Báo cáo Y học: Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A5 A microcalorimetric study pdf

Thermodynamics and kinetics of the cleavage of DNA catalyzedA microcalorimetric study Yi Liang1, Fen Du1, Bing-Rui Zhou1, Hui Zhou1, Guo-Lin Zou1, Cun-Xin Wang2and Song-Sheng Qu2 1 Colle

Thermodynamics and kinetics of the cleavage of DNA catalyzed

A microcalorimetric study

Yi Liang1, Fen Du1, Bing-Rui Zhou1, Hui Zhou1, Guo-Lin Zou1, Cun-Xin Wang2and Song-Sheng Qu2

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 A5

(BLM-A5) 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

number of BLM-A5were calculated by a novel

thermoki-netic method for an enzyme-catalyzed reaction to be

)577 ± 19 kJÆmol)1, 20.4 ± 3.8 lM and 2.28 ± 0.49·

10)2s)1, respectively, at 37.0C This DNA cleavage was a

largely exothermic reaction The catalytic efficiency of

BLM-A5is of the same order of magnitude as that of lysozyme but

several orders of magnitude lower than those of TaqI

restriction endonuclease, NaeI endonuclease and BamHI

endonuclease By comparing the molar enthalpy change for

the cleavage of calf thymus DNA induced by BLM-A5with those for the scission of calf thymus DNA mediated by adriamycin and by (1,10-phenanthroline)-copper, it was found that BLM-A5possessed the highest DNA cleavage efficiency among these DNA-damaging agents These results suggest that BLM-A5is not as efficient as a DNA-cleaving enzyme although the cleavage of DNA by BLM-A5follows Michaelis–Menten kinetics Binding of BLM-A5 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 A2, A5and B2[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

carcinomas, testicular tumors and nonHodgkin’s

lym-phoma [2] The therapeutic effect of BLM is believed to

result from its ability to induce single- and double-strand

breakage of DNA molecules by oxidation of the

deoxyri-bose moiety in the presence of oxygen and a redox-active

metal ion, e.g Fe and Co [2–6] On the other hand, RNA is

also considered as a therapeutically relevant target for BLM

[7,8] It has been found that BLM-induced autoxidation of

ferrous iron follows the Michaelis–Menten kinetics [9,10]

Although a significant number of experimental approaches

have b een used to elucidate the mechanism of DNA

cleavage by BLM in the past two decades [2–6,11–20], thermodynamic information for the scission, which is necessary for a thorough understanding of the mechanism,

is eagerly awaited The purpose of this investigation is to provide detailed thermodynamic and kinetic data for BLM-mediated DNA degradation to furnish insights into the anticancer mechanism of BLM

Microcalorimetry is an important tool for the study of both thermodynamic and kinetic properties of biological macromolecules by virtue of its general applicability, high accuracy and precision [21–24] Recently, this method has yielded a large amount of data on the binding reactions of DNA with DNA-targeting molecules, such as adriamycin (ADM) [25], daunomycin [25,26], Hoechst 33258 [27], ethidium bromide [28], 2,7-diazapyrene [29] and dodecyl trimethylammonium bromide [30] Only a limited number

of authors have, however, paid attention to the energetics of drug-induced cleavage of DNA [31]

In a previous publication from this laboratory [31], 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 A5(BLM-A5), ferrous iron and oxygen A novel thermokinetic method for

an enzyme-catalyzed reaction was proposed and employed

to produce not only the thermodynamic constant (DrHm) but also the kinetic properties (Kmand k2) of the cleavage of DNA catalyzed by BLM-A5with the result that BLM-A5is 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,

Wuhan University, Wuhan, 430072, China.

Fax: + 86 27 8788 2661, Tel.: + 86 27 8721 4902,

E-mail: liangyi@whu.edu.cn

Abbreviations: ADM, adriamycin; BLM, bleomycin; BLM-A 2 ,

bleomycin A 2 ; BLM-A 5 , bleomycin A 5 ; BLM-B 2 , bleomycin B 2 ; BR,

batch reactor; ME, 2-mercaptoethanol; OP, 1,10-phenanthroline;

Vc, vitamin C; UV-vis, ultraviolet and visible.

(Received 17 December 2001, revised 12 April 2002,

accepted 22 April 2002)

have elucidated the binding constant (KB) and the standard

thermodynamic parameters (DbH0m,DbG0m and DbS0m) for

the binding of BLM-A5 to calf thymus DNA using

microcalorimetry The results help understand the binding

mode of BLM-A5to DNA

M A T E R I A L S A N D M E T H O D S

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 200M )1Æcm)1and expressed as

base pair concentrations throughout this paper The

con-centration of BLM-A5 (Hebei Pharmaceutical Factory,

Tianjin, China) was determined at 291 nm using a molar

extinction coefficient of 15 500M )1Æcm)1and the

concen-tration of ADM (Haimen Pharmaceutical Factory,

Zhejiang, China) was determined at 480 nm using a molar

extinction coefficient of 11 500M )1Æcm)1 FeCl2Æ4H2O

(analytical grade) was purchased from Merck’s reagent

Co., Germany Other chemicals used were made in China

and of analytical grade All reagent solutions were prepared

in 10 mM Tris/HCl buffer (pH¼ 7.4) As the FeCl2

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-A5,

ferrous ion and oxygen and the binding of BLM-A5to calf

thymus DNA, were studied in 10 mMTris/HCl buffer at

pH 7.4 and 37.0C 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

2 mL of a FeCl2 solution and compartment II of the reaction cell contained 4 mL of a DNA/BLM-O2mixture This multicomponent system was prepared by mixing DNA and BLM-A5solutions and saturated with purified oxygen before calorimetric experiments To avoid the re-oxidation

of FeCl2solution on exposure to air, purified N2was passed into one compartment of the cell while sample was added to the other As soon as samples were added, the source of N2 was removed and the plug for the reaction cell was closed tightly The same procedure was used for adding samples to the reference cell To avoid the influence of the heat effects

of diluting and mixing, etc on the results, the contents and quantities in both cells were as close as possible except that DNA was not added to the reference cell For the experiments on DNA binding, compartment I of the reaction cell contained 2 mL of a DNA solution and compartment II of the reaction cell contained 4 mL of a BLM-A5solution The heat released by dilution of DNA is negligible

UV-vis spectroscopy

UV and visible spectra were measured using a Shimadzu UV-2401PC spectrophotometer A reaction system contain-ing 21.5 lMBLM-A5, 20 lMferrous iron and 15.2 lMcalf thymus DNA was saturated with purified oxygen and incubated in 50 mM Tris/HCl buffer at pH 7.4 and 20C 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-A5on the spectrum of BLM-A5 The first one was 21.5 lM BLM-A5, the second was a mixture containing 21.5 lM BLM-A5 and 20.0 lM ferric iron, and the third was a mixture containing 21.5 lM

BLM-A5 and 20.0 lM ferrous iron saturated with purified nitrogen The fourth was a solution containing 21.5 lM BLM-A5, 20 lMferric iron and 15.2 lMcalf thymus DNA and the fifth was 15.2 lMcalf thymus DNA These solutions were also incubated in 50 mM Tris/HCl buffer at pH 7.4 and 20C for 30 min and then scanned from 250 to 500 nm

R E S U L T S

Novel thermokinetic models for enzyme-catalyzed reactions

For a simple single-substrate, single-intermediate, enzyme-catalyzed reaction occurring in a batch reactor (BR) with negligible mass-transfer limitations and without self-inacti-vation of the enzyme, from the Michaelis–Menten kinetics,

it follows that

1

tlnð1  xÞ ¼ k2½E0

Km

½S0

Km

x t

 

ð1Þ where t is the reaction time, x the fraction of substrate converted into product at time t, which is nondimensional,

Kmthe Michaelis constant, [S]0and [E]0the initial concen-trations of substrate and enzyme, respectively, and k2, also known as the turnover number of the enzyme [36], the rate constant of breakdown of the enzyme–substrate complex to product

Fig 1 Structure of BLM-A 2 , A 5 and B 2

Under certain conditions, the rate of self-inactivation of

an enzyme may be sufficiently great that it must be taken

into account in the study of the kinetics of the reaction

undergoing catalysis [37] The self-inactivation reactions are

sometimes, although not always, of first order kinetics Even

when first order inactivation is taken into account, other

kinetics schemes, such as the second- or zero-order

self-inactivation, can be accounted for, according to Laidler &

Bunting [37] In the present paper, attention will be confined

to first-order self-inactivation, but the methods are readily

extended to other cases

For a single-substrate enzyme-catalyzed reaction

occur-ring in a BR with the first-order self-inactivation of the

enzyme, the general rate equation is

dơS

dt Ử k2ơEơS

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

ơE Ử ơE0ek1 t đ3ỡ where k1is the first-order rate constant for self-inactivation

of the enzyme

Substituting Eqn (3) in Eqn (2) and performing the

integration between limits [S]0to [S] and 0 to t, we ob tain

xKm

ơS0lnđ1  xỡ Ử

k2ơE0

k1ơS0đ1  e

k 1 tỡ đ4ỡ

If the heat-transfer process in a BR obeys TianỖs equation

[21,33Ờ35], the substrate conversion in a BR may be written

as

Here, D is the calorimetric height at time t (i.e the

thermopile potential difference at times t and 0), a is

the area under the calorimetric curve and the time-axis over

the interval (t Ờ 0), A is the total area under the calorimetric

curve and k is the Newton cooling constant of the

calorimeter system which can be easily determined by

electric calibration [34]

According to the thermo-analytical analog curve method

[38], the calorimetric curve for a reaction occurring in a

conduction calorimeter can be approximately simulated by

the following relationship:

At tỬ tm, dD/dtỬ 0 and D Ử Dm, substituting in Eqn (3),

we get:

where a and b are the analog parameters related to the

thermokinetic system, Dmand tmare the calorimetric curve

characteristic data representing the maximum calorimetric

height and time corresponding to Dm, 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

D Ử t

tm

Substituting Eqn (10) in Eqn (5), we obtain

x Ử a

A ợ bD

eDm

đ11ỡ When a single-substrate enzyme-catalyzed reaction is taking place in a conduction calorimeter, the molar reaction enthalpy is:

DrHm Ử Q1;1=đVT ơS0ỡ đ12ỡ Here, Q1,1is the total heat effect of the reaction, which can be calculated by the integration type of TianỖs equation from the experimental calorimetric curves VTis 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 Ờln(1) x)/t against x/t

is linear with an axis intercept of k2[E]0/Kmand a slope of Ờ[S]0/Km The values of Kmand k2can be calculated from the slope and intercept, respectively, using the calorimetric data from only a single experiment

Eqns (4) and (11) are called the analog calorimetric curve model of a single-substrate enzyme-catalyzed reaction with the first-order self-inactivation of the enzyme The values of

Km, k2 and k1 were obtained from the equations by substituting in at least three sets of experimental data (x and t) and using the MATHSOFT MATHCAD software (version 2001) The value of s, the lifetime of self-inactivation, was calculated using the rate constant k1

A thermodynamic model for the binding of small molecules to DNA

Understanding the thermodynamics of the binding of 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:

where L is a small molecule that binds to DNA and DNAẳL the complex between DNA and L The intrinsic binding constant, KB, is defined by the equation [24,28,29]:

đ1  yỡđơDNA0 nyơL0ỡ đ14ỡ 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

The degree of L binding to DNA, y, can be determined by

the formula:

y Ử DbHm;a=DbH0

where DbH0

m is the standard binding enthalpy per mole of L

and DbHm,ais the apparent molar binding enthalpy which

can be calculated using the equation:

DbHm;a Ử Q2;1=đơL0 VTỡ đ16ỡ

Here Q2,1is the total heat effect of L binding to DNA,

which can be calculated by the integration type of TianỖs

equation from the experimental calorimetric curves

The molar ratio, r, of DNA to L is defined as

r Ử nDNA;0=nL;0 Ử ơDNA0=ơL0 đ17ỡ

where nDNA,0and nL,0are the initial amounts of DNA and

L, respectively Substituting Eqns (15) and (17) into

Eqn (14), we get

rỬ ơDNA0KBnDbHm;ađDbH0m DbHm;aỡ

ơDNA0KBđDbH0

mỡ2 đơDNA0KBợ 1ỡDbHm;aDbH0

m đ18ỡ This thermodynamic model was used to perform a

nonlinear least-squares analysis of the apparent molar

binding enthalpy, DbHm,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, KB, DbH0m and n, were thus obtained The v2

test was used to examine the appropriateness of the model

statistically

The standard molar binding free energy (DbG0m) and the

standard molar binding entropy (DbS0m ) for the binding

reaction were calculated by the fundamental equations of

thermodynamics [28]:

DbG0m Ử RT ln KB đ19ỡ

DbS0m Ử đDbHm0  DbG0mỡ=T đ20ỡ

Thermodynamics and kinetics of the cleavage

of DNA catalyzed by BLM-A5

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-A5, Fe2+and O2returned to the baseline

within 10 min, under the experimental conditions used The

experimental calorimetric curve can be reasonably well

fitted by the simulated analog calorimetric curve between 75

and 210 s at 37.0C The substrate conversion x at time t in

one experiment on the DNA cleavage by BLM-A5can be

calculated using Eqn (11) from the calorimetric data shown

in Fig 2 A plot of Ờln(1) x)/t against x/t in this range is

linear with the value of y-axis intercept being

1.323ở 10)2s)1, the value of slope being)0.7342 and the

linear correlation coefficient being)0.9967 Then, the values

of Kmand k2can be calculated from the slope and intercept

to be 23.6 lM and 2.90ở 10)2s)1, respectively After the

calorimetric experiment on DNA cleavage, the residual

solutions taken from both the reaction cell and the reference one were brownish yellow

Tables 1 and 2 summarize the molar reaction enthalpies and the kinetic parameters for the cleavage of calf thymus DNA by a mixture of BLM-A5, Fe2+and O2at different DNA concentrations and at 37.0C obtained from the analog calorimetric curve models of a single-substrate enzyme-catalyzed reaction without taking self-inactivation

of BLM-A5 into account and with the first-order self-inactivation of BLM-A5 It should be pointed out that Fe2+

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 MichaelisỜMenten kinetics Thus, the observed rate law for the cleavage of DNA catalyzed by BLM-A5at excessive ferrous ion and oxygen concentrations can be expressed as

t0 Ử k2ơBLM0ơDNA0

Here t0is the initial rate for the DNA cleavage by BLM-A5

It should be pointed out that the fact that a reaction can be simulated using the MichaelisỜMenten theory kinetics does not per se imply that a reaction is enzymatic

UV-vis spectra of BLM-A5 Figure 3A compares the UV and visible spectrum from 250

to 500 nm of BLM-A5 after the cleavage of calf thymus DNA by a mixture of BLM-A5, Fe2+and O2with those of the five control systems mentioned in the Materials and methods 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* and n ) 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 , Fe2+and O 2 at 37.0 ồC For curve b,

D Ử 0.03653 te 1Ờt/150

and b Ử 1 The initial concentrations of calf thymus DNA, BLM-A 5 , Fe2+and O 2 are 17.3, 10.8, 340 and 650 l M , respectively.

This result indicates that the absorbing group is unchanged

after this scission The flat peak at 384 nm for curves a and c

in Fig 3B may result from charge transfer transitions

between ferric iron and BLM-A5 The reason why we do not

observe the charge transfer band in curve e is unknown

Thermodynamics of the binding of BLM-A5to DNA

Figure 4 shows two of the calorimetric curves of BLM-A5

binding to calf thymus DNA at different molar ratios of

DNA/BLM-A5 The experimental apparent molar enthalpy

changes for these reactions can be calculated using the

integration type of Tian’s equation and Eqn (16) from the

calorimetric curves The thermodynamic data for the

bind-ing, in which the values of KB, DbH0

m and n are obtained by fitting the apparent molar enthalpy changes to Eqn (18), are

summarized in Table 3 The v2 value of Eqn (18) used to

perform a nonlinear least-squares analysis for the binding of

BLM-A5to DNA is 0.0268, indicating a good

appropriate-ness of the model proposed The remaining standard

thermodynamic parameters for the binding, DbG0m and

DbS0m, 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-A5 to DNA, we

investigated the energetics for both the binding reactions of

ADM and (1,10-phenanthroline)-copper to calf thymus

DNA and found that their thermodynamic binding

param-eters were different from those of BLM-A5 ADM is an intercalator, which inserts its aromatic ring between adjacent base pairs of DNA [25,39] and

(1,10-phenanthro-Table 2 Comparison of the kinetic parameters for BLM-A 5 without

taking its self-inactivation into account and those for BLM-A 5 with

first-order self-inactivation The kinetic data were 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

Inactivation type of BLM-A 5

K m

(l M )

k 2 · 10 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 ± 3.2 kJÆmol )1 Data are expressed as mean ± SD (n ¼ 8) Here, [BLM-A 5 ] 0 ¼ 10.8 l M and R is the correlation coefficient of –ln(1 ) x)/t correlating with x/t.

No.

[DNA] 0

(l M )

[FeCl 2 ] 0

(m M )

[O 2 ] 0

(m M )

–Q1,1 (mJ) R b

–D r H 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 5 after the cleavage of calf thymus DNA by a mixture of BLM-A 5 , Fe2+and O 2

with those of five control systems (a) A reaction system containing 21.5 l M BLM-A 5 , 20 l M Fe2+and 15.2 l M calf thymus DNA sat-urated with purified oxygen, after incubation in 50 m M Tris/HCl buffer

at pH 7.4 and 20 C for 30 min (b) 21.5 l M BLM-A 5 (c) A solution containing 21.5 l M BLM-A 5 and 20 l M Fe3+ (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 , 20 l M Fe3+and 15.2 l M calf thymus DNA (f ) 15.2 l M calf thymus DNA (A) shows the optical spectra between 250 and 550 nm and (B) displays those between 350 and 500 nm.

line)-copper binds to either the major or minor groove of the

double helix [32] The thermodynamic data for these

binding reactions at 37.0C are listed in Table 3 The solid

lines in Fig 5B,C are the predicted apparent molar enthalpy

changes for these binding reactions as calculated using

Eqn (18) and the parameters in Table 3 and in agreement

with the experimental data The v2value of Eqn (18) used to

perform a nonlinear least-squares analysis for the binding of

ADM and (1,10-phenanthroline)-copper to DNA are 4.05

and 0.0139, respectively, indicating that the thermodynamic

model for the binding of small molecules to DNA proposed

in this paper, is reasonable

D I S C U S S I O N

DNA cleavage efficiency of BLM-A5

In Table 4, we compared the molar enthalpy change for the

cleavage of calf thymus DNA induced by BLM-A5 with

those for the scission of calf thymus DNA mediated by two

well-known DNA-damaging agents, ADM [39,40] and

(1,10-phenanthroline)-copper [31,32] Scission of calf

thy-mus DNA induced by BLM in the presence of Fe2+and O2,

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, Fe3+, Vc and O2

and by a mixture of (1,10-phenanthroline)-copper(II), ME

and O2converted 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-ively The experimental temperature is 37.0 C.

Table 3 Thermodynamic parameters for the binding reactions of three antitumor drugs, BLM-A 5 , ADM and (1,10-phenanthroline)-copper, to calf thymus DNA at 37.0 °C These binding reactions were carried out as described in the legend to Fig 5 Thermodynamic parameters were determined using the thermodynamic model for the binding of small molecules to DNA in the results section Data are expressed as mean ± SD.

Drug

K B · 10)4

( M )1 )

n (base pairs/drug)

D b H 0 m

(kJÆmol)1)

D b G 0 m

(kJÆmol)1)

D b S 0 m

(JÆmol)1ÆK)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 Cu2+ 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 Cu2+, to calf thymus DNA at 37.0 °C The initial concentrations of calf thymus DNA are 139 l M

(A,C) and 136 l M (B) Empty circles, experimental data; solid lines, curves predicted by Eqn (18) using the parameters in Table 3.

BLM-A5 possessed the highest DNA cleavage efficiency

among these DNA-damaging agents

BLM-A5is not as efficient as a DNA-cleaving enzyme

BLM has three functional domains (Fig 1) The metal

binding domain is required for metal complexation, oxygen

binding and activation [6] and corresponds to the catalytic

site of DNA-cleaving enzymes, e.g EcoRI endonuclease

[15] The DNA binding domain, encompassing the

bithiaz-ole moiety, can be regarded as the substrate binding site [15]

The carbohydrate moiety is involved in cell permeability

and selective tumor recognition [6] Although BLM is much

smaller than real DNA-cleaving enzymes, it is comparable,

both in size and in domains, to the cleft of the active site of

such type of enzymes, e.g EcoRI endonuclease [15]

Table 5 compares the kinetic parameters for BLM-A5

with those for carbonic anhydrase [41], lysozyme [41], TaqI

restriction endonuclease [42], NaeI endonuclease [43],

BamHI endonuclease [11], blenoxane [11] and BLM-A2

[12] As shown in Table 5, the catalytic efficiency

(repre-sented by k2/Km) of BLM-A5 is of the same order of

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 k2; [11]) of BLM-A5and 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-A2 The catalytic efficiency is

a much better measure for the efficiency of an enzyme than

k2(in this case the cleavage efficiency) Therefore, BLM-A5

is not as efficient as a DNA-cleaving enzyme although the

cleavage of DNA by BLM-A5follows Michaelis–Menten

kinetics The cleavage of calf thymus DNA by a mixture of

ADM, Fe3+, Vc and O2 and by a mixture of (1,10-phenanthroline)-copper(II), ME and O2do not, however, follow the Michaelis–Menten kinetics (data not shown), suggesting that ADM and (1,10-phenanthroline)-copper are unlike DNA-cleaving enzymes

Mode of binding BLM-A5to DNA

As shown in Table 3, the binding of ADM to calf thymus DNA is driven entirely by a large favorable enthalpy reduction but with an unfavorable entropy decrease In contrast, the binding of (1,10-phenanthroline)-copper to calf thymus DNA shows just an opposite thermodynamics of the reaction driven by a large favorable increase in entropy with an unfavorable raise in enthalpy Meanwhile, the binding of BLM-A5to calf thymus DNA seems to be driven

by a favorable entropy change with a less favorable enthalpy change These results indicate that the thermodynamic binding behavior of BLM-A5ranges 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 Fe2+and O2serve as cofactors in DNA cleavage by BLM [2–6] When ferrous BLM is exposed to O2, a transient 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 H m (kJÆmol)1) Product Reference

BLM-A 5 -Fe2+-O 2 )577 ± 20 Free nucleic bases This work, [2,5,13,14] ADM-Fe3+-Vc-O 2b )147.1 ± 6.1 Small DNA fragments This work, [39]

(OP) 2 Cu 2+ -ME-O 2 )35.1 ± 1.8 Linear DNA [31]

a

T ¼ 37.0 C, pH ¼ 7.4.bT 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)1) k 2 /K m ( M )1 Æs)1) Reference Carbonic anhydrase HCO 3 9.6 · 10)3 4 · 10 5 4.2 · 10 7 [41] Lysozyme (NAG) 2 1.75 · 10)4 0.5 2.9 · 10 3 [41] TaqI restriction

endonuclease

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 · 105 [11]

BLM-A 5 DNA 2.04 · 10)5 2.28 · 10)2 1.12 · 10 3 This work

Nakamura & Peisach [47] have suggested that the

bithiazloe structure of BLM-A2 is altered when it is

inactivated It has also been shown that activated BLM-A2

undergoes self-inactivation to a very substantial extent

concomitant with its cleavage of DNA [5,46–49] As some

of the molecules become inactivated and thus are no longer

capable of cleaving DNA, the measured kinetics of cleavage

will lead to underestimating of the cleaving potential of the

remaining molecules The impact of self-inactivation of

activated BLM on the thermodynamics of DNA binding is

more complex As the structural identities of the BLM

degradation products are unknown, it is unclear whether

those products bind to DNA themselves and with what

properties and they might affect BLM binding Although it

is indicated in this paper that the chromophoric group of

BLM-A5 is unchanged when it cleaves DNA, activated

BLM-A5could undergo the first-order self-inactivation to

some extent As shown in Table 2, the lifetime of

self-inactivation of BLM-A5obtained from a model with the

first-order self-inactivation is close to that reported by

Burger and coworkers [46] and the summed v2of 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

37C) Moreover, in this paper, calf thymus DNA is

present when BLM-A5is mixed with Fe2+and O2but not

added after drug activation, and it is well known that DNA

does protect activated BLM against self-inactivation [5,46–

49] Activated BLM-A5may lose its activity slower when

complexed with DNA

In this paper, microcalorimetry and UV-vis spectroscopy

have been combined to study the scission of calf thymus

DNA catalyzed by BLM-A5 A novel thermokinetic

method for an enzyme-catalyzed reaction has been

pro-posed and employed to produce not only the

thermody-namic constant but also the kinetic properties of DNA

cleavage by BLM-A5with the result that BLM-A5is not as

efficient as a DNA-cleaving enzyme The present

thermo-dynamic and kinetic findings have provided further insights

into the mechanism with which BLM functions as both a

DNA-damaging agent and an antitumor drug

A C K N O W L E D G E M E N T S

This work was supported by the 973 Project (G1999075608) from the

Chinese Minister of Science and Technology and the grant (39970164)

from the National Natural Science Foundation of China We are also

grateful to Prof C L Tsou and Prof J M Zhou (Institute of

Biophysics, Academia Sinica, China) for their critical reading of the

manuscript and for their helpful suggestions.

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