Hydrated

Electron-Induced

InactivationofTyrosinase

Aqueous Solution by Exposure to Cobalt-60 Gamma-Rays

HIROAKITERATOinPublication

(1)

of

the

ThesisHydrated-Induced Inactivation of Tyrosinase in Aqueous

Solution by Exposure to Cobalt-60 Gamma-Rays. I. Cresolase
Activity.
Terato, H. and Yamamoto,o. (1994)

Biochemistry and Molecular Biology International, 34, 295300(2)

Hydrated-Induced Inactivation of Tyrosinase in Aqueous

Solution by Exposure to Cobalt-60 Gamma-Rays. II.
Catecholase Activity.
Terato, H. and Yamamoto,o.

(1994)

Biochemistry and Molecular Biology International, 34, 301307

CONTENTS

General Introduction--------------------------------------l

Chapter 1.
Hydrated-Induced Inactivation of Tyrosinase in Aqueous
Solution by Exposure to Cobalt-60 Gamma-Rays. I.
Cresolase Activity-----------------------------------S

Chapter 2.
Hydrated-Induced Inactivation of Tyrosinase in Aqueous
Solution by Exposure to Cobalt-60 Gamma-Rays. II.
Catecholase Activity--------------------------------20

General discussion---------------------------------------38

Acknowledgements-----------------------------------------42

GENERAL

Ionizing

radiation

INTRODUCTION

attacks

organisms

randomly

andinjures various molecules in the cells. For long time DNA
has

been

thoughttobeaprimary

targetonradiation-induced injuries. However the radiation-induced damages ofother cellular components such as membranes

and proteins

including enzymes, must be important for cell death. 1)
Theradiationeffectsonproteinmoleculesare1.

degradation of amino acid residues, 2. breakage of peptide
bonds, 3.

dissociation of hydrogen bonds,

disulfidebonds

molecules. 2)

and

5.

of

these

All

productionof

events4.

incision of

cross

cause

linkinginconformationalchange, fragmentation and aggregation, or raise proteolytic
susceptibilityincellularcondition. 2,

However,3)

thegeneralization of radiation effects on proteins seem to bedifficult, because

proteins have different construction,differentandactivity

variousmechanisms

ofactivity

control. 2)Inthepresent

study,

theauthorsubjected

theradiation effect on tyrosinase. Tyrosinase (EC 1. 12. 18.
1)

is a copper-containing protein,which is one of well-known metalloenzymes. 4-10) The present study is to clarify
what the main active species inactivates tyrosinase. Cells
contain much amount of

H20.

The

weightof

H20

occupies

about 70 % of whole living cells. Therefore, main effects
induced by radiation must be considered as indirect eventswith some kinds of active species derived from H20. It iswellknown that themainradiolytic

1

activespeciesarehydroxyl radicals(. OH :

G =2. 7 )and hydrated electrons(e-aq: G = 2.6).11,12) Other species such as hydrogen atoms( · H: G = 0.6) and hydrogen peroxides

G = O. 7 ) are(H202:

low yields .11, 12)previously many investigators studied the radiationinduced inactivation of enzymes. 2)

All of them presented

that the main active species which inactivates enzymes was·OH.

However,

there

is

no

studies

on

the

focus

into

radiation-induced inactivation of metalloproteins. Then theauthor tried to study on the radiolytic inactivation oftyrosinase. Tyrosinase has two enzymatic activities. One ismonophenolmonooxygenase activity(cresolase activity)

otheractivityiso-diphenoloxidaseand(catecholase

activity) .4-10) Which of ·OH and e-aq is the main species tocause inactivation of this enzyme? The present study is acomparisonofradiation

activities.

Itiseffectinteresting

on

both

whethertheenzymatic
inactivation

mechanism is the same or not for these two activities oftyrosinase.
Tyrosinase participates in the metabolic pathways ofcatechol and catecholamine. 4-10) These pathways are related
with

the

melaninproduction

inanimal

skins

and

plant

tissues. These essential enzyme activities seem to protect
someorganisms

ultraviolet(UV)againstirradiation.

is

able

also

to

In

addition,cause

H20-derived

radicals .13) Sun light containing UV raises amount of H20derived active species in human skin cells. Melanin is a
protector against UV in human skin. Therefore,that produces melanin,tyrosinase

seems to be important to protect2

against UV. From this point of view, inactivation yields ofboth activities of tyrosinase are compared with those ofother enzymes reported.

REFERENCES:

1) Bacq, Z. M. and Alexander, P. (1961) Fundamentals ofRadiobiology. pergamom Press, New York.
2) Yamamoto,o.

(1992) Effect of Radiation on protein

stability; in Stability of Protein Pharmaceuticals, Part
A, Chemical and Physical pathways of Protein Degradation(eds. Ahern, T. J. and Manning, M. C.) pp. 361-421,
Plenum Press, New York.
3) Davies, K. J. A. (1987) Protein damege and degradationby oxygen radicals. J. BioI. Chern., 262. 9895-9901
4) Mason, H. S. (1956) Structure and functions of thephenolase complex. Nature 177, 79-81.
5) Dressler, H. and Dawson, C. R. (1960) On the nature andmode of action of the copperprotein tyrosinase. II.
Exchange experiments with radioactive copper and thefunctioning enzyme. Biochim. Biophys. Acta 45, 515-524.
6) Shimao, K. (1962) Protein purification and kinetic
studies of mammalian tyrosinase. Biochim. Biophys. Acta
62, 205-215.
7) Pomerantz, S. H. (1963) Separation, purification, andproperties of two tyrosinases from hamster melanoma. J.
BioI. Chern. 238, 2351-2357.
8) Jolley, Jr., R. L., Robb, D. A. and Mason, H. S. (1969)
The multiple forms of mushroom tyrosinase: Associationdissociation phenomena. J. BioI. Chern. 244, 1593-1599.

39) Jolley, Jr., R. L., Nelson, R. M. and Robb, D. A.
(1969) The multiple forms of mushroom tyrosinase:
Structural studies on the isozymes. J. BioI. Chern. 244,
3251-3257.
10) Duckworth, H. W. and Coleman, J. E. (1970)
Physicochemical and kinetic properties of mushroom
tyrosinase. J. BioI. Chern. 245, 1613-1625.
11) Buxton, G. V. (1982) Applications of water radiolysis
in inorganic chemistry; in The Study of Fast Processes
and Transient Species by Electron Pulse Radiolysis (eds.
Baxendale, J. H. and Busi, F.) D. Reidel, Dordrecht, pp.
267-28712) Spinks, J. W. T. and woods, R. J. (1976) An
Introduction to Radiation Chemistry, 2nd ed., John Wiley& Sons, New York.

13) Ito, A. and Ito, T. (1983) possible involvement of
membrane damage in the inactivation by broad-band near-uv radiation in Saccharomyces cereviciae cells.
Photochem. Photobiol., 37, 395-402

4

CHAPTER 1
HYDRATED-INDUCED INACTIVATION OF TYROSINASE IN AQUEOUS
SOLUTION BY EXPOSURE TO COBALT-60 GAMMA-RAYS.
I. CRESOLASE ACTIVITY

5ABSTRACT

TyrosinasepHsolution,
activities

(0.2

6.5)

was

mg/ml

which

of

has

irradiatedM phosphate

0.1

cresolase

with

and

60Co

buffercatecholase

gamma-rays.

The

cresolase activity was measured at varying radiation doses
under various atmospheric conditions.

Do wasfound to be

350 Gy in N2-saturated solution. The hydroxyl (OH) radicalhas been shown to be main species involved in radiationinduced

inactivation

of

most

of

enzymes.

However,OHradical scavengers, t-BuOH and MeOH, had no effect in thisstudy. In addition, O2 that acts generally as an enhancerof OH radical-induced inactivation also had little effect.
On

the

other

scavenger,

hand,

N20

as

a

(e-~)

inactivation.

the

main

species

involved in inactivating the cresolase activity,

reducing

results

indicate

inhibited

electron

the

These

and Cu++ markedly

hydratedthat

Cu++ as the active center.

6

e-~

is

INTRODUCTION

Tyros inase (EC 1.
protein

that

12 .

has

18 .

1 ) is a copper -containing

two

enzymaticmonophenolmonooxygenase activity
o-diphenoloxidase

activity

activities,(cresolase activity)(catecholase

activity).

and
The

enzymes are widely distributed in a lot of organisms frommicrobials, plants and animals. It has been reported that
copper contained in this enzyme is the essential moiety of
the active center .1-7)

The present report deals with theeffect of ionizing radiation on tyrosinase in relation to
the amino acid synthesis in cells and melanin production inhuman skin.
Inradiation

biology,

one

of

main

objectsistoclarify radiation-induced effects on molecules constructing
organisms

such asDNA,

proteins(including

enzymes)andothers. When the aqueous systems are exposed to ionizing
radiation,
radicals

two
and

main

e-~

activederivedspeciesfrom waterareproduced;molecules. 8)

It

OH
is

known that enzymes are inactivated mainly by OH radicals
but not bye-~.9)In addition, Cu* has been also shown to

be a sensitizer for the radiation-induced inactivation of
enzymes .10, 11)
The purpose of this study was to determine whether theradiation-induced inactivation of tyrosinase takes place inthe same manner of most of proteins which are inactivatedmainly by OH radicals. As the firstactivity was studied.

7

step,

the cresolase

MATERIALSANDMETHODS

Materials:
MushroomIII,tyrosinase(lyophilized,

electrophoreticallypure)

Chemical Company (U. S. A.
from

Wako

Pure

compounds used;wassalt-free,purchasedfromIndustries(Japan) .

cupric sulfate and chloride,

Copperand cuprous

chloride were from Wako Pure Chemical Industries

distilledwater

used were of

was

used

Sigma

). L-Tyrosine and tyramine were

ChemicalAll the chemicals

grade

analyticalthroughout(Japan).

grade.

these

Triplystudies.

N2 0(Showa Denko, Japan), N2 and O2 (Nihon Sanso, Japan) gases
used were at high grade (>99.99 %).

Irradiation:
Tyrosinase solutions were prepared in 0.1 M phosphatebuffer, pH 6.5, to a final concentration of 1.56 x 10-6 M(0.2 mg/ml). The solutions were saturated with N2,
N20 gasesby bubbling for 15 min,respectively,

02

and

prior togannna-ray irradiation, and t-BuOH (10- 3 M and 10-2 M final
concentrations),

MeOH(10- 3concentrations) ,andcopperconcentrations)

were

also

M

and

compounds
added

to

10-2

M

final

( at

differentthesolutions.

Tyrosinase solutions (1 ml) in Pyrex glass tubes (0 1.2 cm
x 9 cm) were irradiated with 60CO gannna-rays (Shimadzu RT10000S)

at

a

dose

rate

atmospheric conditions

at

of

200

0 °C.

with a Fricke dosimeter .12)

8

Gy/minunder

Dosimetry wasvariousperformed

Enzyme Assay:The

cresolase

activityoftyrosinasewasmeasuredusing tyrosine and tyramine as substrates according to themethodreportedpreviously. 13)

Thereaction

consisted of 2 ml of substrate solution (2 mM).

mixture1.8 ml of

0.1 M phosphate buffer, pH 6.5, and 0.2 ml of the enzyme
solution. After incubation of the mixture at 37°C for 5min, the absorbance of dopachrome produced was measured at
480 nm by Hitachi 124 spectrophotometer. The data presented
here

are

averages

of

not

less

determinations.

9

than

three

individual

RESULTS

Tyrosinasewasexposedtogamma-raysinaqueoussolution under N2 and the cresolase activity was measured
using tyrosine

asamonophenol

substrate

(Fig.

1 ).

The

enzymatic activity decreased in an exponential manner.
( 37% survival dose)value wasfound to be

survival curve with tyramine used as

350

another

Gy.

Do

The

monophenol

substrate was almost the same to that with tyrosine (datanot shown).
No effect of gamma-ray irradiation on Michaelis-Menten
constant,

Km,

was

indicating

noloweringofaffinity

enzyme as shown in Fig.

2.

The experiment with tyramine

found

for

cresolasewithactivitythewithirradiatedused as another monophenol substrate was almost the same tothat with tyrosine (data not shown).
Effectsof02,

t-BuOH,

MeOH

and

N20

on

the

inactivation of the enzyme activity induced by radiation
are also shown in Fig. 1. Oxygen showed little enhancementof the inactivation of the enzyme, and no effect of t-BuOH
and MeOH,

both OH radical scavengers·CH2(CH3)2COH + H20, CH30H + ·OH-+« CH3) 3COH

+ · OH-+·CH20H + H20), were also

found ; that is , these alcohols never protected the enzyme
against inactivation. However, the presence of N20 that is
an e-aq scavenger
blockedtheinvolvedin the(N20 + e-aq

inactivation.

-+

• OH

+ OH- + N2) markedly

Therefore,

inactivation ofmust be e-aq but not OH radicals.

10

this

the

main

species

enzymatic

activityo"o-~i::..
'">..

... 50
>

u

o

-(:)_
--e-..

-0--

.",

•

...o
u

E

>..
c:

N

Q)

Q)

>

.f-

a

Q)

10

~

o

200

400

Dos e, Gy

Pig. 1.
Survival curve of oresolase activity of tyrosinase in H2 saturated solution (0.2 Mg/ml of phoaphate buffer, pH 6.5) irradiatedwith 50Co gamma-rays, and the effects of presence of MeOH (10-2 M), tBuOH (10-2 M), O2 (aaturated), H20 ( ••turated) and Cu++ (10- 4 M). BE:
<2 %.

11

>

"

,
,,

,

,
,
, ,'" '" '",,":- -- ..... -o
",'

6

2

8

10

1/( 5 J. x 10- 3 M- 'Fig. 2.
Lineweaver-Burk plot of cresolase activity of tyrosinase
in N2 -saturated .olution ( 0.2 mq/ml of phosphate buffer, pH 6.5)
irradiated with ~Co qamma-ray ••12

Table. 1.
Effects of Cu++
and
Cu·
on
the
gamma-ray-induced
inactivation of cresolase activity of tyrosinase (0.2 mg/ml of 0.1 M
phosphate buffer, pH 6:5). Tyrosinase a8 substrate: 2 mM; irradiation
dose: 200 Gy; SE: < 2 ,.

Copper ion

Conc. of ions (I) Enzyaatic activity (')

Control (non- irradiated)Ni)
Cu' (chloride)Cu'· .. (chloride)Cu" • (sulfate)
Cu. t· (sulfate)
Cut I (sulfate)

10 . ~
10 ~] O· ~

10 5] 0. 6

13

100.0
47.5
50.3
95.4
95.0
92.0
89.2

Cu* markedly protected against the radiolytic inactivationof cresolase activity(Fig.

1 and Table1).

Cu* gave ahigh degree of protection even at a concentration of 10-6
M,

and there waschloride.

It

inactivation.

no difference

suggestsOn

thecopperother

between thesulfateandions

affectthehand,protection against the inactivation.

14

Cu+

only

conferredlittleDISCUSSION

The ionizing radiation-induced inactivation of many
enzymes has been studied previously. The main participant
in the inactivation in aqueous system was OH radical in all
casespresented. 9)

However,intheinactivationofcresolase activity of tyrosinase in the present study,t-

BuOH and MeOH as the OH radical scavengers did not affectthe inactivation,

while N20 as the e-aq scavenger had amarked protective effect on theinactivation

(Fig.

1).

Therefore the main participant was found clearly to be e-aq
for the inactivation of cresolase activity of tyrosinase.
Figure. 1

also shows that the hit number is one, andfigure. 2 shows that the KmS does not change after various
dose irradiations. Therefore, there must be only one active
center as the inactivation target in the enzyme molecule,which is damaged with the reducing ability of e-aq but notwith the oxidizable OH radical.
In living cells, many kinds of enzymes contain metal
ions. Metal ions play roles such as the ionic binder tokeep molecular conformations and the center of catalytic
activity.

Mushroomtyrosinasemolecule

contains

copperions. Mason 1 ) proposed a configuration of the copper-oxygen
complexoftyrosinase

and

the

oxidationmechanism

monophenol as follows:
Protein-Cu+2 + 02 ~ protein-Cu2-02(I)

Protein-Cu2-02 + monophenol + 2H+~

protein-Cu#2 + o-diphenol + H20

protein-Cu#2 + 2e ~ Protein-Cu+2

15

(II)
(III)

of

In the present study,
Cu H

and

no

protective

activity were founda marked protective effect ofeffect

(Fig.

of

Cu+

on

the

cresolaseand Table 1). Therefore,

1

theabove formula must be replaced as follows:( IV)
Protein-Cu+2-02 + monophenol + 2H+-+

Protein-CuH2 + o-diphenol + H20(V)

The inactivation mechanism of cresolase activity is(VI)
The protection effect of Cu H must be due to reaction (VII)(VII)
or the repair must be due to reaction (VIII).
Protein-CuHCu+ + Cu H

Protein-CuH2 + Cu+-+(VIII)

It is concluded that the inactivation of cresolase activity
is the reduction of Cu H as the active center to Cu+ by e-~.
On the other hand, metal ions such as Fe H and Cu H areable to produce OH radicals by Harber-Weiss cycle (02- + Mn-+

02 + MIl-1,

Therefore
inducedthisMIl-1 + H20 2

and 2.

Cu H

can

be

inactivation

study,

Cu H

a

of

has

-+

MIl + OH- + · OH ) • 11, 12)sensitizer

for

the

non-metalloenzymes.

noeffectonradiationHowever,enhancement

of

in
the

inactivation, but plays a role as a protector of enzymatic
activity against radiation. This result supports OH radical
is

not

the

main

speciestoinactivate

thecresolase

activity of tyrosinase.
The enhancement effect of O2 has been observed in thecase of OH-induced inactivation of enzymes. 9) The schema of
the enhancement is that the protein radical produced by OH
radicalreacts

with

O2

and

16

resultsaprotein-peroxide

radical.

Then

the

protein-peroxide

protein.

In

thisobserved (Fig.

1).

It supports

otherstudy,

radicallittlereacts

with

of

was

effectthat the02

inactivation ofcresolase activity is induced by e-aq but notby OH.

In

addition, 02 reacts with e-aq and produces superoxide (e-aq +O2 -+ 02-). In this case, the main active species is 02- forinactivation. However,

little enhancement of 02 indicateslittle participation of 02- in the inactivation.
Finallyitisconcludedthat theactivecenterofcresolase activity is damaged with the reducing ability of
e-aq but not with the oxidizable OH radical. Especially theactive center as the main target is cupric ion.
tyrosinaseisconstituted

of

two

sets

of

MushroomLand

H

subunits 14 ), each LH of which contains two cupric ions, but
only

one

ion

reduction

by

e-aq

inactivation of cresolase activity.

17

must

be

cause

the

REFERENCES

1) Mason, H. S. (1956) Structure and functions of thephenolase complex. Nature 177, 79-81.
2) Dressler, H. and Dawson, C. R. (1960) On the nature andmode of action of the copperprotein tyrosinase. II.
Exchange experiments with radioactive copper and thefunctioning enzyme. Biochim. Biophys. Acta 45, 515-524.
3) Shimao, K. (1962) Protein purification and kinetic

studies of mammalian tyrosinase. Biochim. Biophys. Acta
62, 205-215.
4) Pomerantz, S. H. (1963) Separation, purification, andproperties of two tyrosinases from hamster melanoma. J.
BioI. Chern. 238, 2351-2357.
5) Jolley,

R. L. Jr., Robb, D. A. and Mason, H. S. (1969)

The multiple forms of mushroom tyrosinase: Associationdissociation phenomena. J. BioI. Chern. 244, 1593-1599.
6) Jolley,

R. L. Jr., Nelson, R. M. and Robb, D. A.

(1969) The multiple forms of mushroom tyrosinase:

Structural studies on the isozymes. J. BioI. Chern. 244,
3251-3257.
7) Duckworth, H. W. and Coleman, J. E. (1970)

Physicochemical and kinetic properties of mushroom
tyrosinase. J. BioI. Chern. 245, 1613-1625.
8) Spinks, J. W. T. and Woods, R. J. (1976) in An

Introduction to Radiation Chemistry, pp. 247-359, John
Wiley & Sons, New York.
9) Yamamoto,o. (1992) Effect of radiation on protein

stability; in Stability of Protein Pharmaceuticals, Part

18

A, Chemical and Physical Pathways of Protein Degradation(eds. Ahern, T. J. and Manning, M. C.) pp. 361-421,
Plenum Press, New York.
10) Anbar, M. and Leitzki, A. (1966) copper-induced
radiolytic dactivation of a-amylase and catalase.
Radiat. Res. 27, 32-40.
11) Winstead, J. A. and Mcnees, D. E. (1974) Effects ofcupric ions on the radiolytic inactivation of lactate
dehydrogenase. Radiat. Res. 59, 466-474.
12) Fricke, H. and Hart, E. J. (1966) Chemical dosimetry;in Radiation Dosimetry Vol.

II

(eds. Attix, F. H. andRoesch, W. C.) Academic Press, New York. 2nd ed. , pp.
202-203.
13) Sharma R. C., Ali, R. and Yamamoto,UVo. (1979) Effect oflight on biological activity of tyrosinase in buffer

solution. J. Radiat. Res. 20, 186-195.
14) Strothkamp, K. G., Jolley, R. L. Jr. and Mason, H. S.
(1976) Quaternary structure of mushroom tyrosinase.
Biochem. Biophys. Res. Commun. 70, 519-524.

19

CHAPTER 2.
HYDRATED-INDUCED INACTIVATION OF TYROSINASE IN AQUEOUS
SOLUTION BY EXPOSURE TO COBALT-60 GAMMA-RAYS.
II. CATECHOLASE ACTIVITY

20

ABSTRACT

Tyrosinase (0.2 mg/ml) was irradiated with 60CO gammarays.

The

catecholase

radiation doses

activity was

under various

measuredatvarying

atmospheric conditions.

Do

was found to be 1.25 kGy and hit number was found to be 2
in N2-saturated solution. OH radical scavengers, t-BuOH and
MeOH, had no effect. 02 that is an enhancer of OH-induced
enzyme inactivation had little effect. However, N20 as a eaq

scavenger

and

Cu++

markedly

protected

againsttheinactivation indicating that e-aq was the main species toinactivatethechromatography,

enzymatic

activity.

By

Ultrogelit was found that the enzymatic activity

was lost when this enzyme dissociated into its subunits.
Thus,

it

was

concluded

that

the

radiation-induced

inactivation was due to the reduction of Cu++ as the active
centerandthechelaterwith

dissociation.

21

e-aq

followedbytheINTRODUCTION

Tyrosinase (EC 1.
protein

that

has

12.

two

18.

1)

enzymaticmonophenolmonooxygenase activity
o-diphenoloxydase

activity

is a copper-containing
activities,

these

are

(cresolase activity)and(catecholase

activity).

The

enzymes are widely distributed in a lot of organisms frommicrobials,
important

plants
roles

in

and

animals,andcontributed

the

the

metabolism

of

amino

and

acids

producing of melanin. 1-7) Radiation-induced inactivations ofthecresolase

activity

has

been

studiedaspreviously

reported. 8)Inaqueoussystems

exposedactive species are produced;

toradiation,

two

main

OH radicals and e-aq derived

from water molecules. 1-7) OH radicals have been reported tobe the main active species responsible for the inactivationof most enzymes on irradiation. 9, 10) In our preceding paper,
however,theauthorshavereported

that

the

cresolase

activity of tyrosinase was inactivated by e-aq but not by OH
radicals.

The present study performed to determine which

species acts as

the main inactivator of the catecholase

activity which is different from the cresolase activity inthe same molecule of tyrosinase.
The preceding study

showedthat

cupricions(Cu++)

protected cresolase activity from inactivation 8 ), contrary
to that Cu++ has been shown to sensitize most enzymes toexposure to radiation. 11, 12)

The authors also studied here

whether Cu++ acts as a protector or a
case

of

catecholase

activity

22

sensitizer in theinactivation.

In

cresolaseinactivation,

Cu++ may play roles

to both scavenge e-aq

and/or repair reduced Cu+ in tyrosinase molecule. B)

23

MATERIALSANDMETHODS

Materials:
L-a-(3, 4-Dihydroxyphenyl) alanine (dopa) and catechol
were purchased from Wako Pure Chemical Industries (Japan).
Mushroom tyrosinase and other chemicals used were the sameones described in our proceeding paper. a) All the chemicals
used were of analytical grade. Triply distilled water wasused throughout these studies.

Irradiation:
Preparation of enzyme solutions and irradiation weremade by the same method described in our preceding paper. 8)

Enzyme Assay:
The catecholase activity was measured using dopa andcatechol as substrates according the previously reported
method. I3 )

By

the

same

method,cresolase

activitywas

measured using tyrosine for comparison. The data presented
here

are

averages

of

not

less

than

22

(LKB,

Sweden)three

individual

determinations.

Gel Chromatography:
Ultrogel

AcA

was

used

for

gel

chromatography. 2.5 ml of tyrosinase solution (0.2 mg/ml of
0.1 M phosphate buffer, pH 6.5) was applied onto the column
(~

0.9

cm

x

45

cm)

after

irradiation

undervariousatmospheric conditions and was eluted with 0.1 M phosphatebuffer, pH 6.5). The enzymatic activity of each fraction24

(2.5 ml) was assayed using dopa for catecholase activity
and

using

tyrosinechromatographytyrosinase

was

mixed

for

cresolase

also

performed

withcysteine

activity.
for

(10-4

M)

a

Ultrogelsolution

withoutofgamma-

irradiation.

Construction of Amino Acids:
Twoirradiated

ml

of

and

tyrosinase

solution

irradiated with

(0.2

gamma-rays

mg/ml)
was

non-

dried

in

vacuo and re-dissol ved in 800 111 of 5. 7 N HCl in a glass
tube. Air in the tube was replaced with N2 gas and was thensuction-sealed using an aspirator. The solution was heatedat 110 °C for 20 h, and the hydrolyzed solution was driedinvacuo.

After

analyzing buffer I

dissolving
pH

2.2 I

in

the

100
sample

111

was

of

amino

acid

examined

with

Beckman 121-M Amino Acid Analyzer. Tryptophan could not bemeasured by this system.

25

RESULTS

After

irradiating tyrosinase

solution under N2,

thecatecholase activity was measured using dopa as substrate
(Fig.

1).

The

enzymatic

activity

decreased

exponential manner after an initial shoulder. Do(37 % surviving dose) and n = 2

inan= 1.25 kGy(hit number) were found.

Catechol was also used as another substrate, the survival
curve with which was

almostthesame asthat with dopa

(data not shown).
No effect of gamma-ray irradiation on Michaelis-MentenKm,

constant,

was

found

for

catecholase

activitywitheither dopa or catechol (data not shown) as also observedin the case of cresolase activity 8) indicating no loweringof affinity with the substrate for the irradiated enzyme.
Effect of O2 , t-BuOH, MeOH and N2 0 on the inactivationof the enzyme induced by irradiation are shown in Fig. 1.
Oxygen,which

enhancestheOHradical-induced

effect,showed little enhancement of inactivation of the enzyme. tBuOH and MeOH, which are OH radical scavengers, were also
found to have no effect. However, the presence of an e-aq
scavenger,

N20,

markedly

inhibitedtheinactivation.

Therefore, the main species involved in the inactivation ofthis enzymatic activity must be e-aq and not OH radicals.
As seenradiolyticin Fig.

1,

inactivation

Cu++ also markedly protected theofcatecholase

activity.

Ataradiation dose as 1 kGy, the catecholase activity decreased
to about 50 % under N2 . In the presence of Cu++, however,

26

o

6'
>

.....
U

C

.-.....u

I

E

MeOH
+N ' "

c

2

>-N

.1,

I

c:

t·BuOH
+N2

."",

(1)
' (1)

.->
10

o

1

2

3

Dos e, kGy

Fig. 1 •Survival curve of catecholase acti vi ty of tyrosinase inN2- saturated solution (0.2 mq/ml of phosphate buffer, pH 6.5)
irradiated wi th ~50CO g8JTl1la-rays, and the effects of the presence of
MeOB (10-2 M), t-BuOB (10-2 M), O2 (eaturated), H 2 0 (saturated) andCu++ (10- 4 M). SEs < 2 ,.

27

the activity was maintained at more than95

% withnodifference between the sulfate and chloride at a concentration of

10~

M, and about 90 % even at a concentration of

10-6 M. However, Cu+ little protected the enzymatic activity(data not shown). These behaviors are almost the same tothose of cresolase activity except for hit-number.
To detect the degradation of enzyme molecules,
enzyme wassubjected Ultrogelthechromatography before andafter irradiation. On the chromatogram, there was found one
peak

beforeirradiation,butanotherpeak

appearedincreasing after irradiation indicating the dissociation of
the enzyme to subunits

(Fig.

2).

In the non-dissociatedenzyme molecule, the cresolase activity was lost but thecatecholase activity was unaffected by increasing radiation
dose. After dissociation, however, the subunits no longer
possessed any enzymatic activity. In addition, it was foundthat tyrosinase dissociated to its subunits in the presenceof cysteine without irradiation (Fig. 2). It indicates thatthe dissociation takes place by reducible reaction.
Table 1 shows amino acid composition of the irradiatedenzyme under different atmospheres. At a dose of 1 kGy,
tyrosine, phenylalanine, histidine and lysine residues weremarkedly modified especially under N20 and under N2 + Cu*.

28

.8
Q)

c:

0'"

.6 Q.~

0>---0 ......

• •o• <lI

o

• •

GO
("4

o .05

.2

•

I

o

.-c
Q)
......

oa. .20oQ)

Nl + cysteine

Nl + 2.5 kGy

~

u
c

.8 >

.15

+-

u

o
u
+-

o

o

-C

A E

L-

o .10

>-N

."

..0
<{

C

W

.05

Fraction

numberFig. 2.
Dissociation of tyrosinase by irradiation under N2 or N20
wi th gamma-rays compared to that only in the presence if cysteine(10- 4 M for 30 min). 2.5 ml of tyrosinase solutions (0.2 mg/ml) were
eluted with 0.1 M phosphate buffer through an Ultrogel AcA 22 column,
are cresolase activity and catecholase activity of each fraction (2.5ml) were measured using tyro.ina and dopa, respectively.

29

Table. 1.
Amino acid catlpOsi tion
fraction after qamma-irradiation.

A.ino acidresidue

CoiIposition ofcontrol (~)

Glu

Thr
Ser
Pro
ProOH
Gly
Ala
Val
Leu

lIe
Cys(half)
let
Trp
His
Phe
1yr
Lys

Arg

12.7
9.5
6. 1
6.4
6.2
8.2
6.4
5.9
7.3
4.9
0.6
1.7
4.2
2.5
4.8
3. 4
4. 6
4.6

tyrosinaseandsurvi vinq

Surviving fraction at a dose of ] kGy(~ of non- irradiated control)

N2

N20

N2 + eu ~ •

]3.8
12.9
6. 7
5. 6
5.1
1.4
8. 2
8.4
6. 7
6. 8
4. 1
o. 1
0.4

99.3
91. 7
92.5
96.6
93.0
90.8
93.3
95.3
94. 1
86.0
84. 7

99.9
90.8
89.2
91. 7
84.9
75.9
95. 7
96.5
91. 1
77. 1
72. 8

95. 7
90. 1
88. 1
91. 7
83. 3
77. 5
100. 0
95. 8
92. 7
75. 1
14. 1

2. 0
4.3
2. 1
6. 1
4. 1

59.8
68. 7
72. 7
72.0
78.5

42.2
53.3
61. 2
62.5
66. 7

48. 7
56. ]
61. 1
59. 2
64.8

Reported(7) This work
Aspof

(Tyrosinase: 0.2 mqlml; Cu++, 10-4 M; H2 and H20, eaturated)

30

DISCUSSION

It has been found that the ionizing radiation-induced
inactivation of cresolase activity of tyrosinase,one ofmetalloenzyme, is due to e-~.8) This mechanism is different
from those of non-metalloenzymes which have been studied as
the attack by OH radical derived from

H20

with radiation.

In the present study, such e-~ participation was also foundin the inactivation of catecholase activity; that is, thee-~scavengers protected the enzymatic activity but the OH

radicalscavengers

concluded that

did

not

(Fig.

1).

It

is

therefore

is a main active species to inactivatee-~

not only the cresolase activity but also the chatecolase
activity.
However,

there

are

some

inactivationofactivity.

catecholaseThecatecholase

tyrosinase

washighlycresolase

activity(Do

differences
activity

activity(Doradio-resistant

=

350

Gy)

betweenandthecresolase

1,250

Gy)

of

compared

to

the

N2

gas

=

under

the

atmospheres (Fig. 1).8) Furthermore, the survival curve of
the catecholase activity showed n
is,= 2, as hit-number; that

this activity is lost when two targets

differing from nare damaged,= 1 for that of cresolase activity.

Inother words, the catecholase activity is different from thecresolase

activity

intheinactivation

mechanism.

Thedifference should be due to their functional structures intyrosinase molecule.
The non-dissociated molecule lost cresolase activity
after

2.5

kGy

gamma-irradiation

31

under

N20

and

N2

atmospheres

(Fig.

2).

However,remained even at considerably
dissociatedmolecule.

Inthecatecholase

higherthedoses

presence

inof

activitythenon-cysteine,tyrosinase molecule dissociated to subunits and no longer
had

the

catecholase

activity

(Fig.

2).

This

finding

indicates that the loss of catecholase activity is due tothe dissociation to the subunits differing from the case ofinactivation of cresolase activity.
Mason 1 ) reported the oxidation mechanism of diphenol
as follows:
Protein-Cu*2 + ~ a-diphenol~

Protein-Cu+2 + a-quinone + 2H+(I)

Reaction (I) must be modified as follows:
subunit-(Cu*2)-subunit + a-diphenol~

2 subunit-Cu+ + a-quinone + 2H+(II)

Therefore, the inactivation mechanism of tyrosinase can be
as follows:
subunit-(Cu*2)-subunit + e-~~

subunit-(Cu*Cu+)-subunit(III)

This step is the cresolase activity loss. It is supportedby that n = 1 shown in cresolase inactivation. a)subunit-(Cu*Cu+)-subunit + e-~~

2 subunit-Cu+(IV)

This step represents the loss of catecholase activity.
issupported

inactivation

by

that

(Fig.

1).

=

n

2

Reactions

shown
(III)

in
and

catecholase(IV)protected or repaired by Cu*.
Cu++ + e-~ ~ Cu+(V)This reaction is the protection mechanism.

32

It

are

subunit-(Cu++Cu+)-subunit + Cu++-+

subunit-(Cu++2)-subunit + Cu+

This reaction is

the

(VI)

repair mechanism forthe cresolase

activity.
2 subunit-Cu+ + Cu++-+

subunit-(Cu++Cu+)-subunit + Cu+(VII)This reaction is the repair mechanism of the catecholase
activity.
As mentioned before,tyrosinase molecule dissociatedto the subunits in the presence of cysteine (Cys-SH)
2).

The

presenceofcysteine

could

bring

(Fig.

out

the

dissociation of the tyrosinase molecule, as follows.
subunit-(Cu++2)-subunit + 2 Cys-SH-+

The

2 subunit-Cu+ + Cys-S-S-Cys + 2H+

dissociationmust

cysteine

like

e-aq.

It

cysteine

participate

resulting

of

the

be

due

is

concluded

in

the

to

reducing
that

of

not

activity but also cresolase activity.

abilitye-aq

tosubunits,

only
If

of

both

dissociation

inactivation(VIII)

andcatecholase

disulfidebondparticipates in the binding of subunits, there must be OHinduced

inactivation

and

02

enhancement

effectastheoxidizable reaction. Indeed, the degradation of cysteine in
the molecule increased in the presence of N20
However,(Table 1).

N20 has little effect on the inactivation(Fig.

1). Therefore, Cu++ must be not only the active center but
also the binder between the subunits to keep the active
form of tyrosinase molecule.

The first

hasinactivation

to cause thefollowed

cresolase

reductionofanother33

Cu++

reduction of Cu++
(III),
to

and

the

cause

the

dissociation

andconsequently toresultthecatecholase

inactivation (IV).
e-~is much larger than OH radical structurally. Thee-~

attack ofwould be difficult in the configuration ofsubunit-(Cu*Cu+)-subunit more than in the configuration ofsubunit-(Cu*2)-subunit
catecholase

activity

becauseisradio-resistibility

higher

than

that

of

of

cresolase

activity. This mechanism can be supported by the higher Do
shown in the survival curve (Fig. 1).
Underprotection

the

dose

effecthigher

(Fig.

than

1 .5

This

would

1).

kGy,

N20

be

lost

the

to

the

due

consumption of N20, as the saturated O2 mostly consume at 1
kGy.
Generally,

radiation-induced inactivation of enzymes

has been explained by the attack of OH

radicals derived

from water molecules. 9, 10) Those previous studies indicatedthat the enzyme inactivation was caused by the oxidation ofhistidine,

tryptophanorcysteine

residues

withOHradicals. Sulfur-containing amino acids and aromatic amino
acids

are

very

radiosensitive,

because

they

have

higher reaction rate constant with OH radicals .10)

much

In thecase of OH radical as main active species, the enhancementofinactivation with 02 or Cu* has

However,inthelittleeffectofinactivations
02

was

of

observed

been reported. 11, 12)tyrosinase
despite

with

the

e-~,

enhanced

damage of some radiosensitive amino acid residues. On theother hand, the presence of N20 or Cu* protected the enzyme
from inactivation despite the enhancement of

amino

acid

residue damages (Table 1). We concluded from these findings34

that theradiation-induced inactivation of tyrosinaseisinduced by reducible ability of e-aq but not by oxidizableability of OH radicals, and that the active center is not
an amino acid residue not only cresolase activity but also
for catecholase activity. At least the catecholase activity
is preserved so long as both Cu* ions as the active centerinthemolecule

molecular

are

not

reducedtostructureispartially

Cu+,

even

broken

if

the

with

the

degradations of some radiosensitive amino acid residues inthepresence

of

N20

or

Cu*.

It

is

notingthatthedegradation of amino acid residues is not critical on the
enzymatic inactivations.
previously,

it

has

been

reportedthathistidine

residues are involved in the configuration of Cu* in theactive center of tyrosinase molecule, and the residues havean interaction with the cysteine residue

located in theactive center .14,15) The present study, however, confirmed no
critical relationship of histidine or cysteine residue tothe active center.
In

previous

study oftyrosinase,

Cu*

been

protector. 13)

UV

derivedaactive

has
is

also

species .16)

uv-induced
also

able

to

inactivation

reported
cause

Therefore,

to

the

to

act

producepresent

of
a

H20-

study

suggests that the protection manner of Cu* is the same in
exposure to both UV and ionizing radiations.

35

REFERENCES

1) Mason, H. S. (1956) Structure and functions of thephenolase complex. Nature 177, 79-81.
2) Dressler, H. and Dawson, C. R. (1960) On the nature andmode of action of the copperprotein tyrosinase. II.
Exchange experiments with radioactive copper and thefunctioning enzyme. Biochim. Biophys. Acta 45, 515-524.
3) Shimao, K. (1962) Protein purification and kinetic
studies of mammalian tyrosinase. Biochim. Biophys. Acta
62, 205-215.

4) Pomerantz, S. H. (1963) Separation, purification, andproperties of two tyrosinases from hamster melanoma. J.
BioI. Chern. 238, 2351-2357.
5) Jolley,

R. L. Jr., Robb, D. A. and Mason, H. S. (1969)

The multiple forms of mushroom tyrosinase: Associationdissociation phenomena. J. BioI. Chern. 244, 1593-1599.
6) Jolley,

R. L. Jr., Nelson, R. M. and Robb, D. A.

(1969) The multiple forms of mushroom tyrosinase:
Structural studies on the isozymes. J. BioI. Chern. 244,
3251-3257.
7) Duckworth, H. W. and Coleman, J. E. (1970)
Physicochemical and kinetic properties of mushroom
tyrosinase. J. BioI. Chern. 245, 1613-1625.
8) Terato, H. and Yamamoto,o. (1994) Hydrated electron-

induced inactivation of tyrosinase in aqueous solution
by exposure to cobalt-60 gamma-rays. I. Cresolase
activity. Biochem. Molecular BioI. International. 34,
295-300

36

9) Spinks, J. W. T. and Woods, R. J. (1976) An

Introduction to Radiation Chemistry"

John Wiley & Sons,

New York.
10) Yamamoto,o.

(1992) Effect of radiation protein

stability; in Stability of Protein Pharmaceuticals, Part
A, Chemical and Physical Pathways of Protein Degradation(eds. Ahern, T. J. and Manning, M. C.) pp. 361-421,
Plenum Press, New York.
11) Anbar, M. and Leitzki, A. (1966) Copper-induced

radiolytic deactivation of a-amylase and catalase.
Radiat. Res. 27, 32-40.
12) Winstead, J. A. and Mcnees, D. E. (1974) Effects ofcupric ions on the radiolytic inactivation of lactate
dehydrogenase. Radiat. Res. 59, 466-474.
13) Sharma R. C., Ali, R. and Yamamoto,o.

(1979) Effect of

UV light on biological activity of tyrosinase in buffer
solution. J. Radiat. Res. 20, 186-195.
14) Pfiffner, E. and Lerch, K. (1981) Histidine at theactive site of Neurospora tyrosinase. Biochemistry, 20,6029-6035.
15) Huber, M. and Lerch, K. (1988) Identification of two

histidines as copper ligands in Streptomyces

glaucescens tyrosinase.

Biochemistry, 27, 5610-5615.

16) Ito, A. and Ito, T. (1983) possible involvement of

membrane damage in the inactivation by broad-band near-uv radiation in Saccharomyces cereviciae cells.
Photochem. Photobiol., 37, 395-402.

37

GENERALTheDISCUSSION

radiation-induced

inactivationoftyrosinase,which is one of metalloenzymes was studied fortwo mainpurposes. 1. There was no report of the radiation-induced
inactivationofmetalloenzymes.

interesting

enzymes

which

has

Tyrosinase

2.

two

kinds

of

is

an

activities,

cresolase activity and catecholase activity.

Cresolase activity( 1) MeOH and t-BuOH as OH radical scavengers did notprotect the inactivation. Oxygen as the oxidation enhancer
hadlittleeffect.

However,

N20

and

Cu*

as

the

e-~

scavengers protected efficiently.
The

(2)

survival

curvesoftheenzymatic

activity

under various conditions showed n = 1 as the hit number.
(3)

Lineweaver-Burk

showed that

had

Km

not

plot

of

variedtheatenzymatic

different

activity

irradiation

doses.
There was no difference between CUS04(4)

and CuCl2

used for the source of Cu* in the protection efficiency.
CuCl used for the source of Cu+ had no protection ability.
The
speciesresultforindicates

the

that

of

(1)

indicates

inactivation is
the

that

e-~.

inactivation was

the

main

activeThe result of(2 )one-hit

The

event.

result of (3) indicates no lowering of affinity with thesubstrate

for

the

irradiatedenzyme.

The

resultof(4 )shows that Cu* is protective but Cu+ is not. Therefore, theenzymatic inactivation must be due to the reducing ability38

of e-~. The protection reaction must be the elimination ofe-~with N20 and Cu++; the reactions are N20 + e-~ -+ N2 +OH + OH- and Cu++ + e-~ -+ Cu+. It is the most reasonable toconclude that the enzymatic

due

to

the

reduction of the active center Cu++ to Cu+ with

e-~

in

tyrosinasesubunit +molecule;
e-~

-+

the

inactivation

reactionisissubunit-(Cu++Cu++)-subunit-(Cu++Cu+)-subunit.

Free Cu++ must

play a role to repair Cu+ to Cu++ in tyrosinase molecule;the reaction is subunit-(Cu++Cu+)-subunit + Cu++ -+ subunit(Cu++Cu++ ) -subunit + Cu+.

Catecholase activity( 1)

Effects of all of OB radical scavengers and e-~

scavengers

were

the

same

as

those

for

cresolase

inactivation.
(2)
variousSurvival curves of the enzymatic activity underconditions

showed

=

n

2

as

the

hit

number,differing from that of cresolase inactivation.
(3)

By

gel

chromatography

after

irradiation,thedissociation of tyrosinase molecule to subunits was shown.
The dissociation was protected with N20. In the presence ofcysteine,

there

was

also

tyrosinase molecule without

found

the

irradiation.

dissociation
Theoffraction ofsubunits no longer had any enzymatic activity.
(4)

Degradation

of

amino

acids

was

observed

after

irradiation. Degradation yield increased in the presence of
N2 0 or Cu++ and O2 enhancing effect on the degradation was
also observed.

39

(5)

Lineweaver-Burk

showed that

had

Km

not

plot

of

variedtheatenzymatic

different

activity

irradiation

doses.
(6)

There was no difference between CUS04 and CuCl 2

used for the source of Cu++ in the protection efficiency.
CuCl usedThe

for the source of Cu+ had no protection ability.
resultof(1)

indicates

that

the

main

activespecies for the inactivation is also e-~. The result of (2)shows that the inactivation is two-hits event; that is, thecatecholase activity is resistant more than the cresolase
activity against
indicatethatradiation.

the

The

enzymatic

results

of

(3)

and

(4)

inactivation

due

to

the

dissociation of tyrosinase molecule to subunits but not tothe degradation of amino acids. The result of (5) indicatesnoloweringofaffinity

irradiated enzyme.
protective

but

The

Cu+

is

with

result of
not.

the

substrate

(6)

shows

Therefore,

forthat Cu++

theand Cu++; the reactions are N20 + e-~
Cu++ +

e-~

~

Cu+.

~

is

enzymatic

inactivations was due to the reducing ability ofprotection reaction must be the elimination of

the

e-~

e-~.

The

with N20

N2 + OH + OH- and

It is most reasonable to conclude thatthe enzymatic inactivation is due to the dissociation oftyrosinase molecule to subunits by thetwo step reductions

with e-~; the reactions are subunit-(Cu++Cu++)-subunit + e-~~ subunit-(Cu++Cu+)-subunit and subunit-(Cu++Cu+)-subunit +

e-aq

~

Cu+

to

2 subunit-Cu+.

Cu++

in

Free Cu++ must play a role to repair

tyrosinasemolecule;

thereactionsUbunit-Cu+ + Cu++ ~ subunit-(Cu++Cu+)-subunit + Cu+.

40

is

2

In the previous studies, OH radical has been known to
main

activeenzymes.

speciesIn thoseforstudies,theinactivationofvariousthe main damaging target

forinactivation was radiosensitive amino acid such as cystein,histidine or tryptophan. However, the present study showed
that

the

main

damaging

targetwasmetalion ( s)inthetyrosinase molecule. Finally, the author concluded that themetalloenzyme which has metal ion(s) as the active center
might be inactivated with e-~ but not with OH radical

41

ACKNOWLEDGMENTS

I would like to express my science gratitude to Dr.
Osamu

Yamamoto,

Professor

of

Gene

Chemistry

Division,

Graduate Department of Gene Sciences, Faculty of Sciences,
Hiroshima

University

for

his

encouragements and criticalinvaluableguidance,

supervision in all

warm

respects.

The author is also grateful to Dr. Yoshihiko Ohyama and Mr.
Takeshi Saito for their kind help in this study and to Mr.
Javed

Kausarfor

hislinguistic

composition.

42

assistancein

English