Báo cáo khoa học: Ascorbic acid induces a marked conformational change in long duplex DNA doc

Ascorbic acid induces a marked conformational change in long
duplex DNA
Yuko Yoshikawa
1,5
, Mari Suzuki
1
, Ning Chen
2
, Anatoly A. Zinchenko
2
, Shizuaki Murata
2,5
,
Toshio Kanbe
3
, Tonau Nakai
4
, Hidehiro Oana
4,5
and Kenichi Yoshikawa
4,5
1
Department of Food and Nutrition, Nagoya Bunri College, Japan;
2
Graduate School of Environmental Studies, Nagoya University,
Japan;
3
Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of
Medicine, Japan;
4
Department of Physics, Kyoto University, Japan;

5
CREST (Core Research for Evolutional Science and
Technology) of Japan Science and Technology Corporation
Ascorbic acid is often regarded as an antioxidant in vivo,
where it protects against cancer by scavenging DNA-dam-
aging reactive oxygen species. However, the detailed mech-
anism of the action of ascorbic acid on genetic DNA is still
unclear. We examined the effect of ascorbic acid on the
higher-order structure of DNA through real-time observa-
tion by fluorescence microscopy. We found that ascorbic
acid generates a pearling structure in single giant DNA
molecules, with elongated and compact regions coexisting
along a molecular chain. Results from electron microscopy
and atomic force microscopy indicate that the compact
regions assume a loosely packed conformation. A possible
mechanism for the induction of this conformational change
is discussed in relation to the interplay between the higher-
order and second-order structures of DNA.
Keywords: ascorbic acid; higher order structure of DNA;
pearling structure; single molecular observation.
Vitamin C (ascorbic acid) is ubiquitous and fundamental in
living cells, where it acts as a water-soluble antioxidant and
an essential cofactor for many enzymes involved in diverse
metabolic pathways. Several epidemiological and experi-
mental studies have shown that the consumption of foods
rich in vitamin C is associated with a decreased risk of
several chronic diseases, including cardiovascular disease
and cancer [1–4]. However, the extent to which vitamin C
contributes to these effects is still unclear [5]. There is
evidence that vitamin C inhibits oxidative DNA damage

in isolated and cultured cells exposed to reactive oxygen
species and UV/visible light [5–9]. On the other hand,
several studies have shown that vitamin C sometimes
increases DNA damage in humans [5,10,11]. These studies
suggest that vitamin C may have anti-oxidative or pro-
oxidative properties depending on the conditions in the cell.
Thus, it may be useful to clarify the mechanism of the action
of vitamin C on DNA [12,13].
It is well known that genomic DNA molecules are very
long, e.g. of the order of 1 cm in human cells. Recently,
it has become clear, from single-chain observation using
fluorescence microscopy together with electron microscopy,
that long DNA exhibits unique responses to different
condensing chemicals [14]. Polyamines, metal cations,
neutral polymers, polypeptides and basic proteins have all
been found to be efficient condensing agents [15]. A variety
of higher-order structures can be generated from the same
long DNA molecule: e.g. toroid, rod, spherical, spool and
intrachain segregated structures [14].
We performed a single-molecule observation of giant
DNA molecules to examine the effect of ascorbic acid at
physiological pH. Surprisingly, we found that ascorbic acid
generates a pearling structure in a giant DNA molecule, in
which elongated and compact parts coexist along a single
molecular chain. A possible mechanism is discussed in
relation to the interplay between the higher-order and
second-order structures of DNA.
Experimental procedures
Materials
T4 phage DNA, 166 kbp with a contour length of 57 lm,

was purchased from Nippon Gene (Toyama, Japan). The
fluorescent dye YOYO-1 was obtained from Molecular
Probes, Inc. (Portland, Oregon, USA). An antioxidant,
2-mercaptoethanol, and
L
-ascorbic acid were purchased
from Wako Pure Chemical Industries (Osaka, Japan).
Fluorescence microscopic observations
T4 phage DNA was dissolved in 10 m
M
Tris/HCl buffer
with 0.1 l
M
YOYO-1 (nucleic acid staining) and 4% (v/v)
2-mercaptoethanol at pH 7.4. Various concentrations of
L
-ascorbic acid (50 l
M
to 10 m
M
) were added. To avoid
intermolecular DNA aggregation, measurements were con-
ducted at a low DNA concentration, 0.3 l
M
in nucleotide
units. Fluorescent DNA images were obtained using a
microscope (Axiovert 135 TV; Carl Zeiss, Jena, Germany)
equipped with a 100 · oil-immersion objective lens and a
Correspondence to Y. Yoshikawa, Department of Food and Nutrition,
Nagoya Bunri College, Nagoya, 451-0077, Japan.

Fax: + 81 52 521 2259, Tel.: + 81 52 521 2259,
E-mail:
Abbreviations: AFM, atomic force microscopy; TEM, transmission
electron microscopy.
(Received 18 March 2003, revised 4 May 2003,
accepted 2 June 2003)
Eur. J. Biochem. 270, 3101–3106 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03699.x
highly sensitive Hamamatsu SIT TV camera, which allowed
recording of images on video tapes. The video image was
analyzed with an image processor (Argus 20; Hamamatsu
Photonics, Hamamatsu, Japan).
Imaging by atomic force microscopy (AFM)
A DNA solution containing ascorbic acid was prepared
as described above, and 5 lL was adsorbed on to freshly
cleaved mica for 1 min. The mica surface was washed with
Milli-Q-purified water, and dried in N
2
gas. An NVB 100
(Olympus, Tokyo, Japan) operated in trapping mode was
used. Images were displayed without modification except
for flattening to remove the background curvature of the
mica surface.
Electron microscopic observations
Samples used for electron microscopy were mounted on
carbon-coated copper grids (No. 200), negative-stained
with 1% uranyl acetate, and observed with a transmission
electron microscope (Jeol 1200EX, Tokyo, Japan) at
100 kV.
CD spectroscopic measurements
Measurements were performed at a T4 phage DNA

concentration of 30 l
M
in 20 m
M
Tris/HCl, pH 7.4. Vari-
ous concentrations of
L
-ascorbic acid (10 l
M
to 150 l
M
)
were added to the DNA solution. CD spectra were recorded
on a Jasco J-720 spectropolarimeter in a 1.0 · 1.0 · 5.0 cm
quartz cell at room temperature.
Results
Figure 1 shows fluorescence microscopic images of individ-
ual T4 DNA molecules in aqueous solution at pH 7.4 in the
absence and presence of ascorbic acid. Depending on the
concentration of ascorbic acid, DNA molecules exhibit
intrachain and translational Brownian motion with differ-
ent conformations. Without ascorbic acid (Fig. 1A), DNA
molecules assume an elongated coil conformation. At
200 l
M
ascorbic acid, DNA remains in a coiled state
(Fig. 1B). At 5 m
M
, folded compact and elongated regions
coexist along a single molecular chain, i.e. intrachain

segregation is observed (Fig. 1C). This segregated confor-
mation appears at concentrations of ascorbic acid of 1 m
M
and above. At 1 m
M
, about 10% of the DNA molecules
show segregated structures, the majority exhibiting the
elongated coil conformation. Above 3 m
M
, most of the
DNA molecules (>80%) are in the segregated state.
To examine the segregated conformation, we used
fluorescence microscopic observation of fixed DNA mole-
cules on a glass surface. Figure 2 shows the fluorescence
images of T4 DNA molecules on a glass slide in 5 m
M
ascorbic acid solution. The DNA molecule was extended on
a glass slide by introducing shear to the solution with a
cover slide. Mini-globules are observed along an extended
DNA molecule on a 2D glass plate. On the other hand,
under the same conditions, the segregated DNA molecules
in the bulk solution show only a few compact regions
(Fig. 1C). As the effective resolution becomes higher on the
fixed DNA, the small mini-globules became visible. From
these observations and the following results obtained by
AFM, it is expected that such small mini-globules observed
A
B
C
0s

3s
5s
0s
3.2s
7s
0s
0.7s
1s
5 µm
Fig. 1. Fluorescence microscopic images of T4 DNA moving freely in
aqueous solution at different ascorbic acid concentrations. (A) Buffer
solution; (B) 200 l
M
ascorbic acid; (C) 5 m
M
ascorbic acid.
5

µm
Fig. 2. Fluorescence microscopic images of T4 DNA fixed on a glass
surface in the presence of 5 m
M
ascorbic acid.
3102 Y. Yoshikawa et al.(Eur. J. Biochem. 270) Ó FEBS 2003
on the fixed DNA may also exist on the segregated DNA in
bulk solution.
Figure 3A,B shows the detailed morphological features
of DNA molecules observed by AFM in the presence of
3m
M

ascorbic acid. The intrachain segregated state is
observed with much higher resolution than those observed
by fluorescence microscopy. It is clear that the mini-globule
part assumes a loosely packed conformation. This AFM
picture corresponds well to the segregated structure
observed by fluorescence microscopy with a lower resolu-
tion (Figs 1 and 2). Figure 3B shows a magnified view of a
condensed part from Fig. 3A. Interestingly, the condensed
part shows the irregular packing of DNA segments.
Figure 3C shows the transmission electron microscopy
1 µm
0.2 µm
0.1 µm
A
B
C
Fig. 3. AFM and TEM images of T4 DNA molecules in the presence of ascorbic acid. (A) and (B) AFM images of T4 DNA molecules in the presence
of 3 m
M
ascorbic acid, where (B) is a magnified view of (A). (C) TEM image of T4 DNA in the presence of 5 m
M
ascorbic acid.
Ó FEBS 2003 Ascorbic acid induces conformational change in DNA (Eur. J. Biochem. 270) 3103
(TEM) image of T4 DNA in the presence of 5 m
M
ascorbic
acid, and indicates that a loosely packed structure is formed.
The morphological features of the condensate observed by
TEM are similar to those observed by AFM, although with
TEM it was difficult to judge whether an elongated coil

region existed in the DNA chain.
It has been well established that single DNA molecules
are packed into a compact toroidal structure with a
diameter of 50–100 nm on the addition of condensing
agents such as polyamines or tervalent metal cations [16,17].
Compared with such a tightly packed state, the folded state
of DNA induced by ascorbic acid is rather swollen.
Next, we measured the changes in the CD spectra of
DNA. Figure 4 shows that the positive Cotton sign at the
% 280 nm band almost disappears when the ascorbic acid
concentration is above 100 l
M
. The change in the negative
band at 246 nm to a positive value is mostly attributable to
the contribution from the added ascorbic acid (see
Fig. 4B). Figure 4C shows the differential CD spectrum
[(30 l
M
DNA + 100 l
M
ascorbic acid) ) (100 l
M
ascor-
bic acid)]. A decrease in the plus Cotton band is observed
at 280 nm, whereas the minus band at 246 nm remains
essentially constant. Interestingly, the band shape in the
difference spectrum resembles that of the C-form of DNA
[18,19].
Discussion
Ascorbic acid induces substantial changes both in the

higher-order and second-order structures of DNA
It is clear that ascorbic acid induces a significant change in
the higher-order structure of DNA. We confirmed the
generation of a segregated structure with multiple mini-
globules using different experimental tools: fluorescence
microscopy and AFM. The mini-globule shows irregular
packing and is very different from previously observed
regular conformations, such as toroid and rod [14,15]. A
similar pearling structure was generated from a long DNA
molecule complexed with histone H1 [20]. However, the
action of ascorbic acid on the pearling structure is thought
to be very different from that of histone H1, as the former is
anionic and the latter is cationic. As ascorbic acid is
negatively charged, it cannot neutralize the charges on the
negative phosphate groups of DNA. Instead, it may interact
with the bases inside the double-stranded structure. Such an
interaction may cause distortion in the double-stranded
structure. Neault et al. [12] performed a Raman and
infrared spectroscopic study on the effect of ascorbic acid
on DNA. They suggested that the OH and C-O groups of
ascorbic acid interact directly with DNA bases.
We shall now discuss the mechanism of the large
conformational change in DNA induced by ascorbic acid,
in relation to the change in second-order structure.
Figure 3C shows the presence of a gnarled conformation
in the condensed part of the chain. This result suggests that
torsional stress is generated along the double-stranded
DNA. It is known that the C-form-like structure of DNA
is found on nucleosome particles [18,21–23], where DNA is
wound around the histone core proteins with high

curvature; the radius is % 5 nm. The similarity of the CD
spectrum to the C-form in Fig. 4C may be explained by the
formation of such an over-wound double-stranded con-
formation, as in a nucleosome. To interpret the CD
spectra, we need to consider the possible effect of
aggregates [24]. It is known that aggregates induce
distortion of the absorption spectrum, as well as the CD
spectrum, through scattering of light. We have confirmed
that, when the concentration of ascorbic acid is increased,
the UV spectra remain on the null level for the region
above 310 nm where both DNA and ascorbic acid exhibit
no light absorption. This indicates that there will be
negligible contribution from aggregates on the CD band.
The above consideration suggests that the unique features
observed by AFM and TEM are closely related to the
change in the secondary structure of DNA.
Theoretical consideration of the stability
of the segregated structure
We now consider the stability of the intrachain segregated
structure induced by ascorbic acid. In general, the free
energy of a condensed object is the result of two different
contributions: bulk and surface energies. For the conden-
sation of DNA induced by ascorbic acid, we have to take
into account the effect of the surviving negative charge,
because negatively charged ascorbate cannot neutralize the
negative charge of DNA.
0
2
220 320240 260 280 300
220

320
240
260
280 300
0
-4
2
-2
4
6
-2
-4
220
320240 260 280 300
0
4
8
CD[mdeg]
CD[mdeg]
CD[mdeg]
Wave length[nm]
Wave length[nm]
Wave length[nm]
A
B
C
0 µM
20
40
60

100
150
Control
Difference
Fig. 4. CD spectra. (A) 30 l
M
T4 DNA in the presence of different
concentrations of ascorbic acid. (B) 100 l
M
ascorbic acid. (C) Solid
line, difference spectrum [(30 l
M
T4 DNA + 100 l
M
ascorbic acid)
solution ) (100 l
M
ascorbic acid) solution]. Broken line, 30 l
M
T4
DNA in the absence of ascorbic acid.
3104 Y. Yoshikawa et al.(Eur. J. Biochem. 270) Ó FEBS 2003
It is well established that, when DNA is folded into a
tightly packed structure accompanied by parallel ordering
of the segments, the negative charge on the DNA molecule
almost completely disappears. This is similar to the com-
paction by spermidine(3+) at low salt concentrations
[25]. On the other hand, when DNA is compacted with
spermidine(3+) at high salt concentration, its volume
becomes one order larger than that of the tightly packed

and ordered state, suggesting the survival of negative charge
in the volume part of compact DNA [26]. Thus, the
remaining negative charge would make a significant contri-
bution to the stability of the compact state when the packing
is less dense, as in the case of DNA complexed with ascorbic
acid.
In the situation of less dense compaction, the free energy
of a single giant DNA in the compact globular state with
respect to the elongated state is given as
F ¼ÀaN þ bN
2=3
þ cQ
2
=R ð1Þ
where N is the number of Kuhn segments on the DNA. It is
well established that a single Kuhn segment in double-
stranded DNA is composed of 300 base pairs [14,27,28]. In
eqn (1), the first and second terms correspond to the volume
and surface energy of a globular compact state, respectively.
The third term represents the instability due to the
remaining charge in the globule. Q and R are the remaining
electronic charge and radius of the condensed particle. The
constants a, b, and c depend on the manner of compaction
or condensation, and are all positive. It is reasonable to
expect that Q$N and R$N
1/3
.Insuchaframework,the
following relationship is deduced [14,27,28], where c
1
is a

constant.
F ¼ÀaN þ bN
2=3
þ c
1
N
5=3
ð2Þ
Equation (2) implies that a condensate with a surviving
electronic charge in the bulk should be destabilized above a
critical number N
c
of segments. When N is larger than N
c
,
the single-globule conformation is destabilized and, as a
result, multiple mini-globules are formed. Thus, the fully
compact state becomes unstable when the residual charge
becomes large, which may correspond to the present case.
It has been found that polycations, such as histone H1
[20] and aminated poly(ethylene glycol) [29], induce a similar
intrachain segregated structure in giant DNA molecules.
The stability of a segregated structure induced by poly-
cations has been interpreted in terms of the incomplete
charge neutralization [28,29]. Although the mechanism of
the compaction induced by ascorbic acid is quite different
from that caused by polycations, the contribution of the
surviving negative charge to the stability of the segregated
state will be the same.
It has been reported that giant DNA molecules are folded

into a compact state by the addition of a negatively charged
polymer, polyglutamic acid [30]. In this case, DNA com-
paction is induced by the Ôcrowding effectÕ, similar to the
mechanism of compaction induced by neutral hydrophilic
polymers such as poly(ethylene glycol) [15]. The concentra-
tion of polyglutamic acid required in monomer units to
induce compaction is very high, of the order of 1
M
. Thus,
ascorbic acid induces compaction of DNA in a very
different way from the negatively charged polymer.
Biological significance of the action of ascorbic acid
on DNA
Ascorbic acid is present in human blood at a concentration
of % 50 l
M
[31,32]. Moreover, the concentration of ascorbic
acid in human cells and tissues can exceed that in the blood
by one order of magnitude [31–33]. In particular, human
circulating immune cells, such as neutrophils, monocytes
and lymphocytes, accumulate ascorbic acid in millimolar
concentrations [32,33]. Therefore, the ascorbic acid concen-
tration that induced the large conformational change in
DNA in this study may be of physiological significance.
It is thought that the folded compact state of DNA is
resistant to external stimuli, such as reactive oxygen species
and restriction enzymes. For example, it has been reported
that the tight and ordered DNA packing in the bacterium
Deinococcus radiodurans promotes resistance to environ-
mental stress [34]. It has also been shown that compacted

DNA is highly resistant to the action of a restriction enzyme
[35]. It is possible that ascorbic acid may reduce oxidative
damage by changing the higher-order structure of DNA. To
our knowledge, this possible biological effect of ascorbic
acid has not previously been considered.
In this study, it has become clear that ascorbic acid has a
dramatic effect on the conformation of giant DNA mole-
cules. At present, the physicochemical mechanism of the
conformational transition of DNA remains an open ques-
tion. Presumably, direct interaction of ascorbic acid with
DNA bases and distortion of the double-stranded structure
may explain the large change in the higher order structure. It
may be of importance to clarify the biological significance of
the action of ascorbic acid, in relation to its effects on both
the higher-order and second-order structures of DNA.
Acknowledgements
This work was supported in part by a Grant-in-Aid from the Ministry
of Education, Science, Sports, and Culture of Japan.
References
1. Hanck, A.B. (1988) Vitamin C and cancer. Prog. Clin. Biol. Res.
259, 307–320.
2. Block, G. (1991) Vitamin C and cancer prevention: the epidemi-
ologic evidence. Am.J.Clin.Nutr.53, 270S–282S.
3. van Poppel, G. & van den Berg, H. (1997) Vitamins and cancer.
Cancer Lett. 114, 195–202.
4. Jacob, R.A. & Sotoudeh, G. (2002) Vitamin C function and status
in chronic disease. Nutr. Clin. Care 5, 66–74.
5. Carr, A. & Frei, B. (1999) Does vitamin C act as a pro-oxidant
under physiological conditions? FASEB J. 13, 1007–1024.
6. Noroozi, M., Angerson, W.J. & Lean, M.E.J. (1998) Effects of

flavonoids and vitamin C on oxidative DNA damage to human
lymphocytes. Am.J.Clin.Nutr.67, 1210–1218.
7. Fischer-Nielsen, A., Loft, A. & Jensen, K.G. (1993) Effect of
ascorbate and 5-aminosalicylic acid on light-induced 8-hydroxy-
deoxyguanosine formation in V79 Chinese hamuster cells.
Carcinogenesis 14, 2431–2433.
8. Pflaum, M., Kielbassa, C., Garmyn, M. & Epe, B. (1998)
Oxidative DNA damage induced by visible light in mammalian
cells: extent, inhibition by antioxidants and genotoxic effects.
Mutat. Res. 408, 137–146.
9. Catani, M.V., Ross, A., Costanzo, A., Sabatini, S., Levrero, M.,
Melino, G. & Avigliano, L. (2001) Induction of gene expression
Ó FEBS 2003 Ascorbic acid induces conformational change in DNA (Eur. J. Biochem. 270) 3105
via activator protein in the ascorbate protection against
UV-induced damage. Biochem. J. 356, 77–85.
10. Podmore, I.D., Griffiths, H.R., Herbert, K.E., Mistry, N., Mistry,
P. & Lunec, J. (1998) Vitamin C exhibits pro-oxidant properties.
Nature (London) 392, 559.
11. Halliwell, B. (1999) Vitamin C: poison, prophylactic or panacea?
Trends Biochem. Sci. 24, 255–259.
12. Neault, J.F., Naoui, M. & Tajmir-Riahi, H.A. (1995) DNA–drug
interaction. The effects of vitamin C on the solution structure of
calf-thymus DNA studied by FTIR and laser Raman difference
spectroscopy. J. Biomol. Struct. Dyn. 13, 387–397.
13. Lutsenko, E.A., Carcamo, J.M. & Golde, D.W. (2002) Vitamin C
prevents DNA mutation induced by oxidative stress. J. Biol.
Chem. 277, 16895–16899.
14. Yoshikawa, K., K. & Yoshikawa, Y. (2002) Compaction and
condensation of DNA. In Pharmaceutical Perspectives of Nucleic
Acid-Based Therapy (Mahato, R.I. & Kim, S.W., eds), pp.

136–163. Taylor & Francis, London.
15. Bloomfield. V.A. (1996) DNA condensation. Curr. Opin. Struct.
Biol. 6, 334–341.
16. Plum, G.E., Arscott, P.G. & Bloomfield. V.A. (1990) Condensa-
tion of DNA by trivalent cations. 2. Effects of cation structure.
Biopolymers 30, 631–643.
17. Yoshikawa, Y., Yoshikawa, K. & Kanbe, T. (1996) Daunomycin
unfolds compactly packed DNA. Biophys. Chem. 61, 93–100.
18. Ivanov, V.I., Minchenkova, L.E., Schyolkina, A.K. & Poletayev,
A.I. (1973) Different conformations of double-stranded nucleic
acid in solution as revealed by circular dichroism. Biopolymers 12,
89–110.
19. Reich, Z., Ghirlando, R. & Minsky, A. (1991) Secondary con-
formational polymorphism of nucleic acid as a possible functional
link between cellular parameters and DNA packaging processes.
Biochemistry 30, 7828–7836.
20. Yoshikawa, Y., Velichko, Y.S., Ichiba, Y. & Yoshikawa, K.
(2001) Self-assembled pearling structure of long duplex DNA with
histone H1. Eur. J. Biochem. 268, 2593–2599.
21. Matsuyama, A., Tagashira, Y. & Nagata, C. (1971) A circular
dichroism study on the conformation of DNA in rat liver chro-
matin. Biochim. Biophys. Acta 240, 184–190.
22. Weischet, W.O., Tatchell, K., Van Holde, K.E. & Klump, H.
(1978) Thermal denaturation of nucleosomal core particles.
Nucleic Acids Res. 5, 139–160.
23. Van Holde, K.E. (1988) The DNA in the core particle. In Chro-
matin, pp. 231–241. Springer-Verlag, New York.
24. Bustamante, C., Tinoco, I. Jr & Maestre, M.F. (1983) Circular
differential scattering can be an important part of the circular
dichroism of macromolecules. Proc. Natl. Acad. Sci. USA 80,

3568–3572.
25. Yamasaki, Y., Teramoto, Y. & Yoshikawa, K. (2001) Dis-
appearance of the negative charge in giant DNA with a folding
transition. Biophys. J. 80, 2823–2832.
26. Yoshikawa, Y., Yoshikawa, K. & Kanbe, T. (1999) Formation of
a giant toroid from long duplex DNA. Langmuir 15, 4085–4088.
27. Grosberg, A.Y. & Khokhlov, A.R. (1994) Biopolymers. In
Statistical Physics of Macromolecules, pp. 289–344. American
Institute of Physics Press, New York.
28. Yoshikawa, K. (2002) Field hypothesis on the self-regulation of
gene expression. J. Biol. Phys. 28, 701–712.
29. Yoshikawa, K., Yoshikawa, Y., Koyama, Y. & Kanbe, T. (1997)
Highly effective compaction of long duplex DNA induced by
polyethylene glycol with pendant amino groups. J. Am. Chem.
Soc. 119, 6473–6477.
30. Ichiba, Y. & Yoshikawa, K. (1998) Single chain observation on
collapse transition in giant DNA induced by negatively-charged
polymer. Biochem. Biophys. Res. Commun. 242, 441–445.
31. Rose, R.C. (1988) Transport of ascorbic acid and other water-
soluble vitamins. Biochim. Biophys. Acta 947, 335–336.
32. Guaiquil, V., Farber, C.M., Golde, D.W. & Vera, J.C. (1997)
Efficient transport and accumulation of vitamin C in HL-60 cells
depleted of glutathione. J. Biol. Chem. 272, 9915–9921.
33. Rumsey, S.C. & Levine, M. (1998) Absorption, transport, and
disposition of ascorbic acid in humans. J. Nutr. Biochem. 9,
116–130.
34. Levin-Zaidman, S., Englander, J., Shimoni, E., Sharma, A.K.,
Minton,K.W.&Minsky,A.(2003)Ringlikestructureofthe
Deinococcus radiodurans genome: a key to radioresistance? Science
299, 254–256.

35. Oana, H., Tsumoto, K., Yoshikawa, Y. & Yoshikawa, K. (2002)
Folding transition of large DNA completely inhibits the action of
a restriction endonuclease as revealed by single-chain observation.
FEBS Lett. 530, 143–146.
3106 Y. Yoshikawa et al.(Eur. J. Biochem. 270) Ó FEBS 2003