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)

Conformational studies of hepatitis B surface antigen (HBsAg) by amino acid analysis.

The amino acid composition of hepatitis B surface antigen (HBsAg) purified from plasma of asymptomatic carriers was examined for further structural an...
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