESULTS AND DISCUSSION The of that
of
when
of
DTT Ellman
almost
in
S-CM the
with of
data
half-cystine
acid
intact in
cysteine and
composition
HBsAg
cysteine
alkylated
content
are
was
was
summarized
5.2
residues
HBsAg
was
4.5
by
the
determined
5,5•Œ-dithio-bis-(2-nitrobenzoic)
(1958), 3
amino
disulfide
no
cysteine
bonds
exist
was per
detected. 100
in
per
100
Table molar
residues.
On
(DTNB)
according
It
is apparent
from
amino
The
content acids
the
other
spectrophotometric
acid
molar
1. amino
and hand,
technique to
the
these
results
method that
acids.
Purified HBsAg has a high absorbance at 280 nm and a characteristic shoulder at 285 nm indicating a high molar concentration of tyrosine and/or tryptophan. Amino acid analysis has revealed only 2.5-2.8 moles percent tyrosine (Table 1). Therefore, the content of tryptophan was estimated by magnetic circular dichroism (MCD) through the courtesy of Dr. C. Djerassi of Stanford University. As a result, tryptophan content was determined to be 6.42 residues per 100 molar amino acids. Another noteworthy finding is that proline was found in a larger quantity (12.3 moles %) when compared with other viral proteins. Unusual large hydro phobic forces due to high tryptophan and proline contents could render the protein TABLE 1.
* Values •õ
are expressed
S-earboxymethylcysteine,•ö
Amino acid composition of HBsAg
as pmoles
of amino
determined
acid by
per
MCD.
100 µmoles
recovered
.
The
Amino
Acid
Composition
of Hepatitis
B Surface
Antigen
181
highly resistant to a variety of chemical treatments and enzymatic cleavages (Kim and Bissell 1971).
On the other hand, the content of a-helix in HBsAgprotein was calculated to be 70-80% based on ORD and CD measurements (Sukeno 1972a; Hirschman et al. 1973). However, the amino acid compositionof the HBsAg protein component is not concordant to the high a-helix content, since HBsAg contained 12.3 moles % of proline, a known disrupter of the a-helix, as well as a high percentage (50 moles %) of non-a-helical amino acid residues (Val, Ile, Ser, Cys, Thr, Gly, Pro) (Fassman 1963). Two possibilitiescan be consideredfor the explanation of such inconformity. One is a possible a-helical structure in ordinary sense in spite of its higher content of non-a-helical amino acid residues. Another is an a-helical structure estimated by means of CD does not necessarilymean true helical structure, as the high amount of covalent bonds and noncovalent interactions in HBsAg polypeptide may give such specificspectra like true a-helix. It is true that the tertiary structure of HBsAg appears to play an important role for the serologic reactivity, since chemical modifications such as reduction and alkylation of disulfide bonds with DTT (Sukeno et al. 1972b) or denaturation with sodium dodecyl sulfate (SDS) (Kim and Bissell 1971) or succinylation of tryptophan with N-bromosuccinimide(NBS) (Rao and Vyas 1974) resulted in a loss of antigenicity. Thus, the tertiary structure seems to be more important in maintaining the antigenic intergrity of HBsAg than the role of secondary structure. Nevertheless ORD and CD spectra of HBsAg was not influenced by DTT or 8 M urea treat ment (Sukeno et al. 1972a). Thus it seems probable that HBsAg might have true a-helical structure in spite of its higher content of non-a-helicalamino acid residues. The same kind of observation was recently made with fd phage which contained almost 100% a-helical structure (Nakashima et al. 1975). Acknowledgment This work was supported Health
and Welfare,
by grants
from
the Ministry
of Education
and the Ministry
of
Japan.
References
1) Dressman, G.R., Hollinger, F.B., Suriano, J.R., Fusioka, R.S., Brunschwig, J.R. & Melnick, J.L. (1972) Biophysical and biochemical heterogeneity of purified hepatitis B antigen. J. Virol., 10, 469-476. 2) Ellman, G.L. (1958) A colorimetric method for determining low concentrations of mercaptans. Arch. Biochem. Biophys., 74, 443-450. 3) Fassman, G.D. (1963) Optical rotatory dispersion. In: Method in Enzymology, 6, Edited by W.P. Colowcik, & N.O. Kaplan, Academic Press, Inc., New York, pp. 928-957. 4) Hirschman, S.Z., Schwartz, J., Vernace, S., Schaffner, F. & Ganz, C. (1973) An electron microscopic study of the structural polymorphism of hepatitis B antigen. J. infect. Dis., 128, 605-617. 5) Kim, C.Y. & Bissell, D.M. (1971) Stability of the lipid and protein of hepatitis associated (Australia) antigen. J. infect. Dis., 123, 450-476. 6) Nakashima, Y., Wiseman, R.L., Konigsberg, W. & Marvin, D.A. (1975) Primary structure and sidechain interactions of PFL filamentous bacterial virus coat protein.
182
N. Sukeno
et al.
Nature, 253, 68-71. Rao, K.R. & Vyas, G.N. (1974) Hepatitis B surface antigen (HBsAg): tryptophan content and biological activity. J. gen. Virol., 54, 571-573. 8) Sukeno, N., Shirachi, R., Shiraishi, H. & Ishida, N. (1972a) Conformational studies of Australia antigen by optical rotatory dispersion and circular dichroism. J. Virol., 10, 157-158. 9) Sukeno, N., Shirachi, R., Yamaguchi, J. & Ishida, N. (1972b) Reduction and reoxida tion of Australia antigen: loss and reconstitution of particle structure. J. Virol., 9, 182-183. 10) Vyas, G.N., Williams, E.W., Klaus, G.G.B. & Bond, H.E. (1972) Hepatitis associated Australia antigen. Protein, peptides and amino acid compositions of purified antigen with its use in determining sensitivity of the hemagglutination test. J. Immunol., 108, 1114-1117. 7)