Superoxide dismutase mimetic activity of cytokinin-copper(II) complexes

Vol. 137, No. 1, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

May 29, 1986

Pages 372-377

SUPEROXIDE DISMUTASE MIMETIC.ACTIVITY OF CYTOKININ-COPPER(I1) COMPLEXES Hideshi Faculty

Inoue

and Masaaki

of Pharmaceutical Hongo, Bunkyo-ku,

Received April

Hirobe*

Sciences, University Tokyo 113, Japan

of

Tokyo

10, 1986

SUMMARY: Dissociation constants of cytokinins, derivatives of Eirwhich form complexes with cupric ion, were determined by spectrophotometry and the stability constants of their copper complexes by pH titration. The values found for kinetin were 3.76, 9.96, 7.8, and 15.3 for pK1, pK2, logkI, and logB2, respectively, and those for 6-benzylaminopurine were, in the same order, 3.90, 9.84, 8.3, and 15.9. The copper(I1) complexes with kinetin and 6-benzylaminopurine had superoxide dismutase mimetic activity, and the reaction rate constants which were dete in_fd Jq polarography, were for kinetin and 1.5x10 rp M s for 6-Qenzylaminopurine at pH 9.8 and 25'C. 0

1986

Academic

PfeSE,

Inc.

Senescence particularly aging

and

(1).

include

accelerated

associated (CK),

radical compounds

radicals,

seem to play

an important

In higher

plants,

radical-associated

postharvest of

with

two

free

fruit cut

can

of plant

demonstrated

scavenger;

and free

parameters

of cytokinin

in

syndrome,

wilt

a group

occur

a multifactorial

oxy free

processes

phenomena

al.

is

spoilage,

flowers

(2-6),

be effectively

senescenceby cytokinins

Thus,

free

radicals

needs

to be elucidated.

that

CK interaction CK may

(ii)

CK may serve

formation

by

by enzyme

act

such

$1.50 0 I986 by Academic Press, of reproduction in any form

Inc. reserved.

372

Leshem

as a direct

free

oxidation

as xanthine

interaction

radicals

as an incipient

inhibiting

the

free

oxidase

*To whom correspondence should be addressed. Abbreviations: SOD, superoxide dismutase; DIPS, 3,5-diisopropylsalicylic acid; 6-BAP, 0006-291X/86 Copyright All riphrs

and

regulators.

(i)

in

senescence,

growth

ways:

role

leaf

retarded

with

radicals,

could radical

preventative of (7-9).

et

of

plant Frimer

CK, cytokinin; 6-benzylaminopurine.

free

purine et

3,5-

BIOCHEMICAL

Vol. 137, No. 1, 1986

al.

(8)

amines

demonstrated

that

by extraction

amine

bond in

superoxide

of

with

NOW we investigated with

copper

the

copper

complex

is

in

mimetic

activities

it

that

remove

chloroplast

is

formed

has

and

retardation

describes of CK-copper

Materials

the

with

the

CK so efficiently

senescence

communication

of but

solvent,

RESEARCH COMMUNICATIONS

reacts

hydrogen

and effectively

Since

This

the

the possibility

activity.

participate

superoxide

an aprotic

reacts

AND BIOPHYSICAL

(arylmethyl)-

a-carbon is

not

atom in the

clear

whether

in an aqueous

solution.

CK might

superoxide abundant SOD-like

form

by SOD mimetic

in copper, activity,

by quenching stability

a complex

a CK-

if it

may

of superoxide.

constants

and SOD

complexes.

and Methods

Materials and apparatus: Kinetin was obtained from Tokyo Chemical Industry Co., Ltd., 6-benzylaminopurine (6-BAP) from Wako Pure Chemical Industries, Ltd., -and copper(I1) sulfate (99.6 %) from Kanto Chemical Co., Inc. UV and visible spectra were obtained with a Hitachi 557 double-beam spectrometer. The pH was determined with a pH meter, Model PT-30 (Tokyo Kagaku Sangyo Co., Ltd.). Determination of dissociation constants: The -constants of kxetin and 6-BAP were determined photometric method. Solutions containing 0.1 mM of BAP and various concentrations of HCl or NaOH were ionic strength of the solution was maintained at NaCl. The UV spectra and pH were measured at 25'C.

dissociation by spectrokinetin or 6prepared. The 0.1 by adding

Determination of stability constants: The stability constants of CK-copper complexes were determined by Bierrum's method (10,111. Solutions containing 0.02 mM CuSO4 and 0.2 mM kinetin or 6-BAP were prepared and titrated with standard NaOH (0.01 N) at 25'C. The ionic strengths of the solutions were maintained at 0.1 by adding NaCl. Determination SOD mimetic activities --of SOD mimetic activity: of copper complexes were polarographically determined (12,13) using a Yanaco voltammetric analyzer Model P-1000. A copper complex was prepared in solution by mixing the -$ igand an-$ vy$; A copper complex concentration of between 10 and 10 achieved by subsequent addition of the copper complex to a buffered solution of 0.1 M sodium borate containing 0.5 mM triphenylphosphine oxide. After each addition, the height of the polarographic wave was recorded. The rate constant of dismutation was calculated by measuring the increment of the polarographic wave as a function of the copper complex concentration, according to the equation of Koutecky et al. (14). ((7.421+,)-7.42)/(2.25-l.25(Il/Id))=k[C]t 11 and Id are the mean limiting currents eithe 2in the presence 373

Vol. 137, No. 1, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

or absence of the copper complex, respectively, of mercurY electrode, [Cl the concentration of and k the rate constant of the reaction.

Results The

formation

was observed formed

in

of

identified

composition of

determine

of

the

Cu*+

copper

was 2:1,

a complex

by 6-BAP

dissociation

1).

measurement,

release complex

the

of proton

constants

variation

(15,16).

The

of

a compound

having

the

In

aqueous

was also

absorption

the

observed was too

continuous

variation

6-BAP

with

the

when the

ratio

of

6-BAP

of kinetin

by spectrophotometric and the

used

the

of Bjerrum

2),

to draw and the

and 6-

(Table

I).

results

for

solutions

the

formation

stability

curves

constants

V(632nm)

CuCl2 6BA

Fig.1,

Continuous variation

purine complex in Total

0 1~3

concentration

2 .6

:t :: mole fraction

plot

:i

for

copper(6-benzylamino-

ethanol. of CuClz and 6-BAP was 1.0

374

and

method

pH titration

Cu*+ and CK were

small

formation

(pKI

and pK2)

by

method.

to be Cu(6-BAP)2.

constants

(Fig.

solution,

was expected

containing method

was

from

was maximum

composition

determined

and Cu*+

the

by the

The compound

6-BAP

and Cu*+ but

composition

The dissociation BAP were

existence (Fig.

as the

However,

of

continuous

Cu(6-BAP)*

the

CK and Cu*+ in ethanol

measurements.

of

the

spectrophotometric to

between

by reaction

method

indicated

formation

complexes

solution

by the

results

and Discussion

by spectrophotometric ethanol

tg the drop time the copper complex

mM.

The

by were

Vol.

137,

No.

BIOCHEMICAL

1, 1986

Table I. constants

Dissociation of their

AND

BIOPHYSICAL

+

9.96 +

3.76

6-Benzylaminopurine

3.90 + 0.10

equilibria B2 is

1 and the

overall

(Table 2 are

the

inhibited

(data

superoxide

polarographic

(i.e.,

the

procedure

logkl

lW2

7.8

15.3

8.3

15.9

the

copper (12,13)

and

K2 = klk,).

(1) (2)

SOD mimetic The rate

following

respectively,

nitrobluetetrazolium

shown).

and

and k2,

[cu (CK) I + k2 CK- +---=CU(CK)~

the

not

by kl

of

kl

of

by CU(~-BAP)~,

expected

between

reduction

constants

constant

+ CK-M-?

0.04

9.84 + 0.19

The

expressed

(Cu(CK))+'+

As

0.02

I).

stability

Cu2+

was

PK2

Kinetin

(10,ll)

COMMUNICATIONS

constants of cytokinins and stability copper complexes (25"C, ion strength 0.1) PK1

calculated

RESEARCH

by activity

of

constants

complexes (Table

of were

II).

the the

Ko2

was

complex reaction

determined

The

SOD

by

mimetic

ii

Fiq..

Formation

25-C and ionic

curves

strength

of copper(cytokinin 0.1. 375

complexes

at

Vol. 137, No. 1, 1988

BIOCHEMICAL

Table

II.

AND BIOPHYSICAL

Rate constants for reaction with coppercompounds

SOD

cuso4

of superoxide

k, M-is-l

PH 9.8

(1.8 + 0.2)

x 10'

this

work

7-9

2

x 109

ref.

22

9.8

(1.7 + 0.1)

x 107

this

work

5.0

x 108

ref.

23

6 C~(kinetin)~

9.8

(2.3 + 0.1)

x lo7

this

work

Cu(6-benzylaminopurine)2

9.8

(1.5 + 0.2)

x 10'

this

work

Cu(salicylic

9.8

(2.0 + 0.2)

x lo7

this

work

activity

acid)2

of

complexes

at pH 9.8 to that SOD-like The

rate

is pH

more

of

living

core

copper

complexes.

superoxide

is more

site,

than of

Native

SOD is

weight

and as a result

low

affect

is

at

action,

it

did

of

of

stable

a membrane

(20)

in

a

enzyme thought

376

to

therefore,

in

in

at

a hydrophobic

SOD oxygen

has a high

to be less

effective

catalyzes toxicity. molecular against

site. lipophilic

weight, such

or

depresses which

solvent

and reactive

cytosol.

and

may be

considered

superoxide,

may be more

the

complex

is

the

affect

in an aprotic

protonation

of

at pH 7

because

The CK-copper

a hydrophobic

molecular

but

(17).

decay

105M-1s-1

SOD

(19),

to have

pH 7.5)

and reactive

superoxide

a water-soluble

present

SOD-mimetic

in

e.g.,

at

pH 9.8 and

and longevity

protein,

Recently,

M-is-l

at pH 9.8.

The

superoxide

is known

at pH 7 than

stable

solvent.

disproportionation

superoxide

not

the

systems,

of

does

reactivity

hydrophobic

, which

equivalent

second-order at

residues

with

the

M"s"

lysine

a protic

control

acid)2

of

reactive

in

lo3

level

Superoxide than

CK and Cu2+ were

for'spontaneous

about

participation activities

between

(k = (1.64+0.15)x10g

constant

The

(18).

formed

of Cu(salicylic

activity

superoxide

with

RESEARCH COMMUNICATIONS

as

CLI(~,~-DIPS)~

copper

complexes have

been

a

BIOCHEMICAL

Vol. 137, No. 1, 1986

attracting

have

Sorenson

attention.

complexes

including

copper

anti-inflammatory

found

that

activity

CK forms and is

CU(~,~-DIPS)~

a complex

be

with

that

aspirinate than

copper,

complex to

RESEARCH COMMUNICATIONS

reported

stronger

the able

(21)

anthranilate,

activity

lipophilic, and

AND BIOPHYSICAL

may

participation

the

which

and ligands.

copper 3,5-DIPS As we

has SOD mimetic

possess

activity in

like

senescence

retardation. References 1. Fridovich, I., 1976, "Free Radicals in Biology" Vol. 1, Edited by W.A. Pryor, Academic Press, New York, pp. 239-277 2. Apelbaum, A., Wang, S., Burgoon, A.C., Baker, J. and Lieberman, M., (1981) Plant Physiol., 67, 74-79 3. Chia, L.S., Thompson, J.E. and Dumbroff, E.B., (1981) Plant Physiol., 67, 415-420 4. Physiol., 49, 757-763 Frenkel, C., (1972) Plant 5. Grossman, s. and Leshem, Y., (1978) Phisiol. Plant., 43, 359362

6. Legge, R-L., Thompson, J.E. and Baker, J.E., (1982) Plant Cell Physiol., 23, 171-177 7. Leshem, Y.Y., (1984) Can. J. Bot., 62, 2943-2949 8. Frimer, A.A., Aljadeff, G. and Ziv,?., (1983) J. Org. Chem., 48, 1700-1705 9. Leshem, Y.Y., Wurzburger, J., Grossman, S. and Frimer, A.A., (1981) Physiol. Plant., 53, 9-12 10. Bjerrum, J., "Metal Ammine Formation in Aqueous Solution," P. Hasse and Son, Copenhagen, 1941 11. Calvin, M. and Wilson, K.W., (1945) J. Am. Chem. Sot., 67, 2003-2007 12. 13. 14. 15. 16.

Rigo, A., Viglino, P. and Rotilio, G. (1975) Anal. Biochem., 68, 1-B Brunori, M. and Rotilio, G., (1984) Methods in Enzymology, 105, 22-35 fitecky, J., Bridcka, R. and Hames, V., (1953) Collect. Czech. Chem. Commun., 18, 611-615 Job, P., (1928) Ann. Chim., 2, 113-203 Vosburgh, W.C. and Cooper, G-R., (1941) J. Am. Chem. Sot., 63,

437-442

Eunes, M., Lengfelder, E., Zienau, S. and Weser, U., (1978) Biochem. Biophys. Res. Commun., 81, 576-580 18. Bielski, B-H-J., (1978) PhotocheF Photobiol., 28, 645-649 19. Argese, E., Rigo, A., Viglino, R., Orsega, E., Marmocchi, F., Cocco, D. and Rotilio, G., (1984) Biochim. Biophys. Acta, 17.

787, 20.

21. 22. 23.

205-207

Asada, K., Takahashi, M. and Hayakawa, T., 1983, “Oxy Radicals and Their Scavenger Systems”, vol. I, Edited by G. Cohen and R.A. Greenwald, Elsevier Biomedical, New York, PP. 240-245 Sorenson, J.R.J., (1984) Chem. Britain, 1110-1113 Forman, H.J. and Fridovich, I., (1973) Arch. Biochem. Biophys., 158, 396-400 Klug-Roth, D. and Rabani, J., (1976) J. Phys. Chem., 80, 588591

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