Study on the interaction of new water-soluble porphyrin with DNA

Article abstract of DOI:10.1016/j.saa.2004.08.009

A porphyrin meso-tetrakis{[4-(1-pyridyl)propoxy]phenyl}porphyrin (TPyPP) and its Ni complex (TPyPP(Ni)) have been synthesized and characterized by ¹H NMR, UV-vis spectra. The interaction of two porphyrins with calf thymus-DNA (CT-DNA) has been

Full text of DOI:10.1016/j.saa.2004.08.009

Spectrochimica Acta Part A 61 (2005) 2041–2047
Study on the interaction of new water-soluble porphyrin with DNA
Jingwan Kang∗, Haixia Wu, Xiaoquan Lu, Yongsheng Wang, Lin Zhou
Department of Chemistry, Northwest Normal University, Gansu China, Lanzhou 730070, P.R. China
Received 1 May 2004; accepted 18 August 2004
Abstract
A porphyrin meso-tetrakis{[4-(1-pyridyl)propoxy]phenyl}porphyrin (TPyPP) and its Ni complex (TPyPP(Ni)) have been synthesized and
characterized by 1H NMR, UV–vis spectra. The interaction of two porphyrins with calf thymus-DNA (CT-DNA) has been explored by UV–vis,
fluorescence and circular dichroic spectroscopy and viscosity measurements. The results suggest that these porphyrins can bind to DNA by
the same binding mode. TPyPP outside binds by self-stack with DNA both at low drug load r (=[porphyrin]/[DNA]) and high drug load.
Though TPyPP(Ni) has center metal nickel, binding mode with DNA has little difference compared with TPyPP, dominating out-binding
mode with different direction along DNA. The binding constants of the TPyPP and TPyPP(Ni) to DNA were 4.65 × 105 M−1 and 3.2 ×
105 M−1, respectively. A colored precipitate was found after time in two porphyrin’s viscosity measurement. The reasonable interpretation is
the porphyrins with alkyl connected N-position of pyridine can strongly interact with the anionic phosphates of DNA and lead to hydrophobic
complex.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Water-soluble porphyrin; DNA; Out-binding
1. Introduction
interaction of cationic porphyrins with synthetic and natu-
ral DNA has been widely studied using visible absorption
spectroscopy and circular dichroism (CD) [11–13], fluores-
cence [14], Raman [15], NMR [15,16], ESR [17], viscometry
[18,19], footprinting [20,21], kinetic methods [22] and X-ray
crystallography [23].
Interaction of porphyrins and metalloporphyrins with
DNA has a considerable interest due to their medical appli-
cations. Their special properties: high optical absorption, rel-
atively high quantum yields of triplet state and fluorescence,
or paramagnetism of some metal complexes, provide the use
of porphyrin in medicine, as active compounds in radiolog-
ical [1,2], and magnetic resonance imaging [3,4] of cancer
detection and as photosensitizers in photodynamic therapy
(PDT) of cancer [5,6]. Porphyrin demonstrates the photody-
namic activity against psoriasis atheromatous plaque, viral
and bacterial infections including HIV as well [7].
Cationic porphyrins are considered as double functional
compounds that strongly bind to DNA and photo dynamically
modify the target site of a DNA molecule by a mechanism
similar to that of anti-cancer antibiotics such as bleomycin
and daunomycin based on the DNA cleavage [8–10]. The
There are a number of advantages of studying the DNA
binding interactions of cationic metalloporphyrins. First,
that these molecules bind to nucleic acids in ways similar
to“real” anti-tumor drugs, i.e., by intercalative or external
binding modes, makes these porphyrins powerful probes of
drug-DNA interactions, as well as DNA structure. Second,
their high solubility and weak tendency to aggregate in water
(except at very high porphyrin concentrations) make them
suitable for investigation under a wide variety of solution
conditions. Third, by varying the metal center and peripheral
substituents, the porphyrin’s binding mode to nucleic acid
duplexes can be easily “tuned” to be the intercalative or
external type. For example, the thin square planar CuII;
NiII derivatives of meso-tetrakis (N-methylpyridinium-4-
yl)porphyrin (H2P(2)) are capable of intercalation, since
∗
Corresponding author. Tel.: +86 931 7972613; fax: +86 931 7971697.
E-mail address: jwkang@nwnu.edu.cn (J. Kang).
1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2004.08.009

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J. Kang et al. / Spectrochimica Acta Part A 61 (2005) 2041–2047
from purchased from Sino-American Biotechnology Com-
pany (Beijing, China). UV–vis spectra were recorded in
solutions on UV-3400 spectrophometer (Hitachi, Japan).
Fluorescence spectra were carried out using RF-540
spectrofluorophometer (Hitachi, Japan). CD spectra were
recorded on a Jasco-20C spectropolarimeter (Japan). 1H
NMR spectra using Brucker AM-400. Meso-tetrakis[4-(3-
bromopropoxy)phenyl]porphyrin was obtained by method
[27].
Scheme 1. Structure of porphyrin.
these metalloporphyrins do not ligate H2O molecules at their
vacant axial sites and can be inserted into the pocket between
two adjacent, closely spaced (3.4 A) B-DNA base pairs.
2.1.1. Synthesis of meso-tetrakis{[4-(1-pyridyl)-
propoxy]phenyl}porphyrin
˚
Meso-tetrakis[4-(3-bromopropoxy)phenyl]porphyrin
(50 mg) was dissolved in 50 ml anhydrous chloroform.
The solution was heated to refluxing and 1.3 ml pyri-
dine was added. The mixture was refluxed for 48 h,
and then removed solvent. The residue was washed by
acetone for three times. The yield was 85% 1H NMR
(DMSO-d6): ␦ 9.33(d,8H, ␣-py-H) 8.82(s,8H, ␤-pyrrole)
8.69(t,4H, ␤-py-H) 8.29(t,8H,r-py-H) 8.17–8.10(m,8H,ph-
H) 7.24(d,8H,ph-H) 5.01–4.98(t,8H,Ar-OCH2-) 4.42(t,8H,-
CH2py) 2.65–2.49(m,8H, ArOCH2CH2-) −2.94(s,2H,NH)
UV–vis (phosphate buffer pH = 7.0) λmax (nm): 421, 517,
554.4, 590, 646.6.
Three major binding modes have been proposed for the
binding of cationic porphyrins to DNA: intercalation, out-
side groove binding and outside binding with self-stacking
in which porphyrins are stacked along the DNA helix
[12,24–26]. One motivation for these spectroscopic inves-
tigations has been to establish empirically reliable crite-
ria for differentiating between the intercalative and outside
binding modes of porphyrins with various synthetic and na-
tive DNAs. A thorough, initial investigation conducted in
Pasternack’s laboratory showed [11]: that intercalating por-
phyrins are characterized by: (i) large red (ꢀλ ≥ 15 nm) and
hypochromic (H ≥ 35%) shifts of their Soret maxima, (ii)
negative (−) induced CD activity in the Soret region, and
(iii) high selectivity for GC-rich DNA sequences. In con-
trast, outside binders displayed: (i) much smaller red shifts
(ꢀλ ≤ 8 nm) and little hypochromicity (H ≤ 10%), and some-
times hyperchromicity) of their Soret maxima, (ii) positive
(+) induced CD bands in the Soret region, and (iii) a distinct
preference for AT-rich minor groove segments.
The extensive exploration of cationic porphyrins has
been hitherto limited to cationic porphyrins with alkyl con-
nected 4-position of pyridine and its various derivates. In
the present study, we synthesized a cationic porphyrins with
alkyl connected N-position of pyridine: meso-tetrakis{[4-(1-
pyridyl)propoxy]phenyl}porphyrin (TPyPP) and its Ni com-
plex (the structure as shown in Scheme 1). The interaction of
the porphyrins with calf thymus-DNA (CT-DNA) was stud-
ied on the basis of their spectral changes in the Soret band
and induced CD in the visible region as well as fluorescence
spectra and viscosity measurement.
2.1.2. Synthesis of meso-tetrakis[4-(3-bromopropoxy)-
phenyl]porphyrin nickel
Meso-tetrakis[4-(3-bromopropoxy)phenyl]porphyrin
(50 mg) was dissolved in 50 ml toluene and heated to reflux.
Then (1 g) nickel acetate was added. The mixture was
refluxed for 5 h then removed solvent. The residue was
washed by water and obtained a purple crystal of porphyrin
with yield of 80%.
2.1.3. Synthesis of meso-tetrakis{[4-(1-pyridyl)-
propoxy]phenyl}porphyrin nickel
The complex was prepared by a procedure similar to that
TPyPP.
1H NMR(DMSO-d6): ␦ 9.36(d,8H, ␣-py-H) 8.77-
8.72(m,12H, ␤-pyrrole, ␤-py-H) 8.32–8.29(m,8H, r-py-H)
8.13(m,8H,ph-H) 7.92(m,8H,ph-H) 7.34–7.18(m,8H,ph-H)
4.98(t,8H, ArOCH2-) 4.38(t,8H, CH2py-) 2.64(m,8H,
ArOCH2CH2-).
We find TPyPP bind DNA by self-stacked outside bind-
ing and electrostatic interaction. While TPyPP(Ni) has center
nickel which do not ligate H2O molecules at its vacant axial
site but it bind DNA by an outside binding mode and elec-
trostatic interaction.
UV–vis(phosphatebufferpH=7.0)λmax(nm):419, 525.6,
648.8.
2.2. Spectral measurements
All experiments, except where specially indicated, were
performed at room temperature in a phosphate buffer pH 7.0
consisting of 6.1 mM Na2HPO4, 2 mM NaH2PO4, and I =
0.15 M NaCl. A stock solution of CT-DNA was prepared and
stored in phosphate buffer. The concentration of DNA was
determined by UV absorbance at 260 nm using the molar
absorption coefficient (6600 M−1 CM−1) [28]. The interac-
tion of cationic porphyrins with DNA were determined by
2. Experimental
2.1. Materials and instruments
All chemicals were analytical grade. Pyrrole was freshly
distilled before use. Calf thymus-DNA was purchased

J. Kang et al. / Spectrochimica Acta Part A 61 (2005) 2041–2047
2043
absorption spectro-photometric titrations at a room tempera-
ture. Emission spectra of EB bound to DNA in the absence
and presence of the TPyPP and TPyPP (Ni) were determined.
The induced CD spectra were recorded after the addition of
DNA solution to the solution of porphyrin.
2.3. Viscosity measurement
The viscosity of DNA solutions was measured at 30
0.1 ◦C using an Ubbelohde viscometer. Typically, 10.0 ml of
phosphate buffer was transferred to the viscometer to ob-
tain the reading of flow time. For determination of solution
viscosity, 10.0 ml of buffered solution of 160 ␮M DNA was
taken to the viscometer and a flow time reading was obtained.
An appropriate amount of porphyrin was then added to the
viscometer to give a certain r (r = [porphyrin]/[DNA base
pair]) value while keeping the DNA concentration constant,
and the flow time was read. The flow times of samples were
measured after a thermal equilibrium was achieved (30 min).
Each point measured was the average of at least five readings.
The data obtained were presented as (η/η0) versus r, where
η is the reduced specific viscosity of DNA in the presence
of porphyrin and η0 is the reduced specific viscosity of DNA
alone [18,19].
Fig. 1. Absorption spectra of TPyPP (5.8 ␮M) in the absence (top) and
presence of calf thymus-DNA (1.6 ␮M, 3.2 ␮M, 4.8 ␮M, 6.4 ␮M, 8.0 ␮M,
9.6 ␮M, 11.2 ␮M,) in phosphate buffer pH 7.0 (I = 0.15 M NaCl). Arrow
shows that the absorbance changes upon increasing DNA concentration.
hypochromicity of Soret band of TPyPP upon binding to
DNA was found to be 32% (hypochromicity, H% = {[(εf
− εb)/εf] × 100}, where εf and εb are the molar absorption
coefficients for the free and bound forms of the porphyrin).
Red shift was 6.7 nm. The percentage hypochromicity of
TPyPP(Ni) with DNA was 35.5%, red shift was 7.4 nm.
From above mentioned changes (large hypochromicity
and moderate red shift), we considered the interaction
TPyPP and its Ni complex with DNA were both out-binding
modes.
2.4. Electrophoresis test
The compound TPyPP(or TPyPP(Ni) was mixed with
plasmid DNA (pBR322) at different molar ratios, after 1.5 h
of incubation in the dark at 37 ◦C. Then electrophoresis ex-
periments were carried out on an electrophoresis system and
the gels were stained with EB for 0.5 h after electrophoresis,
and then photographed.
Binding constants for the interaction of cationic por-
phyrins with DNA were determined by absorption spectro-
photometric titrations at a room temperature. The fixed
3. Results and discussion
3.1. UV–vis spectral studies
The binding of certain complex to DNA produces
hypochromism, a broadening of the envelope, and a red shift
of the complex absorption band. These effects are partic-
ularly pronounced for intercalators, with groove binders,
a large wavelength shift usually correlates with a complex
conformational change on binding or complex–complex
interactions. A spectral change of TPyPP and TPyPP(Ni)
with addition of DNA shown in Figs. 1 and 2. They exhibited
the hypochromism on the incremental addition of DNA with
varying degrees of bathochromic shift, indicating interaction
of porphyrin with DNA. The intercalative binding of
porphyrin to a DNA helix has been characterized [11] by
large changes in the absorbance (hypochromism ≥35%) and
an appreciable shift in wavelength (red shift ≥15 nm) due to
the interaction of a DNA ␲ stack and porphyrin ␲ system.
While outside binders displays smaller red shifts (ꢀλ ≤
8 nm) and little hypochromicity (H ≤ 10%). The percentage
Fig. 2. Absorption spectra of TPyPP(Ni) (5.2 ␮M) in the absence(top) and
presence of DNA (1.6 ␮M, 3.2 ␮M, 4.8 ␮M, 6.4 ␮M, 8.0 ␮M, 9.6 ␮M,
11.2 ␮M, 12.8 ␮M) in phosphate buffer pH 7.0 (I = 0.15 M NaCl). Arrow
shows that the absorbance changes upon increasing DNA concentration.

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J. Kang et al. / Spectrochimica Acta Part A 61 (2005) 2041–2047
amount of cationic porphyrin in phosphate buffer was titrated
with a stock solution of DNA. The changes in absorbance of
the Soret band upon addition of DNA were monitored at the
maximum of the Soret band. The apparent binding constant,
K
app of cationic porphyrins to DNA was calculated using Eq.
(1):
ꢀ
ꢁ
[DNA]total
1
=
[DNA]total
(|εapp − εf|)
(|εb − εf|)
1
+
(1)
{Kapp(|εb − εf|)}
Fig. 3. Effect of increasing amounts of TPyPP (ꢀ),TPyPP(Ni) (ꢁ) on the
relative viscosities of CT-DNA at 30 0.1 ◦C and [DNA] = 160 ␮M and r
= [porphyrin]/[DNA].
where εapp, εf and εb correspond to Aobsd/[porphyrin], the
extinction coefficient for the free porphyrin and the extinc-
tion coefficient for the porphyrin in the fully bound form,
respectively. In the plot of [DNA]total/(|εapp − εf|) versus
DNA]total, Kapp is given by the ratio of the slope to the inter-
cept [29–31]. The apparent binding constants of TPyPP and
TPyPP(Ni) were calculated to be 4.65 × 105 M−1and 3.2 ×
[(3-(trimethylammonio)propyl)-oxy]phenyl] porphine), they
have similar structure [12,36]. But Marzilli et al. [36] have not
reported T␪OPP bind DNA and induce colored precipitate in
viscosity measurement. The porphyrins in this investigation
have peripherally substituent with N+ in ring of pyridine, in-
teraction between N+ and carbon atom in pyridine form a big
p–␲ conjugate system, positive charge distribute the whole
pyridine ring, so they have strong ability that attract electron.
When they bind negatively charged phosphate oxygen atom
which has strong offering electron ability, due to the pyri-
dine small space hindrance, oxygen atom could easily near to
nitrogen atom and form a hydrophobic complex whose cova-
lence increase. These measurements show that the long flexi-
bletentaclearmsofTPyPPandTPyPP(Ni)probablyfacilitate
favorable outside self-stacking interactions while simultane-
ously permitting near-optimal electrostatic interaction with
the DNA phosphate groups.
105 M−1
.
The porphyrin TPyPP and TPyPP(Ni) have the flexible
tentacles. The flexible tentacle arms of the porphyrin repre-
sent a unique characteristic for an outside binder. The ten-
tacles potentially give TPyPP and TPyPP(Ni) a relatively
large “footprint” compared to other porphyrins, such that the
outside-bound TPyPP and TPyPP(Ni) are able to span several
base pairs when bound in either an edge-on or face-on man-
ner [32]. McMillin and co-workers [33] have suggested that
outside groove binding produces a larger porphyrin footprint
than intercalation.
3.2. Viscosity
Spectroscopic data are necessary, but not sufficient to sup-
port a binding mode. As a means for further clarifying the
binding of the porphyrins, viscosity measurements were car-
ried out on DNA by varying the concentration of the added
porphyrins. A classical intercalative mode causes a signifi-
cant increase in viscosity of DNA solution due to increase
in separation of base pairs at intercalation sites and hence
an increase in overall DNA length. By contrast, complexes
those bind exclusively in the DNA grooves by partial and/or
non-classical intercalation, under the same conditions, typ-
ically cause less positive or negative or no change in DNA
solution viscosity [34]. Fig. 3 shows the effect of increasing
the concentration of porphyrin on the relative viscosity of
the DNA. The results reveal that TPyPP and TPyPP(Ni) de-
creases the relative viscosity of DNA. The decreased relative
viscosity of DNA may be explained by an outside-binding
mode, which produced bends or kinks in the DNA and thus re-
duced its effective length and concomitantly its viscosity. The
results suggest that non-classical intercalative or out-binding
interaction could be ruled out for TPyPP and TPyPP(Ni).
We found that the red precipitations were observed visu-
ally after time. This phenomenon was observed by Hoff-
man [35]. Compared TPyPP with T␪OPP(meso-tetrakis[4-
3.3. Fluorescence spectroscopic studies
Ethidium bromide (EB) emits intense fluorescence light
in the presence of DNA, due to its strong intercalation be-
tween the adjacent DNA base pairs. It was previously re-
ported that the enhanced fluorescence can be quenched by
the addition of a second molecule [37,38]. The quenching
extent of fluorescence of EB bound to DNA is used to de-
termine the extent of binding between the second molecule
and DNA. The emission spectra of EB bound to DNA in
the absence and the presence of the porphyrins are given in
Figs. 4 and 5. According to the classical Stern–Volmer equa-
tion [38]:
I0
= 1 + Kr
I
where I0 and I are the fluorescence intensities in the
absence and the presence of complex, respectively, K a
linear Stern–Volmer quenching constant, r the ratio of total
concentration of complex to that of DNA. The fluorescence
quenching curves of EB bound to DNA by the porphyrins are
shown in Figs. 4 and 5. The quenching plots illustrate that
the quenching of EB bound to DNA by the porphyrins are

J. Kang et al. / Spectrochimica Acta Part A 61 (2005) 2041–2047
2045
Fig. 4. Emission spectra of EB bound to DNA in the presence of the TPyPP (left), Fluorescence quenching curve of EB bound to DNA by TPyPP (right), [EB]
= 5 ␮M, [DNA] = 12 ␮M, λex = 435 nm. Arrows show the intensity changes upon increasing concentration of the porphyrin, 0, 0.32 ␮M, 0.64 ␮M, 0.96 ␮M,
1.28 ␮M, 1.5 ␮M.
in good agreement with the linear Stern–Volmer equation,
which also proves that the porphyrins bind to DNA. In the
plot of I0/I versus [porphyrin]/[DNA], K is given by the
ratio of the slope to intercept. The K values for TPyPP
and TPyPP(Ni) were 3.01 and 2.17, respectively. The data
suggests that the interaction of TPyPP with DNA is stronger
than TPyPP(Ni), which is consistent with the above absorp-
tion spectral results. In the presence of DNA, the excitation
spectrum of EB+ undergoes a large red shift and the excited-
state electron becomes easily removable. Many experiments
have indicated long-range electron transfer between EB+ as
electron donor or acceptor and drug molecules as electron
acceptor or donor, respectively [39,40]. Long-range electron
transfer is an important factor, but it is not the only one.
Pasternack [41] once suggested the possibility of energy
transfer between EB+ and porphyrins in the presence of
DNA. Here the experiments indicate that with increasing
concentration of TPyPP, the fluorescence of EB+ decrease,
while the fluorescence of TPyPP appears and increases at
655 nm. The appearance of isoemissive point at 634 nm in
the presence of DNA, may indicate complex connection in
porphyrin-EB [41]. So interpretation of quenching fluores-
Fig. 5. Emission spectra of EB bound to DNA in the presence of the TPyPP(Ni) (left); Fluorescence quenching curve of EB bound to DNA by TPyPP(Ni)
(right), [EB] = 5 ␮M, [DNA] = 12 ␮M λex = 435 nm. Arrows show the intensity changes upon increasing concentration of the porphyrin 0, 0.32 ␮M, 0.64 ␮M,
0.96 ␮M, 1.28 ␮M, 1.6 ␮M.

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J. Kang et al. / Spectrochimica Acta Part A 61 (2005) 2041–2047
cence of EB is porphyrin self-stacking along DNA helix
and strong interaction electrostatic interactions between the
positively charged N substituent on the porphyrin periphery
and the negatively charged phosphate oxygen atom of DNA
and lead to porphyrin shield EB+ fluorescence and energy
transfer between EB+ and cationic porphyrin [41].
3.4. Induced CD
In general, DNA binding porphyrins do not possess
a chiral center and are optically inactive. However, the
CD spectrum in the drug absorption region, especially in
the Soret band, is induced when it forms a complex with
polynucleotides. Although the origin of induced CD of the
achiral porphyrin–DNA complex is not clear, it is believed
to be induced by the coupling of the transition moments of
achiral drug and chirally arranged nucleobase transition or
by excitonic interaction of the DNA-bound drug. The shape
and magnitude of induced CD depends on the binding mode
and location of the drug, and the nature of the nucleobases
[12,42,43]. As shown in Figs. 6 and 7, the TPyPP and
TPyPP(Ni) used here do not yield CD spectra in the absence
of DNA, but CD spectra were induced for the porphyrins
in the presence of DNA, due to the interaction between the
transition moments of the achiral porphyrin and chirally
arranged DNA base transitions. In Fig. 6, TPyPP displays
negative ICD spectra in the 400–430 nm region and positve
ICD spectra around 430–460 nm when bound to CT-DNA
(r = 0.2), when r = 0.09 the molar ellipiticity ([θ]) both
positive([θ] = 1.03 deg dm2 mol−1) and negative band ([θ]
= −1.23 deg dm2 mol−1) increase slightly. Conservative CD
pattern is consistent with an outside binding mode with self-
stacking [12,13]. As above studies have shown no increase
in DNA solution viscosity upon addition of these two por-
phyrins. But TPyPP(Ni) display a same behavior as TPyPP,
Fig. 7. Induced CD spectra of TPyPP(Ni) 7.8 ␮M in the absence (dashed
line)andpresenceofCT-DNA(solidline) r=0.2(a), r=0.09(b)inphosphate
buffer pH 7.0 (I = 0.15 M NaCl).
448 nm appear in Fig. 7. When r = 0.2, positive band blue
shift to 426 nm ([θ] = 0.3 deg dm2 mol−1), negative band
blue shift slightly to 446 nm ([θ] = −0.44 deg dm2 mol−1).
These results are indicative that TPyPP and its Ni complex
bind DNA by out-side self-stacking along the DNA helix,
but they self stack by different direction. The porphyrin do
not intercalate because of the high electron density in the
porphyrin core [33]. These changes in CD spectrum in Soret
band reflect binding mode is not obvious varying depend on
porphyrins concentration.
3.5. Interaction of porphyrin with plasmid DNA pBR322
Fig. 8 is shown the interaction of porphyrins with pBR322
DNA. Control experiments indicated that no cleavage of
DNA happened in the presence porphyrins. But as shown
in Fig. 8, with increasing concentration of TPyPP, the lane
1–3 run slower than lane 0 that pBR322 DNA alone. Due to
positively charged N substituent on the porphyrin periphery
and negatively phosphate oxygen atom of DNA and this inter-
action is so strong that prevent EB staining. With increasing
concentration of TPyPP, the blocking effect is so obvious as
to the light of EB in UV lamp disappear (the photo is not
shown). While with concentration of TPyPP(Ni) increasing,
the lane 4–6 behavior is the same as that of lane 1–3. The
rational interpretation is that porphyrin–DNA adduct is tight
when r = 0.09 a positive band ([θ] = 0.54 deg dm2 mol−1
)
at 432 nm, a negative band([θ] = −0.58 deg dm2 mol−1) at
Fig. 8. Interaction of supercoiled pBR322 DNA by porphyrin TPyPP
TPyPP(Ni) and 12 ␮L reaction mixtures contained 40 ng of plasmid DNA.
Lane 0: DNA alone; lane 1: DNA + TPyPP (1 ␮M); lane 2: DNA + TPyPP
(5 ␮M); lane 3: DNA + TPyPP (10 ␮M); lane 4: DNA + TPyPP(Ni) (1 ␮M);
lane 5:DNA + TPyPP(Ni) (5 ␮M); lane 6: DNA + TPyPP(Ni) (10 ␮M).
Fig. 6. Induced CD spectra of TPyPP (6.4 ␮M) in the absence (dashed line)
and presence of CT-DNA (solid line) r = 0.2 (a), r = 0.09 (b) in phosphate
buffer pH 7.0 (I = 0.15 M NaCl).

J. Kang et al. / Spectrochimica Acta Part A 61 (2005) 2041–2047
2047
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