Chemiluminescence in L-tyrosine-H2O2-horseradish peroxidase system: Possible formation of tyrosine cation radical

Vol. 128, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

April 30, 1985

Pages

936-941

CHEMILUMINESCENCE IN L-TYROSINE-H202-HORSERADISH PEROXIDASE SYSTEM: POSSIBLE FORMATION OF TYROSINE CATION RADICAL Yoshio

Ushijima,Minoru

Nakano*, Choichi

Takyu

and Humio Inab

Clinical Laboratory, Gunma University Hospital * College of Medical Care and Technology, Gunma University, Maebashi, 371, Japan tResearch

Institute of Electrical Tohoku University, Sendai,

Communication Japan

Received March 13, 1985 Summary: Tyrosine-H 02-horseradish peroxidase system at pH 7.4 emitted light in visi 3 le region. Phenolic compounds other than tyrosine were also emissive? whereas methoxy phenylalanine and phenyl compounds were not, in H 0 -peroxidase systems. Chemiluminescence spectrum of tyrosine 03% yrosine-H 0 -horseradish peroxidase system showed two prominent peaks a 32 78 nm and 500 nm (Luminescence 1) and additional two or three peaks near 550 and 610 nm (Luminescence 2). Luminescence 1 is quite similar to the phosphorescence originated from an excited tyrosine in triplet state, while Luminescence 2 is quite similar to the phosphorescence originated from an indole in triplet state. Possible formation of tyrosine cation radical (a precursor of the excited tyrosine) and indole cation radical in the enzyme protein (a precursor of the excited tryptophan residue) were discussed. 0 1985 Academic

Press,

Inc.

The peroxidases, lactoperoxidase oxidation

of

to be bityrosine rescence

(2) tyrosine

to a highly

(1,4).

compound from

tyrosine

dimer

we isolated

in o,o-biphenyl

BT formation,

radical,

as suggested

by Gross and Sizer

tyrosine-H202

-HRP system

emits should

a phenoxy (1). the

compound, the highly

proposed fluo-

0 1985 by Academic Press, Inc. of reproduction in any form resewed.

radical,

The present light

936

(BT),

by mass

of peroxidase-catalyzed

in visible

be addressed.

0006-291X/85$1.50 Copyright All rights

the

catalyze

linkage

In the process

(5).

To whom correspondence

will

-HRP system and identified it tyrosine-H202 , -bis (B-carboxy-@-aminoethyl) -diphenyl,

linked

tyrosine

(3),

(HRP) (1,2),

fluorescence

Recently,

and NMR spectrometries

*

peroxidase

and ovoperoxidase

to be 2,2'-dihydroxy-5,5 the

horseradish

could work

be formed

reports

region,

that

Vol. 128, No. 2, 1985

BIOCHEMICAL

suggesting

the

generation

-catalyzed

oxidation

of

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tyrosine

cation

radical

by the

enzyme

tyrosine.

Material and Methods HRP was purchased from Toyoboseki K.K. The concentration was determined at 403 nm using a (R.Z.22.9). millimolar extinction coefficient of 103 (6). All chemicals were reagent grade. All reactions were carried out at 25' C. BT formation was followed on Hitachi 200-20 spectrophotometer. The increase in the absorbance at 315 nm was used as an index of BT formation (2). Chemiluminescence intensities and spectra in visible region were measured and taken by a luminescence reader (Aloka BLR-101) (7) and a filter type spectrometer (7,8), respectively, Results

When L-tyrosine

was incubated

sence of H 0 under aerobic 22 emitted the light in visible molecular

oxygen

for

or anaerobic region,

luminescence

With

the

linearly fixed

intensity

(data

increasing

only integrated

with with

tyrosine

light

not

the pre-

the system no requirement

shown). light

With

enzyme concentration,

intensity

was not parallel

fixed in-

(Fig.

maximal

enzyme concentrations

of

the

intensity

concentration

concenrations,

increasing lower

condition,

maximal

H202 and tyrosine

increased

a linearlity However,

with

HRP in

suggesting

H202 and the enzyme concentrations, creased

with

(Fig. with

light showing 1,Bj. the

5

s

\

.5

4 :

0 k

IO 2030405060 INCUBATION,

MIN

Fig. l.A. Effect of tyrosine concentration on HRP-induced luminescence. The incubation mixture contained 1.67 mM H 0 3.54 i.lM HRP, and L-tyrosine in 100 mM K-phosphate buffer at 8 H2i.4 in a total volume of lml. L-tyrosine concentrations were: 1, 0.04 mM; 2, 0.08 mM; 3, 0.2 mM; 4, 0.5 mM; 5, 1.0 mM. Fig. 1.B. nescence.

Effect of enzyme concentration on HRP-induced lumiThe incubation mixture contained 0.2 mM L-tyrosine, HRP and 100 mM K-phosphate buffer at pH 7.4 in a :,:~lm~o%~a~of lr;ll . HRP concentrations were: 1, 0.44 ~.IM; 2, 0.89 uM. i.lM; 3, 1.77 nM; 4, 3.54 PM; 5, 7.08 PM; 6, 14.16 937

1,A).

for-.

Vol. 128, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL

INCUBATION,

RESEARCH COMMUNICATIONS

MIN

Fig. 2. Relationship between BT formation and integrated light intensity. The incubation mixture was the same as in Fig. l,B.4, except that total volume was 3 ml. All values are normalized to be expressed as percentage of the final value.

mation

of a highly

no direct

relation

Of the

fluorescent of

product,

the luminescence

compounds tested

other

pounds,

such as monoiodotyrosine,

benzoic

acid,

were emissive

phenolic

compound with

alanine,

and phenyl

importance

with

than

L-tyrosine,

OH group,

in phenols

for

(Table

suggesting

phenolic

com-

and p-hydroxy-

in H202-HRP systems,

inactivated

2),

BT formation.

diiodotyrosine,

compounds were not

of OH group

Table 1.

BT (Fig.

whereas

a

p-methoxyphenyll),

HRP-induced

indicating

the

luminescence.

Chemiluminescence of the compounds in H202-HRP systema

Compounds in 1 mM

Chemiluminescence Maximal light Relative intensity to tyrosine counts/min

L-Tyrosine Monoiodotyrosine 3,5-Diiodotyrosine p-Methoxyphenylalanine Phenylalanine p-Hydroxybenzoic acid Benzoic acid

2350 1860 710 neg. neg. 290 neg.

100.0 79.1 30.2 0 0 12.3 0

a The incubation mixture contained 1.67 mMH 0 3.54 PM HRP compounds and 100 mM K-phosphate buffer at pii 8:4, in a totai volume of 1 ml. 938

Vol.

128,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

WAVELENGTH,

Fig. 3. tyrosine cences except started tained

type 3).

in

the

the

visible

peaks at

3,B,

of

of

the peak intensity

electron

originated

from

electron

interaction

(9).

indole

acetic

dependent

tyrosine

is

spectra

the

(9) quite

indole

showed two

1) and additional

Luminescence

hydroperoxidase

formed

as a precursor 939

from (Fig.

to the

490 nm, 1.1, tyrosine 3,A to

cation

and B).

On

the phospho-

radical-solvated

2 were also system

(7)

system

(10).

have first

2) in the

similar

of

similar

cation

(Fig.

of 500 nm to that

that

originated

2 is

Gross and Sizer radical

spectra

is quite

acid-H202-myeloperoxidase

prostaglandin

Discussion

which

interaction

Luminescence

rescence

by a filter

reference

516 nm to

the phosphorescence

hand,

taken

the peak intensity

ratio

the other

some

chemiluminescence

550 and 610 nm (Luminescence

The ratio

radical-solvated

were

the luminescence

1.25,

for

system

with

of 478 nm was approximately

reported

the

478 and 500 nm (Luminescence

peaks near

region.

of

nm

species,

-HRP

and compared

As shown in Fig.

two or three

excited

tyrosine-H202

spectrometer

prominent

COMMUNICATIONS

Comparison of chemiluminescence spectra of excited and trvptophan (A) with those of HRP-induced lumines(B). The-incubationSmixture was the same as in Fig l,B.4, that total volume was 15 ml. Spectral measurement was at the time at which the maximal light intensity was oband continued for 15 min. Exp 1 -, Exp. 2 _-__.

To investigate spectra

RESEARCH

obtained

from

and tryptophan

suggested of BT during

that the per-

Vol. 128, No. 2, 1985

BIOCHEMICAL

“q-&y-y-

GDf--JRL (c4nmiFcm3~I

e

. 0

HOOR-

AND BIOPHYSICAL

HOR

(TYR,T’)

RESEARCH COMMUNICATIONS

-DOR (NEUTRAL

b hV

wIcAL)

R (ST)

mfR,So)

TYRCSINE= TYR GROUND STAEEXCITED STA~ITRIPLETIT‘

R

Sa

Possible mechanisms of the production of excited tyro4. sine and BT in the process of HRP-catalyzed oxidation of tyrosine. Fig.

oxidase-catalyzed portance

of OH group

luminescence obtained likely plex

oxidation

in phenolic

and a greate

from that

excited

similarity

losses

tyrosine

to tyrosine

in a triplet an electron

cation

once in a triplet

cation

would

be deprotonated

probably

radical

abstracts

protein

yielding

2 after

producing

ing electron

electron

Thus tyrosine enviroment,

Since the

cation

of

the

by the heme iron

residue

enzyme should

the

seems

residue which

complex

of

with

to

the the A part formed

in the

emits

enzyme

Luminescence

state

by catchof HRP upon

tyrosine

(2).

in some hydrophobic the cation

and back donation tyrosine

com-

to BT.

of tyrosine

can be formed

of

those

same figure,

in a triplet

deprotonation

excitation

it

emits

is a streospecificity

Abstraction

protected.

the process

radical,

there

radical

in which

and then

radical

is

radical of electron

probably

carried

in HRP.

References 1

Gross,A.J. 1614.

and Sizer,I.W.

(1959) 940

J:

im-

an electron

and dimerized

neutral

1 with

(9),

gains

As shown in the

cation

tryptophan

again.

which

from tryptophan

a indole

D- and L-tyrosine,

be highly

or the

the

the enzyme induced

stste

state

(Fig.

the cation

4).

from

by the enzyme-H202

radical,

light

of

Judging

of Luminescence

characteristic radical

(1).

compounds for

tyrosine

tyrosine

yielding

return

of tyrosine

Biol.

Chem. 234,

1611-

would in out

Vol.

2 3 4 5 6 7 8 9 10

128,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Bayse,G.S., Michaels,A.W., and Morrison,M. (1972) Biochim. Biophys. Acta, 284, 34-42. Deits,T., Farrance,M., Kay,E.S., Medill,L., Turner,E.E., Weidman,P.J., and Shapir0,B.M. (1984) J. Biol. Chem. 259, 13525-13533. Lehrer, S.S., and Fasman,G.D. (1967) Biochemistry 6, 757-767. lJshijima,Y., Nakano,M., and Goto,T. (1984) Biochem. Biophys. Res, Commun. 125, 916-918. Schonbaum,G.G., and Lou,S. (1972) J. Biol. Chem. 247, 33533360. Kobayashi,S., Sugioka,K., Nakano,M., Takyu,C., Yamagishi,A., and Inaba,H. (1980) Biochem. Biophys. Res. Commun. 93, 967973. Nakano,M., Noguchi,T., Sugioka,K., Fukuyama,H., Sato,M., Shimizu,Y., Tsuji,Y., and Inaba,H. (1975) J. Biol. Chem. 250, 2404-2406. Vladimirov,Y.A., Roshchupkin,D.I. and Fesenk0,E.E. (1970) Photochem. Photobiol. 11, 227-246. Yoshimoto,T., Yamamoto,S., Sugioka,K., Nakano,M., Takyu,C., Yamagishi,A., and Inaba,H. (1980) J. Biol. Chem. 255, 1019910204.

941