Thermodynamics and kinetics of the cleavage of DNA catalyzed
by bleomycin A
5
A microcalorimetric study
Yi Liang
1
, Fen Du
1
, Bing-Rui Zhou
1
, Hui Zhou
1
, Guo-Lin Zou
1
, Cun-Xin Wang
2
and Song-Sheng Qu
2
1
College of Life Sciences and
2
College of Chemistry and Molecular Science, Wuhan University, China
Microcalorimetry and UV-vis spectroscopy were used to
conduct thermodynamic and kinetic investigations of the
scission of calf thymus DNA catalyzed by bleomycin A
5
(BLM-A
5
) in the presence of ferrous ion and oxygen. The
molar reaction enthalpy for the cleavage, the Michaelis–
Menten constant for calf thymus DNA and the turnover

suggest that BLM-A
5
is not as efficient as a DNA-cleaving
enzyme although the cleavage of DNA by BLM-A
5
follows
Michaelis–Menten kinetics. Binding of BLM-A
5
to calf
thymus DNA is driven by a favorable entropy increase with
a less favorable enthalpy decrease, in line with a partial
intercalation mode involved in BLM-catalyzed breakage of
DNA.
Keywords: bleomycin; DNA cleavage; kinetics; microcalor-
imetry; thermodynamics.
The bleomycins (BLMs, Fig. 1) are a family of naturally
occurring, structurally related, glycopeptide-derived antitu-
mor antibiotics discovered by Umezawa and coworkers
from cultures of Streptomyces verticillus in 1966 [1], which
have more than 200 members, such as A
2
,A
5
and B
2
[2].
BLMs consist of an unusual linear hexapeptide, a disac-
charide and a terminal amine (the R group in Fig. 1).
Mixtures of BLMs are presently used for the clinical
treatment of a variety of cancers, notably squamous cell

microcalorimetry and agarose gel electrophoresis were
applied to check the oxidative degradation of DNA induced
by (1,10-phenanthroline)-copper, a well-known DNA-dam-
aging agent [32]. In the present paper, microcalorimetry and
UV-vis spectroscopy were combined to study the scission of
calf thymus DNA by a mixture of bleomycin A
5
(BLM-A
5
),
ferrous iron and oxygen. A novel thermokinetic method for
an enzyme-catalyzed reaction was proposed and employed
to produce not only the thermodynamic constant (D
r
H
m
)
but also the kinetic properties (K
m
and k
2
) of the cleavage of
DNA catalyzed by BLM-A
5
with the result that BLM-A
5
is
not as efficient as a DNA-cleaving enzyme. In order to gain
insights into the nucleotide binding interactions of BLM, we
Correspondence to Y. Liang, College of Life Sciences,

,D
b
G
0
m
and D
b
S
0
m
)for
the binding of BLM-A
5
to calf thymus DNA using
microcalorimetry. The results help understand the binding
mode of BLM-A
5
to DNA.
MATERIALS AND METHODS
Materials
Calf thymus DNA (Sigma Chemical Co., MI, USA) was
purified by ethanol precipitation and centrifugal dialysis and
sheared by sonication at ice bath temperatures for 30 min.
The absorbances at 260 and 280 nm for purified DNA were
measured at room temperature. DNA concentrations were
determined spectroscopically at 260 nm using a molar
extinction coefficient of 13 200
M
)1
Æcm

Tris/HCl buffer (pH ¼ 7.4). As the FeCl
2
solution is easily oxidized by oxygen, it was placed in a
brown bottle and then flushed with purified nitrogen for
10 min, sealed and stored in a refrigerator until use.
Moreover, it was freshly prepared on each occasion.
Isothermal microcalorimetry
The cleavage of calf thymus DNA by a mixture of BLM-A
5
,
ferrous ion and oxygen and the binding of BLM-A
5
to calf
thymus DNA, were studied in 10 m
M
Tris/HCl buffer at
pH 7.4 and 37.0 °C. The heat effects of the reactions
mentioned above were determined using a LKB-2107 batch
microcalorimeter (Stockholm, Sweden), which consists of a
microbatch reactor with a heat-conduction isothermal
calorimeter [31,33–35]. For the experiments on DNA
cleavage, compartment I of the reaction cell contained
2mL of a FeCl
2
solution and compartment II of the
reaction cell contained 4 mL of a DNA/BLM-O
2
mixture.
This multicomponent system was prepared by mixing DNA
and BLM-A

5
,20l
M
ferrous iron and 15.2 l
M
calf
thymus DNA was saturated with purified oxygen and
incubated in 50 m
M
Tris/HCl buffer at pH 7.4 and 20 °C
for 30 min and then scanned from 250 to 500 nm. Five
control systems were chosen to investigate the effect of
DNA cleavage by BLM-A
5
on the spectrum of BLM-A
5
.
The first one was 21.5 l
M
BLM-A
5
, the second was a
mixture containing 21.5 l
M
BLM-A
5
and 20.0 l
M
ferric
iron, and the third was a mixture containing 21.5 l

it follows that
À
1
t
lnð1 À xÞ¼
k
2
½E
0
K
m
À
½S
0
K
m
x
t

ð1Þ
where t is the reaction time, x the fraction of substrate
converted into product at time t, which is nondimensional,
K
m
the Michaelis constant, [S]
0
and [E]
0
the initial concen-
trations of substrate and enzyme, respectively, and k

¼
k
2
½E½S
K
m
þ½S
ð2Þ
where [S] and [E] are the concentration of substrate and the
total concentration of active enzyme at time t, respectively.
The decay law for the first-order self-inactivation is
½E¼½E
0
e
Àk
1
t
ð3Þ
where k
1
is the first-order rate constant for self-inactivation
of the enzyme.
Substituting Eqn (3) in Eqn (2) and performing the
integration between limits [S]
0
to [S] and 0 to t,weobtain
x À
K
m
½S

D ¼ ate
Àkbt
ð6Þ
At t ¼ t
m
,dD/dt ¼ 0andD ¼ D
m
, substituting in Eqn (3),
we get:
a ¼ eD
m
=t
m
ð7Þ
b ¼ 1=kt
m
ð8Þ
where a and b are the analog parameters related to the
thermokinetic system, D
m
and t
m
are the calorimetric curve
characteristic data representing the maximum calorimetric
height and time corresponding to D
m
, respectively. For a
fast reaction, the value of b turns out to be 1. For a slow
reaction, however, the value of b is 2/3 [38].
Combining Eqns (6), (7) and (8), we get

1;1
=ðV
T
Á½S
0
Þð12Þ
Here, Q
1,1
is the total heat effect of the reaction, which
can be calculated by the integration type of Tian’s
equation from the experimental calorimetric curves. V
T
is
the total volume of the reacting system, 6 mL in the present
case.
Eqns (1), (11) and (12) are called the analog calorimetric
curve model of a single-substrate enzyme-catalyzed reaction
without taking self-inactivation of the enzyme into account.
It is a novel application of the thermo-analytical analog
curve method and suitable to both fast and slow enzyme-
catalyzed reactions. A plot of –ln(1 ) x)/t against x/t
is linear with an axis intercept of k
2
[E]
0
/K
m
and a slope of
–[S]
0

small molecules to DNA is of practical interest, because
many small molecules that bind to DNA are effective
pharmaceutical agents, especially in cancer chemotherapy
[25].
From these experiments, it is found that the interactions
of DNA with many small molecules, such as BLM and
ADM, are at rapid equilibrium:
DNA þ L Ð DNA Á L ð13Þ
where L is a small molecule that binds to DNA and DNAÆL
the complex between DNA and L. The intrinsic binding
constant, K
B
, is defined by the equation [24,28,29]:
K
B
¼
y
ð1 À yÞð½DNA
0
À ny½L
0
Þ
ð14Þ
Here, [DNA]
0
and [L]
0
are the initial concentrations of
DNA and L, respectively, n is the exclusion parameter
which presents the number of base pairs covered by each L.

m;a
¼ Q
2;1
=ð½L
0
Á V
TÞ
ð16Þ
Here Q
2,1
is the total heat effect of L binding to DNA,
which can be calculated by the integration type of Tian’s
equation from the experimental calorimetric curves.
The molar ratio, r, of DNA to L is defined as
r ¼ n
DNA;0
=n
L;0
¼½DNA
0
=½L
0
ð17Þ
where n
DNA,0
and n
L,0
are the initial amounts of DNA and
L, respectively. Substituting Eqns (15) and (17) into
Eqn (14), we get

Àð½DNA
0
K
B
þ 1ÞD
b
H
m;a
D
b
H
0
m
ð18Þ
This thermodynamic model was used to perform a
nonlinear least-squares analysis of the apparent molar
binding enthalpy, D
b
H
m,a
, as an explicit function of the
molar ratio r using the
MICROCAL ORIGIN
software
(ver. 6.0) and the values for three unknown binding
parameters, K
B
, D
b
H

S
0
m
¼ðD
b
H
0
m
À D
b
G
0
m
Þ=T ð20Þ
Thermodynamics and kinetics of the cleavage
of DNA catalyzed by BLM-A
5
From the spectroscopic results, the ratio of the absorbance
at 260 nm to that at 280 nm for purified DNA used in the
present study is about 2.07. As shown in Fig. 2, the
calorimetric curve for the cleavage of calf thymus DNA by a
mixture of BLM-A
5
,Fe
2+
and O
2
returned to the baseline
within 10 min, under the experimental conditions used. The
experimental calorimetric curve can be reasonably well

and the kinetic parameters for the cleavage of calf thymus
DNA by a mixture of BLM-A
5
,Fe
2+
and O
2
at different
DNA concentrations and at 37.0 °C obtained from the
analog calorimetric curve models of a single-substrate
enzyme-catalyzed reaction without taking self-inactivation
of BLM-A
5
into account and with the first-order self-
inactivation of BLM-A
5
. It should be pointed out that Fe
2+
is used in about 30-fold molar excess relative to BLM
despite the fact that only 2.28 turnovers (presumably
corresponding to DNA cleavage events) per 100 second
are observed (Table 1). From Fig. 2 and Table 1, it can also
be seen that this DNA cleavage was a largely exothermic
reaction and followed Michaelis–Menten kinetics. Thus, the
observed rate law for the cleavage of DNA catalyzed by
BLM-A
5
at excessive ferrous ion and oxygen concentrations
can be expressed as
t

2+
and O
2
with those of
the five control systems mentioned in the ÔMaterials and
methodsÕ and Fig. 3B shows those between 350 and
500 nm. It can be seen from Fig. 3A that the large
underlying peak at 291 nm for BLM, which has been
ascribed to the bithiazole p) p*andn ) p* transitions [19],
does not shift after this scission, provided that the absorb-
ance for calf thymus DNA has been subtracted from the
total absorbance for the reaction system after the cleavage.
Fig. 2. Experimental calorimetric curve (a) and the corresponding
simulated analog calorimetric curve (b) of the scission of calf thymus
DNA by a mixture of BLM-A
5
,Fe
2+
and O
2
at 37.0 °C. For curve b,
D ¼ 0.03653 te
1–t/150
and b ¼ 1. The initial concentrations of calf
thymus DNA, BLM-A
5
,Fe
2+
and O
2

m
and n are obtained by
fitting the apparent molar enthalpy changes to Eqn (18), are
summarized in Table 3. The v
2
value of Eqn (18) used to
perform a nonlinear least-squares analysis for the binding of
BLM-A
5
to DNA is 0.0268, indicating a good appropriate-
ness of the model proposed. The remaining standard
thermodynamic parameters for the binding, D
b
G
0
m
and
D
b
S
0
m
, are calculated by Eqns (19) and (20), respectively.
Thermodynamics of the binding of ADM
and (1,10-phenanthroline)-copper to DNA
To establish the action mode of BLM-A
5
to DNA, we
investigated the energetics for both the binding reactions of
ADM and (1,10-phenanthroline)-copper to calf thymus

2
(s
)1
) s (s)
Non-self-inactivation 20.4 2.28
First-order self-inactivation 4.22 1.70 188
Table 1. Thermodynamic and kinetic data of the cleavage of calf thymus DNA by a mixture of BLM-A
5
,Fe
2+
and O
2
at 37.0 °C. The
thermodynamic and kinetic data were obtained from the analog calorimetric curve model of a single-substrate enzyme-catalyzed reaction without
taking self-inactivation of BLM-A
5
into account. The molar enthalpy change for the reaction of BLM-A
5
,Fe
2+
and O
2
has been determined by
microcalorimetry to be )34.4±3.2kJÆmol
)1
. Data are expressed as mean ± SD (n ¼ 8). Here, [BLM-A
5
]
0
¼ 10.8 l

m
(kJÆmol
)1
)
K
m
(l
M
)
k
2
· 10
2
(s
)1
)
1 3.47 0.34 0.66 12.00 ) 0.9916 577 24.1 1.77
6.93 0.34 0.66 25.23 ) 0.9968 607 21.9 1.83
2 6.93 0.68 0.66 23.28 ) 0.9931 560 15.7 1.75
17.3 0.34 0.65 59.65 ) 0.9967 574 23.6 2.90
3 17.3 0.34 0.65 62.62 ) 0.9912 602 23.3 2.21
17.3 0.68 0.65 59.50 ) 0.9970 572 14.8 2.30
4 34.7 0.34 0.65 115.5 ) 0.9957 556 22.4 2.49
34.7 0.68 0.65 117.3 ) 0.9989 564
577 ± 19
17.0
20.4 ± 3.8
2.98
2.28 ± 0.49
Fig. 3. A comparison of the UV and visible spectrum of BLM-A

BLM-A
5
and 20 l
M
Fe
3+
. (d) A mixture con-
taining 21.5 l
M
BLM-A
5
and 20 l
M
Fe
2+
saturated with purified
nitrogen. (e) A mixture containing 21.5 l
M
BLM-A
5
,20l
M
Fe
3+
and
15.2 l
M
calf thymus DNA. (f ) 15.2 l
M
calf thymus DNA. (A) shows

2+
and O
2
,
converted calf thymus DNA to free nucleic bases [2,5,13,14].
From electrophoresis experiments, it was found that nicking
of pBR-322 DNA by a mixture of ADM, Fe
3+
,VcandO
2
and by a mixture of (1,10-phenanthroline)-copper(II), ME
and O
2
converted pBR-322 DNA to small DNA fragments
[39] and linear DNA [31], respectively. As is seen in Table 4,
the higher the degree of DNA strand scission by drugs, the
larger the molar enthalpy change for the DNA cleavage.
Fig. 4. Calorimetric curves of BLM-A
5
binding to calf thymus DNA.
The initial concentration of calf thymus DNA is 139 l
M
and the initial
concentrations of BLM-A
5
are (a) 43.0 l
M
and (b) 86.0 l
M
,respect-

(kJÆmol
)1
)
D
b
S
0
m
(JÆmol
)1
ÆK
)1
) Action mode
BLM-A
5
4.19 ± 0.94 5.31 ± 0.12 ) 10.2 ± 0.4 ) 27.4 ± 0.6 55.5 ± 3.2 Partial intercalation
ADM 10.9 ± 1.6 4.83 ± 0.92 ) 46.3 ± 0.9 ) 29.9 ± 0.4 ) 52.9 ± 4.2 Intercalation
(OP)
2
Cu
2+
21.6 ± 5.7 3.07 ± 0.10 16.3 ± 0.2 ) 31.7 ± 0.7 155 ± 3 Groove binding
Fig. 5. Apparent molar enthalpy changes for the binding reactions of (A)
BLM-A
5
(B) ADM and (C) (OP)
2
Cu
2+
, to calf thymus DNA at

2
[12]. As shown in Table 5, the catalytic efficiency (repre-
sented by k
2
/K
m
)ofBLM-A
5
isofthesameorderof
magnitude as that of lysozyme but several orders of
magnitude lower than those of TaqI restriction endonuc-
lease, NaeI endonuclease and BamHI endonuclease. As can
also be seen from Table 5, the cleavage efficiencies (repre-
sented by k
2
; [11]) of BLM-A
5
and of some DNA-cleaving
enzymes, such as TaqI restriction endonuclease, NaeI
endonuclease and BamHI endonuclease, are of the same
order of magnitude but one order of magnitude higher than
those of blenoxane and BLM-A
2
. The catalytic efficiency is
a much better measure for the efficiency of an enzyme than
k
2
(in this case the cleavage efficiency). Therefore, BLM-A
5
is not as efficient as a DNA-cleaving enzyme although the

binding behavior of BLM-A
5
ranges between those of ADM
and (1,10-phenanthroline)-copper and are in line with a
partial intercalation mode involved in BLM-catalyzed
breakage of DNA [44,45]. The partial intercalation given
here is a threading intercalation mode [6,44,45] in which the
bithiazole moiety is partially intercalated between DNA
base pairs and the C-terminal substituent has been threaded
through the helix to the major groove. The partial interca-
lation of BLM induces the relaxation of supercoiled DNA
[4], resulting in a moderately favorable increase in entropy.
About the self-inactivation of activated BLM
Both Fe
2+
and O
2
serve as cofactors in DNA cleavage by
BLM [2–6]. When ferrous BLM is exposed to O
2
,atransient
complex of drug, iron and oxygen, which is kinetically
competent to initiate DNA degradation and commonly
termed activated BLM, is formed [2,4,5,13,14,16,18,46].
Table 4. Comparison of the molar enthalpy change for the cleavage of calf thymus DNA induced by BLM-A
5
and those for the scission of calf thymus
DNA mediated by two DNA-damaging agents, ADM and (1,10-phenanthroline)-copper. Data are expressed as mean ± SD (n ¼ 5–8).
Cleavage system D
r

T ¼ 25.0 °C, pH ¼ 7.4, [ADM]
0
¼ 5.75 l
M
, [FeCl
2
]
0
¼ 340 l
M
, [Vc]
0
¼ 650 l
M
and oxygen was in excess.
c
T ¼ 37.0 °C, pH ¼ 7.0.
Table 5. Comparison of the kinetic parameters for BLM-A
5
and those for carbonic anhydrase, lysozyme, TaqI restriction endonuclease, NaeI
endonuclease, BamHI endonuclease, blenoxane and BLM-A
2
. Here, NAG is N-acetylglucosamine.
Enzyme Substrate K
m
(
M
) k
2
(s

DNA 5.3 · 10
)8
2.2 · 10
)2
4.2 · 10
6
[42]
NaeI endonuclease DNA 1.0 · 10
)8
4.5 · 10
)2
4.5 · 10
6
[43]
BamHI endonuclease DNA 8.9 · 10
)9
7.0 · 10
)3
7.9 · 10
5
[11]
Blenoxane DNA 1 · 10
)3
[11]
BLM-A
2
DNA 2.39 · 10
)3
[12]
BLM-A

5
is unchanged when it cleaves DNA, activated
BLM-A
5
could undergo the first-order self-inactivation to
some extent. As shown in Table 2, the lifetime of self-
inactivation of BLM-A
5
obtained from a model with the
first-order self-inactivation is close to that reported by
Burger and coworkers [46] and the summed v
2
of the fit
using this model is of the same order of magnitude as that of
the model without taking self-inactivation into account
(data not shown). A first-order self-inactivation could be
due to denaturation of the peptide part of the compound
leaving the bithiazloe unit intact but uncoupling the DNA
binding part of the metal complexation part (feasible at
37 °C). Moreover, in this paper, calf thymus DNA is
present when BLM-A
5
is mixed with Fe
2+
and O
2
but not
added after drug activation, and it is well known that DNA
does protect activated BLM against self-inactivation [5,46–
49]. Activated BLM-A

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