Effect of uv laser radiation on copper-proteins

Journal of Molecular Structure, 79 (1982) 443-446 Ekevier ScientiEcPublishingCompany,Amsterdam-PrintedinThe

EFFECT

OF

UV

LASER

RADIATION

N.EACCI.

P.FABENI,

Istituto

di Ricerca

ON COPPER-PROTEINS

R.LINARI

and

sulle

443 Netherlands

G.P.PAZZI

Onde

Elettromagnetiche

C.N.R..

de1

Firenze

(Italy)

mushroom

tyrosinase

ABSTRACT The and

results

of

Limulus

irradiation

polyphemus

In all the proteins

studies hemocyanin

we

have

of with

human a

considered

ceruloplasmin.

nitrogen

the

laser

absorption

(A= band

337.1 at

of dimer Type 3 copper centres,

decreases,

while an increase

at 280 nm is observed_

Moreover

a new hand

at ahout

ceruloplasmin.

sharp oscillations

are recorded

Finally

in ceruloplasmin.

400

are

presented.

nm, characteristic

330

of the absorbance

nm appears

of the absorbance

when the irradiation

nml

in irradiated

at 610 and 794 nm

is switched off.

INTRODUCTION

interaction

The

between

UV

and biological

radiation

Furthermore

of a great deal of investigations. lasers in biology and medicine of high photon densities the

Here we report

laser copper-proteins

we

containing

by an antiferromagnetically

macroscopic have

and

obtained

coupled copper[IIl

emission

337.1 nml lies Just inside the above band.

I A=

253.7 nml showed

conformational 330 nm with lasers typical

studies of mushroom a

changes

a mercury

lref.33. Moreover lamp

[ref. 51 produces of

Type

1

ph&oinactLvation

centres,

a

tyrosinase of

the

of UV

[ref. 11.

with a nitrogen

pair and show a characteristic

laser

irradiation

object

These centres are constituted

hand around 330 nm [ref. 21, so that the

Previous

the

diffusion

level

by irradiating

Type 3 centres.

been

the studies on the effect

molecular

absorption CA=

has

the increasing

has even more stimulated

at both

results

systems

line

of the nitrogen

with a mercury

enzyme molecule

the irradiation

lamp

mainly due

of ceruloplasmin

to

at

(ref.

41 or in the range 360 + 514.5 nm with different

sharp

decrease

so

suggesting

0022-2860/82/000O+l000~$02.76~1982

of

the

610

nm absorption

a direct energy transfer

Ekevier Scientific Publishing Company

band, between

which Type

is 3

444

and Type 1 capper sites [ref. 41. while the Type 2 centres by the photochemical

process, as it -an be deduced

seem to be unaffected

by epr measurements

[ref. 51.

RESULTS AND DISCUSSION Buffered

Limulus polyphemus

of human ceruloplasmin, mushroom tyrosinase and -5 hemocyanin (concentration -ICI M, pH = 71 were irradiated

at room temperature

using a nitrogen

frequency,

aqueous

solutions

and anaerobic

The three proteins nm:

an increase

during irradiation observed,

10 ns; repetition

IO+15 Hz; peak energy, 0.3Gl.O mJ1. The experiments

in both aerobic

~330

laser (pulse duration,

conditions

behave similarly of

the

former

and

(Fig.11. A partial

without

observing

as for the absorption a decrease

of the

permanent modifications

substantial

differences.

bands at. 280 and

latter

are

recorded

recovery of the Type 3 centre band is

while the increase of the 280 nm absorbance

so indicating

were performed

last s also after irradiation,

of the protein.

w

0

20

4b

60

60

100

t tmin)

Fig.1. Effect of nitrogen laser irradiation [peak power 40 kW1 on the absorbance at 280 nm [curve al and at 344 nm (curve bl in mushroom tyrosinase 10.4 mg/ml, pH = 7.111. The arrow indicates when the irradiation was switched off.

445

In irradiated

ceruloplasmin

could be assigned

a new absorption

to some copper chromophores

band arises at about 400 nm, which

arising from the partial denaturation

of the protein. Besides authors

the bleaching

of the 610 nm chromophore.

[refs. 4,51. we observed

the recovery behaviour

an oscillating

period after irradiation

I

I

20

40

by other

of the absorbance although

band at 794 nm. The oscillations

an hour and the latest ones show a periodlczty

I

behaviour

[Fig. 21. An analogous,

is shown by the absorption

0

already described

during

less evident. vanish within

of 657 minutes.

I

I

60

*

t(min)

80

Fig. 2. Time dependence of the 610 nm absorbance of Irradiated human CerUlOPlaSmln. Laser peak power, 45 kW; protein concentration, 1.6 A 10 M; pH = 7.03. The arrow indicates when the irradiation was switched off.

Reaction mechanisms equilibrium

Macroscopic

fluctuations

because the oscillations

and not for

behaviour

require

two

are attributable

only

redox process

should be observed for each absorptzon

to sulphur-to-copper

to

can be band

bands. On the other hand these two bands at 610 and 794 nm

involving

is required

using an intense

in the bulk of the solution

charge transfer transitions

so that Cu-S bond seems to be involved in the oscillation

work

systems far from

Crefs. 6,71 and it is likely we induce such a condition

source of radiation. excluded,

that give oscillatory

both Type 1 and Type

identify

the

main

species

3 centres taking

in Type 1 centres

mechanism.

could a part

A fluctuating

be suggested. in the process.

but further

446

REFERENCES 1 R. Pratesi and C.A. Sacchi cEds.1, Lasers in photomedicine and photobiology, Springer-Verlag, qerlin, 1980. 2 R. Malkin and B.G. Malmstrb'm, Adv. Enzymol., 33[19701177-244. 3 R.C. Sharma. R. Ali and 0. Yamamoto. J. Radiat. Res.. 20~19791166-195. 4 Y. Henry and J. Peisach. J. Biol. Chem.. 253C197837751-7756. 5 M. Herve, A. Garnier. L. Tosi and M. Steinbuch. Biochem. Biophys. Res. Comm.. aot197e1797-804. 6 G. Nicolis and .I. Portnow, Chem. Rev., 73[19731365-364. 7 A. Nitzan and .J. Ross, J. Chem. Phys.. 59[19731241-250.