Thiols liberate covalently bonded flavin from monoamine oxidase

Vol.

94, No.

May

30,

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 579-585

1980

THIOLS LIBEPATE Kazuho

COVALENTLY BONDED FLAVIN FROM MONOAMINE OXIDASE

Watanabe,

Noshi

Minamiura

and Kerry

T. Yasunobu*

From the Department of Biochemistry-Biophysics, Medical School, University of Hawaii, Honolulu, Received

March

John A. Burns Hawaii 96822

31,198O

SlIMMARY Although it was recommended that Z-mercaptoethanol should be added to all buffers used in monoamine oxidase purification, purification of the enzyme in the absence of thiols has yielded double the amount of In fact the presence of 0.1 1< 2-mercaptoethanol or dithioerythrienzyme. tol-SDS1 was found to liberate about 50% of the covalently bonded FAD. This observation is surprising since the covalently bonded flavin has There is a thioether linkage been reported to be B-a-cysteinyl-FAD. between the flavin and the protein and mild reducing agents such as merThe imcaptoethanol would not normally cleave the thioether linkage. portance of the present finding is that the thiols may be used possibly to liberate suicide substrate-flavin adducts from the enzyme for structural studies without isolating pure flavin peptides. The flavin can be removed simply by treatment of the enzyme with 0.1 M thiol solution containing 1% sodium dodecylsulfate followed by chromatography on Sephadex G-25. Bovine bound

liver

monoamine

FAD by Igaue

and Kearney

et al.

et al.

(3)

oxidase (1).

zyme which

inactivate

enzyme bound and

flavin

flavin

adducts,

derived

from

it the

chemical

reagents

structure

of the

procedure

for

*

(6).

pargyline In order

inactivated

flavin

adduct. the

for

of this

of suicide

substrates

reprints

covalent

Flavin

the task

from

Abbreviations

used

is:

SDS, sodium

in the

with

the (5)

of the pure

peptides

the enzyme by

of determining

have been

found

in the present

the and the report.

be addressed.

This work was supported in part by grant MH 21539 Institutes of Mental Health and RIAS-SER-77-06923 Science Foundation. 1

(2)

of the en-

structure

and isolate

release

is discussed should

flavin

adducts

the

to obtain

Such reagents flavin

et al.

3-N,N-dimethylpropyne-1

to determine

simplify

covalently

of Walker

structure

(4),

enzyme.

greatly

liberating

To whom requests

the

has been necessary

would

studies

enzyme by forming

include

phenylhydrazine

that A number

the

shown to contain

The elegant

concluded

enzyme was G-a-cysteinyl-FAD.

was first

docecyl

from from

the National the National

sulfate. 0006-291X/80/100579-07$01.00/0

579

Cop.vrighl (3 I980 bv Academic Press, Inc. All righrs ofreproducrion in any form reserved.

Vol.

94, No.

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

NATCRIALS AND METHODS Enzyme. Purified bovine liver monoamine oxidase was prepared by the procedure reported by Plinamiura and Yasunobu (7) but P-mercaptoethanol was omitted from all solutions used to purify the enzyme. 2-Mercaptoethanol, dithioerythritol and FAD were purchased from Sigma Chemical Co. Biogel A0.5m was purchased from Bio Rad Labs. All common reagents used were of reagent grade quality. Enzyme Determinations. The activity and specific activity of the enzyme were measured by the procedure of Tabor et al. (8) in which the oxidation of benzylsmine to benzaldehyde is monitored at 250 nm. Protein concentrations were determined by the procedure of Lowry et al. (9) using bovine serum albumin as the standard. Other Measurements. All spectral data reported were obtained in a Cary Model 14 Spectrophotometer. All pH measurements were made with the Corning Digital 112 pH meter. PaSULTS AND DISCUSSIONS -Is Mercaptoethanol Oxidase.

i+inamiura

be added

to all

increase

the yield

of the

initial

fragments.

one month noted

that

and was judged electrophoresis, the remainder nevertheless Spectral purified

around

I.

However,

the initial

Uowever,

it

prevents

it

still not

weight

of the enzyme showed

Properties enzyme

for

certain

-of Purified

(specific

350 nm, a significant

of the mitochondria plus

high

outer

specific

It

for should

activity

be

(11,400) gel

to be 52,000. activity

about

it

Although should

of enzyme experiments.

11,400)

The absorption showed

spectrum

a slight

peak at 412 nm and a shoulder

580

mem-

enzyme activity

specific

Enzyme.

activity

Triton

to add 1 x 10e5 M thiol

was determined

types

purifi-

of the

of SDS-polyacrylamide

a lower

of

with

the freezing

maintains

by the criterion

molecular

be suitable

of the yield

the enzyme from precipitating.

to be pure its

of the enzyme

the enzyme from

of the enzyme has a very

to

of enzyme during

desirable only

in order

purification

yields

washing

?lonoamine

2-mercaptoethanol

in a doubling

to liberate

is

that

a recent

of the mitochondria appears

of Purified

enzyme purification

The increased

enzyme since

part

recommended

in the

which

but also

(7)

the Yield ---

has resulted

to disruption

to the purified

the

(1)

homogenate

brane

thiol

in Table

are due to: prior

used

of enzyme.

enzyme as shown

X-100

to Improve

and Yasunobu

solutions

in the absence

cation

--Needed

of

shoulder

at about

450 nm

Homogenate

0.25-0.45

2.

3.

'

b

a

Pure

I.

‘78

138

377

1,600

1,000

2,000

2,000

Volume

16660

80% pure

electrophortic

by SDS-disc analysis.

electrophoretic

8goo

go00

2250

5250

2900

460

Activity (units/ml)


to yield

RESULTS OF WZYMZ PURIFICATION

in 3 days starting from mitochondria or 48.3 mg of enzyme per kg liver.

by SDS-disc

At least

Prepared of liver

0.5% CholateC

0.5 M Buffer-

0.2% cholateb

0.2 M Buffer-

k;r Eluate

DEAE Eluate

5.

i 1.

CaP04 Gel Eluate

wash

4.

AmSO4PPt

C. 5% Triton

i.7

Step

Table

13 lbs

43% of initial

1.44

1.2

1.78

0.90

5.95

12.3

8.72

Protein hdlul)

(About

activity

11528

7295

5~56

2500

882

237

52.8

Spec . iiC t .

Bovine

4

130 x lo4

123 x 104

33s x 10

360 x 10 4

525 x 10’

10~

x IO4

580 x

92

Total units

1x2

168

67 1

1,440

5,950

24,520

17,440

Total Frotein

or 280 mg of enzyme from

Liver)a

5.8 kg

22

21

55

62

91

100

Yield

Vol.

94, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Figure 1. Visible absorption spectrum of monoamine oxidase, specific activity of 11,000. A, native enzyme at concentration of 1.78 mg per ml in 0.1 M potassium phosphate buffer, pH 7.4, which contained 0.2X potassium cholate G, identical to A except that the solution contained 1X SDS, enzyme: SDS ratio of 1:5.9. C, identical to B except that the solution was heated at 100" for 10 minutes. D, same as C except for the addition of mercaptoethanol to 0.1 M.

(Fig.

1).

Although

contaminant

this

has the highest far

this appears

specific

and shows a single

trophoresis. for

shoulder

is

for

activity

of any preparation

When the

largely

resembles content

the absorbance

at 450 nm of the oxidized

The flavin

(10) of flavin

per

Thiols showed

that

Liberate

shoulder

gel this

the

the spectrum spectrum

elec-

peak as

forms

enzyme was found

persists

but

after

of flavoproteins

can be determined

and reduced

thus

in SDS, a slight

at 450 nm still

oxidase

of the purified

bleach

heated

Therefore,

of the monoamine

content

110,000

enzyme is

extent

and the flavin

1) The enzyme

we have isolated

does not

abolished.

to a greater

reasons:

by SDS-polyacrylamide

of dithionite

at 355 nm and the

the peak at 412 nm is treatment

the following

band when examined

heme-proteins. observed

be due to a heme containing

unlikely

2) The addition

reported

this

412 nm peak could

from

of the enzyme to be one mole

g of enzyme. --Flavin

from Monoamine

the FAD in monoamine

oxidase

582

Oxidase. was bonded

Igaue to the

et al.

(1)

enzyme covalently.

Vol.

94, No.

BIOCHEMICAL

2, 1980

P-I .P-IS ,

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1 LO-

.p-2.

-2 =088’ N

“oosa c” fo.4

-

G 0.2r

Effluent

(ml)

Effluent

(ml)

Figure 2. Separation of liberated flavin from monoamine oxidase by niogel A-O. 5x11 column chromatography. About 380 mg of enzyme in 0.02 11 YH4HC03 was first treated with 1.25% SDS-O.1 M mercaptoethanol (final concentrations) and then chromatographed on a 2.6 x 62 cn column of Biogel A-0.5m. The eluant was 0.02 PI NII4HC03. Figure 3. Rechromatography of liberated flavin on Sephadex G-25. The concentration flavin obtained from the Biogel A-O.% chromatography step was applied to a 2 x 113 cm column which was eqllilibrated and eluted with 0.02 M Nh4liC03.

Walker

al.

et

oxidase

is

erythritol.

surprise,

liberated

pH 7.4.

solution

0.1 M with first

by thiols

Sufficient

dissolved

to the A-0.5m

thiol. (Fig.

was separated

from

absorbance,

at 450 nm, it

was determined

liberated

attached flavin

the

the

enzyme,

was concentrated FAD (Fig.

Concluding

Remarks.

liberated

at least

it

the

was then

The solution

flavin

flavin

in mono-

showed

and dithio-

added

about

the remainder

on Sephadex

chromatoG-25

3.

flavin

of the flavin form.

and

From the

50% of the

altered

was recorded

to make the

was then

as shown in Fig. that

spectrum

The present half

as 8-u-cysteinyl-

in 1% SDS in 0.1 M potassium

in a spectrally

and the 4),

that

2) and then

while

to the enzyme although

of authentic

thiol

from

was bound

such as 2-mercaptoethanol

the protein

been

flavin

2-mercaptoethanol

respect

on a Eiogel

the

we have observed

The enzyme was first

phosphate,

graphed

that

demonstrated

Much to our

FAD (4). amine

have

had

was still The liberated

and compared

with

that

maxima at 365 and 453 nm. investigation

of the flavin

583

has shown

from monoamine

that

oxidase.

excess There-

Vol.

94, No,

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

it1 WAVELENGTH

(nm)

Figure 4. Ultraviolet-visible absorption spectrum of liberated flavin (P-2) from monoamine oxidase after Sephadex G-25 column chromatography. authentic FAD and 3, flavin liberated from enzyme. fore,

caution

must be excercized

Eut the addition if

of thiol

the concentration

a number

with it

peptides

which

vided

a simpler

high

concentrations

in denaturing thioether are

several

of suicide (4-6).

has been necessary must

then

linkages possible

for

of thiols

enzyme purification.

Thiol

the purified

concentrations Recent

50% yield. substrates

inactivate

In order

to characterize

to use proteolytic The present

liberating

by Sephadex

cleaved

explanations

monoamine

to liberate

namely

flavin

has proby the use of

or dithioerythritol

chromatography.

reducing

agents

the cleavage

of the

584

oxidase

the flavin

G-25

by mild for

investigations

investigation

the flavin,

enzyme in the range

enzymes

such as Z-mercaptoethanol

followed

are not

during

in storing

M.

be purified.

procedure

solvents

-5

in about

the flavin

usage

useful

at 10

the flavin

have shown that

adducts,

still

is kept

of 0.1 I< liiierate

by reacting

is

in its

A,

Normally

as thiols. flavin

from

There the

Vol.

94, No.

BIOCHEMICAL

2, 1980

enzyme.

1) The form

S-a-cysteinyl-FAD protein

but

in which possibly

or a thiohemiacetal

that

the structure

that

the

benzenoid

the

flavin

is bound

linkage.

of the bound

adjacent

BIOPHYSICAL

a disulfide

S-C bond can be cleaved ring

AND

to the

linkage

RESEARCH

to the between

2) A more appealing

flavin because

is

instead

COMMUNICATIONS

enzyme is not the flavin explanation

8-a-cysteinyl-FAD

of the activating

effect

and is but

of the

S-C bond.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Igaue, I., Gomes, B., and Yasunobu, K.T. (1967). Biochem. Biophys. Res. Commun. 2, 562-570. -Walker, W.H., Kearney, E.B., Seng, R.L., and Singer, T.P. (1971). Eur. J. Biochem. 6, 328-331. G&y, E-B., Salach, J.I., Walker, W.H., Seng, R.L., Kenney, W., Zeszotek, E., and Singer, T.P. (1971). Eur. .I. Biochem. 2, 321-327. Chuang, H.Y.K., Patek, D.R., and HellerG, L. (1974). E. --J. Biol. w, 2381-2384. Maycock, A.L., Abeles, R.H., Salach, J.I., and Singer, T.P. in and Flavoproteins (1976). Elsevier Press, T.P. Singer (ed). Flavins Amsterdam, pp. 218-224. Nsgy, .I., Kenney, W.C., and Singer, T.P. (1979). J. --Biol. Chem. 254, 2684-2688. Minamiura, NT., and Yasunobu, K.T. (1978). Arch. Biochem. Biophys. 189, -I__ 481-489. Tabor, C.W., Tabor, H., and Rosenthal, S.M. (1954). e. s-, --3. Biol. 645-661. Lowry, O.B., Rosebough, N.J., Farr, A.L., and Randall, R.J. (1951). J. --Biol. Chem. 193, 265-271. Kearney, E.B. (1960). J. Biol. Chem. 235, 865-877. -___-

58.5