Vol. 91, No. 2, 1979 November

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

28, 1979

Pages

REACTION OF FeBlm WITH DNA: W. E. Antholine'

528- 533

Fe(II)Blm-NO

and D. H. Petering2

1 Radiation

Biology and Biophysics Section, The Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, U.S.A. 2 Laboratory for Molecular Biomedical Research, Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201, U.S.A.

Received

October

8, 1979

Summary. The structure of the iron bleomycin nitric oxide complex is altered in the presence of calf thymus DNA as determined from epr studies. This altered structure predominates for one iron bleomycin nitric oxide molecule per coil of the DNA helix. In the absence of nitric oxide, as the pH is lowered, iron bleomycin dissociates in two steps, supporting the hypothesis that in-plane nitrogens may be easily perturbed. Introduction. mechanism which it

Bleomycin of action,

occurs

in

has been

this

than

conditions,

attack

and cleave developed

bound

Fe(II)Blm

directly

at the To probe

Fe(III)Blm

with

Sugiura

complex

and that addition

changes

binds

site

of DNA.

Abbreviations. FeBlm - iron IHP - inositol

the reaction epr

thymus to the

with

oxygen

the

plane

@ 1979

Recently,

interaction

orientation (8).

with that

which has now

Fe 2+ .

oxygen

by Academic Press. Inc. in any form reserved.

528

The

radicals

of Fe(II)

and

communication DNA is

(7).

considered.

Co(II)Blm

is

a pentacoordinate

- O2 adduct

is

formed

(8).

bound

oxygen

of the axially Cobalt

bleomycin

appears

epr - electron paramagnetic resonance, Blm - bleomycin, bleomycin, FeBlm-NO' - iron bleomycin nitric oxide complex, hexaphosphate.

of reproduction

in

under

mechanism

in a previous

evidence

0006-291X/79/220528-06$01.00/0 Copyright Alf rights

oxidizes

to produce

catalytic

of 02 a Co(II)Blm

Co(II)

(1).

to DNA and chelates

of iron-bleomycins,

DNA, the

of DNA

is much more effective

A more elaborate binds

spectral

scission

reagents

As Fe(II)Blm

was investigated

in the presence

respect

first

As a possible

such as OH' are generated,

(3-6).

in detail

O2 and thiols

presented

(2).

radicals

and reacts

mechanism

of calf

with

bleomycin

then

study

Earlier

oxygen

the DNA backbone

this

In the present

After

reduced

Fe(II)Blm,

ligand

agent.

on the strand

and sulfhydryl

bleomycin,

the metal-free

antitumor

focused

of bleomycin

ferrous

in which

useful

has been

the presence

aerobic

been

a clinically

attention

shown that

reaction

is

to be in-

Vol. 91, No. 2, 1979

active

both

in the

the present oxide

strand

study

adduct

and 0

BIOCHEMICAL

scission

examines

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

reaction

the binding

to DNA as a more direct

and as a cytotoxic

of the stable model

for

agent

ferrous

the

(9).

Thus,

bleomycin-nitric

interaction

of Fe(II)Blm,

DNA

2'

Materials

and Methods.

Materials. Bleomycin, a clinical mixture containing about 70% bleomycin A2 and significant B2, was supplied by Bristol Laboratories. Nitric oxide gas was purchased from either Matheson or Union Carbide Gas Products. Calf thymus DNA was purchased from Sigma. Dithionite, NaCl, HCl, NaOH and Fe(NH4)2(S04)2*6(H20) were reagent grade. Calf thymus DNA and bleomycin were deoxygenated with argon before adding Methods. an appropriate aliquot of an Fe(NH4)2(S04)2*6(H20) solution which was under nitrogen. The nitric oxide bound bleomycin complex, FeBlm-NO', was prepared in the presence and absence of DNA by flushing the system anaerobically with NO'. The FeBlm-NO. solution in a tonometer was transferred anaerobically to an argon-filled glove box where samples were frozen in liquid nitrogen for epr measurements (7). Fe(Z+)Blm rapidly oxidizes in air to give stock solutions of Fe(3+)Blm. All stock solutions of FeBlm and DNA were adjusted to pH 7.0 with 2N NaOH. W-visible absorption spectra were obtained with an Acta V Spectrophotometer. A few grains of dithionite were added to curvettes to keep Fe(2+)Blm fully reduced before scanning the visible spectrum and recording the pH with a Radiometer PHM-26 meter. Results nitric

and Discussion. oxide

of DNA the binding

Figure

complex, FeBlm-NO

into

nine

derivatives.

complex

perturbs

ligation

trans In the

resolved

axis, possibly

is

which

consists

a trans

oxide

is

of DNA.

bleomycin In the

spectra

absence

(10-12). further

Upon re-

hemoglobin-nitric in a modified

the

of the

of a large

the high

simulations.

field field Both

approximately

a amino nitrogen

ligand

suggesting

environment

of the nitrogen

ferrous

oxide FeBlm

existence

of axial

is altered

(Figure

(10).

the result

with

the

of NO' is not

as seen with

of the axial

of FeBlm-NO

for

reported

structure

nitrogen

of DNA, the high

further which

to recently

hyperfine

of DNA the

comparison

in-plane

similar

the lack

nitric

change

without

and absence

triplet

the spectrum

presence

but

in the presence

by an axial

In the presence

difficult to the

lines

to the

The predominant 2.06.

the

is

Nevertheless,

complex

spectra

FeBlm-NO,

to Fe(II)Blm

solved

1 shows the epr

iron

shift

in the

g-value shoulder

perpendicular

from bleomycin.

529

at 1.967

g-values

the iron

Bleomycin

well

of DNA is

may be attributed

to the iron oxide,

g-value

is also

in the absence

of these

from nitric

low field

1).

is

nitric molecule, thought

oxide and to bind

to

Vol. 91, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

g,=2.006

I g,4.967

Figure 1. The epr spectrum of a frozen (-196OC) solution of 0.1 mM FeBlm-NO' in the absence (upper trace) and the presence (lower trace) of 18 mgms/ml of calf thymus DNA and -15 M phosphate buffer at pH 7.2..

to DNA through binding

the dithiazole

site

nitrogen,

(13).

which

closest

direct

be likely

are link

sites

for

and terminal

The histidine four

peptide

imidazole bonds

to the DNA bound the in-plane

amine

at a site

nitrogen

from

the

and the

dithiazole

dithiazole

perturbation

distant

the metal

deprotonated

moiety,

and terminal of the

from

appear

amine

Fe(II)Blm

peptide to be the

and would

structure

thus

observed

here. There

is

the absence

little

change

in the

triplet

hyperfine

of DNA; aN = 23 Gauss in the presence

perturbation

of the axial

nitric

derivatives

oxide

in the presence

C, and NO ferrous

presence

of IHF' where

weakened

(18).

oxide

ferrous

in the presence

of DNA is

chrome

nitric

the

parameters

of DNA) suggesting

bond

in contrast

little

to hemoglobin

of IHP (14-16).

The spectrum for

similar

to the

spectra

reported

myoglobin

(17),

and for

hemoglobin

interaction

(aN = 25 Gauss in

with

530

the

imidazole

NO ferrous nitric

trans

oxide

to nitric

of FeBlm-NO cytoin

the

oxide

is

BIOCHEMICAL

Vol. 91, No. 2, 1979

2'0 40

0

60

AND BIOPHYSICAL

CiO 160 Xi0 340 160 Iti0 [DNA]/ [FeBlmNOl

RESEARCH COMMUNICATIONS

260

Figure 2. The epr signal intensity in the presence of DNA is pfotted versus the concentration ratio of DNA bases to FeBlm-NO. The parameters A and D are defined in Figure 1 for FeBlm-NO and FeBlm-NO in the presence of DNA. The DNA concentration was measured spectrophotmetrically at 260 rims (~-6.6 x 103KI). The FeBlm concentration was determined from the dilution of a stock solution. All solutions were made up in .05 M phosphate buffer at pH 8.0. The DNA conX; the DNA concentration is centration is 0.8 mM as indicated by the 25 mM as indicated by the circled symbol

Figure

2 gives

per FeBlm-NO The ratio ratio

for

a major

of the

parameters

centration.

results

10 to 30 base pairs a single

coil

helix.)

Strong

binding

is a substantially with

structure,

of calf

thymus

in Figure

1 are

in Figure

per molecule

larger

2 appears

and suggest and review

to DNA.of binding

FeBlm-NO

(Thirty

about

with

2 - 5 molecules ratio

than

DNA con-

20 and 60 bases

base

pairs

bleomycin

here

or

correspond

per

coil

A2 showing

for

to

of the

of Blm per base pair

observed

the

[DNA]/[FeBlm-NO]

and variable

one FeBlm-NO

data

versus

was 0.8 mM when of

to be between

of FeBlm-NO.

report

ligand

constant

structure.

plotted

The DNA concentration

with

DNA needed

to be in the altered

~10 and 25 mM when the ratios obtained

and Crooke of free

the

a (19).

FeBlm-NO'

DNA.

In seeking the

concentrations.

of the DNA helix

ratio

complex

A and D as defined

were

The endpoint

concentration

of the FeBlm-NO

of [DNA]/[FeBlm-NO]

Similar

>lO.

of the

fraction

of DNA and FeBlm-NO

the ratios

This

an estimate

an explanation the generality

for

the predominately

of the effect

531

is

recalled.

in-plane Both

effect thiols,

of DNA on such as

Vol. 91, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/N\ c= FeBlm

Fe + Blm z+ FeBI$

0.16

‘N

TC

0.14

60

‘N’

0

-

70

0.10 i4 a

.06

.06

30

E

.04

20

z a

.02

10

0

1

2

3

5

6

PH Figure and at versus at g=4 NaCl.

cysteine

3. 480 the and The

The pH dependence of the absorbance at 413 nm and 480 nm for Fe(3+)Blm nm for Fe(2+)Blm (left ordinate) in addition to the pH dependence peak to peak signal height (right ordinate) for high spin Fe(3+)Blm low spin Fe(3+)Blm at g=2.18. All solutions are made up in a .l M concentration of both Fe(3+)Blm and Fe(2+)Blm is approximately 0.2 UK

and glutatione

perturbin-plane at the

structural

pH dependence

(Figure

3).

high

(11,

(Figure

3).

nmr evidence and amine

nitrogen

process

and Fe(III)Blm.

(3)

states

in two steps

as interpreted

from

The low

spin

form

to Fe(II1)

relaxation.

Other it

is

which authors

linked

at pH 7 is is not have

also

Thus we looked

two oxidation

and then

We show here

oxidative

of FeBlm epr

converted

detected

reported

by reversible

spectra to

by epr a high

spin

equilibrium

structure.

However,

that

the

to these

changes.

fast

Fe(II)Blm

to observe

of iron

form

20).

to the native

Similarly,

length

spin

during

of Fe(II)Blm

dissociates

due to its

of FeBlm

changes

of binding

absorbance

an intermediate spectroscopy

and oxygen features

Fe(III)Blm

and corresponding

form

(7),

in the presence

the

dithionite

the

second

the

iron

ligands

step.

of dithionite

prevents Nevertheless,

center

of Fe(II)Blm

(20).

The reduced

532

shows

spectral

the same first

observation

Gupta

has presented

at pH 5.2 has lost number

at lower

of ligands

both leads

step wave-

recent

13C

imidazole to the

spin

Vol. 91, No. 2, 1979

state

change.

other

three

groups.

Their

are

weakly that plane

to the

in

iron;

to the

the

interaction

metal-ligand

That and are

Acknowledgement. This Cancer Institute Grant by NIH Grant RR-01008.

is

research CA-22184.

data

is

only

and the primary

dissociated

readily

of FeBlm with structure

these

the pyrimidine half

of Fe(II)

and the primary

interpret

are

iron

the binding

the pyrimidine

respectively.

bound

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

support

however,

and Fe(III)Blm

5.7 and 4.8

also

of Blm,

et al.,

of two groups Fe(I1)

data

ligands

Gupta

which

BIOCHEMICAL

the

imidazole

displaced DNA and with

conformationally

in this

and Blm to the and secondary in terms amine

first

and peptide by H+. thiols

Thus, this

amine

of the binding

(20).

The pHs at

dissociation nitrogen it part

is

step are

only

proposed

of the in-

perturbed.

was carried out with funds from The National The ESR work was done at a facility supported

References. 1. Sausville, E. A., Peisach, J., and Horwitz, S. G. (1978) Biochem. l7, 27402746. 2. Sausville, E. A. Stein, R. W., Peisach, J., and Horwitz, S. B. (1978) Biochem. 17, 2746-2754. T. (1978) J. Antibiot. 2, 1310-1312. 3. Sugiura, Y., and Kikuchi, G. R. (1979) FEBS Lett. 97, 47-49. 4. Oberley, L. W., and Buettner, 5. Sugiura, Y. (1979) Biochem. Biophys. Res. Commun. 82, 649-653. D., Rao, E. A., Petering, D. H., Sealy, R. C., and Antholine, W. E. 6. Solaiman, Int. J. Rad. Oncol. Biophys. (in press). 7. Antholine, W. E., and Petering, D. H. Biochem. Biophys. Res. Commun. (in press). 8. Sugiura, Y. (1978) J. Antibiot. 31, 1206-1208. 9. Nunn, A. D., and Lunec, J. (1978) European J. of Cancer 16, 857-900. 10. Sugiura, Y. (1979) Biochem. Biophys. Res. Commun. 88, 913-918. 11. Burger, R. M., Peisach, J., Blumberg, W. E., and Horwitz, S. B. (1979) Abs. Biophys. J. (2, part 2): 37a. D. H. (1979) Abs. Am. Chem. Sot., Sept. 12. Antholine, W. E., and Petering, Meeting. 13. Taketa, T., Muraoka, Y., and Nakatani, T. (1978) J. Antibiot 31, 1073-1077. 14. Antholine, W. E., Mauk, A. G., and Taketa, F. (1973) FEBS Letts 2, 199-202. 15. Rein, H., Ristau, D., and Scheler, W. (1972) FEBS Letts. 2, 24. 16. Trittelvitz, E., Sick, H., and Gersonde, K. (1972) European J. Biochem. 3, 578. 17. Yonetani, T., Yamamoto, H., Erman, J. E., Leigh, J. S., and Reed, G. H. (1972) J. Biol. Chem. 247, 2447-2455. 18. Perutz, M. F., Kimartin, J. V., Nagai, K., Szabo, A., and Simon, S. R. (1976) Biochem. 15, 378-387. 19. Strong, J. E. and Crooke, S. T. in Current Status and New Developments (1978) (edit. by Carter, S. K., Crooke, S. T., and Umezawa, H.) Academic Press. 20. Grupta, R. K., Ferretti, J. A., and Caspary, W. J. (1979) Biochem. Biophys. Res. Commun. 3, 534-541.

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