Production and identification of bityrosine in horseradish peroxidase-H2O2-tyrosine system

Vol. 125, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 28, 1984

Pages 916-918

PRODUCTION AND IDENTIFICATION OF BITYROSINE IN HORSERADISH PEROXIDASE-H202-TYROSINE SYSTEM Yoshio

Ushijima*,

Minoru

Nakano+

and Toshio

Goto#

*Clinical Laboratory, Gunma University Hospital +College of Medical Care and Technology, Gunma University, Maebashi, 371 , Japan #Laboratory of Organic Chemistry, Faculty of Agriculture, Nagoya University, Nagoya, 464 , Japan

,

Received October 12, 1984

Summary: Production of bityrosine (2,2'-dihydroxy-5,5'-bis (8carboxy-8-aminoetyl)-diphenyl) was established in horseradish peroxidask-H202-tyrosine system at pH 9.2 by mass and NMR spectral metries. 0 1984 Academic Press. Inc.

Horseradish oxide

has been

product

peroxidase

reported

bityramine,

phenyl

(1).

to the

with

fluorescence

data

also

during

produce

under

anaerobic

product tant

to oxidize

similar

has been

product

undertaken tyrosine

suggested

respects (1,2).

the

yielding

to

to identify using

the

their

of

of

tyrosine

of bityramine

ultraviolet

absorption

bityrosine of

(PBT)

and could

tyrosine

solution

The identification

of

the main

tyrosine

is

very

the

PBT formed

a main

convertion

irradiation (3).

per-

(@-amino-etyl)-di-

by similarity

ultraviolet

mechanism

of hydrogen

tyramine,

Such a proposed

conditions

system,

presence

conditions,

in peroxidase-catalyzed to solve

the

2,2'-dihydroxy-5,5'-bis

Under

to bityrosine

in

oxidation

reaction.

The present

in horseradish

imporwork was

peroxidase-H202-

mass and NMR spetrometries.

Material and Methods: Horseradish peroxidase (R.Z.22.5) was purchased from Toyoboseki K.K. and used without further purification. FAB mass spectrum and NMR spectrum were obtained using JEOL, JMSD300, JEOL, JNM-FX200 respectively, 0006-291X/84 Copyright All rights

$1.50

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

916

Vol. 125, No. 3, 1984

Results

BIOCHEMICAL

and Discussion:

ml of water,

A 1.0

and 50 ml of

of horseradish

shaking. with

acid

powder was then

dissolved

with

a clear

supernatant

column acid,

by Anderson

lected

and transferred

(Merck

product

acetic

acid/H20

(Rf=O.lS)

carried

out

mental tive

(2).

No 5745).

had been equilibrated

After

for

data

as those

reported

purification

approximately was obtained.

by others

with

thin

layer

0.2 N acetic acid

as

was col-

chromatoplates

the plates

in butanol/

strong

fluorescence

water.

and extraction

With

the

procedure

of PBT. Under the

experi-

(Table

absorption I)

and fluo-

were essentially

the same

(3,4).

Table I ULTRAVIOLET ABSORPTIONAND FLUORESCENCE PROPERTIESOF PBT Ultraviolet absorption maximum

Fluorescence Excitation Emission maximum maximum

3.6

283

(283)a'b

287

(284)b

410

(410)b

10.7

317

(317)a

320

(315)b

410

(410)b

PH

a: Data from ref. b: Data from ref.

was

10 mg of PBT, a ninhydrin-posiUltraviolet

of PBT obtained

phosphate

fluorescence

with

was

centrifugation,

with

and extracted

to 6.0

solution

0.2 N acetic

showing

same chromatography

conditions,

rescence

the area out

further

compound,

with

developing

(4:1:l,v/v),

the

After

to a cellulose

The eluate

6 N

The lyophilized and the

(1).

on preparative

was scratched

extract

the pH was adjusted

charcoal

was performed

by 40 mg

to 9.2 with

was lyophilized.

was transferred

elution

followed

in 910

12 h at 37O C without

in 50 ml of water,

1 g of activated

and the

for

reaction,

(16 x 350 mm) which

described

water

was incubated

and the mixture

treated

was dissolved

The pH was adjusted

At the end of the

acetic

g of L-tyrosine

0.1% H202 was added,

peroxidase.

NaOH, and the mixture

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(316)b

4. 3. 917

Vol. 125, No. 3, 1984

BIOCHEMICAL

7

6

Fig.1

FAB mass spectrum +l)

corresponding

spectrum

7.8 & 5.3

Hz,

Hz, Nd),

indicating

Ha),

dd, 6.98

and 7.22 that

substituted

ortho

NMR spectrum of PBT.

of bityrosine

signals:6

(lHx2,

benzene in one half

ring of

3.08

J=14.5 (lHx2,

(lHx2,

PBT is

(data

d, J=8.3

a symmetric

aromatic

consideration protons.

dihydroxy-5,5'-bis Thus, conversion

It

is,

of tyramine

Hz,

dimer

and an ABX type the molecule.

Hc),

dd,

Hf),

That hydroxy

therefore,

established

from

to bityramine,

(lHx2,

system

the coupling

as the chemical

dd,

d, J=

is

position

is apparent shifts that

of

PBT is

is from

the 2,2'-

-diphenyl. tyrosine,

identical

by the peroxidase-H202

to the sys-

tem (1). References 1. 2. 3. 4.

Gross, A.J. and Sizer, I.W. J. Biol. Chem., 234, (1959) 1611-1614. Anderson, S.O. Biochim. Biophys. Acta, 93, 213-215. (1964) Lehrer, S.S. and Fasman, G.D. (1967) Biochem., 6, 757-767. Anderson, S.O. (1963) Biochim. Biophys. Acta, 69, 249-262. 918

J=

a 1,2,4,-tri-

spin

group

& 7.8

He) (Fig.l),

in which three

DSS)

(lHx2,

7.16

as well

be formed

lH-NMR

J=14.5

3.96

& 2.2 Hz,

(R-carboxy-R-aminoethyl) PBT could

not shown).

ppm (lHx2,

dd, J=8.3

and (M/2

in 2H20 (standard:

& 5.3 Hz,

and not meta to the phenolic

mechanistic

3

of PBT showed peaks at 361 (M+l)

to that

following

3.25

Hz, Hb),

present

4

5

(200 MHz at 23OC) of PBT taken

showed the

2.2

AND BIOPHYSICAL RESEARCH COMMUNICATIONS