Separation of the apoprotein and reconstitution of the holoprotein from the long-lived intermediate in bacterial bioluminescence

BIOCHEMICAL

Vol. 58, No. 1, 1974

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

SEPARATION OF THE APOPROTEIN AND RECONSTITUTION OF THE HOLOPROTEIN FROM THE LONG-LIVED Charles

INTERMEDIATE IN BACTERIAL

L. Murphy,

George

J. Faini

BIOLUMINESCENCE

and John

Lee

Department of Biochemistry University of Georgia hthens, Georgia 30602

Received

March

8,1974

Summary : A long-lived intermediate in bacterial bioluminescence, which has been suggested to be an FMN flavoprotein, has been separated as an apoprotein plus free FMN and the holoprotein reconstituted by addition of FMN (Ka = 7 x lo5 M-1). The apoprotein preparation reacts with long-chain aldehyde to give the full quantum yield observed for the complete system. Only after removal of all remaining FMN in the apoprotein preparation by prior dialysis of luciferase against KBr and inclusion of apoflavodoxin in the reaction mixture, can a dependence of the light output on FMN be observed. Bacterial bioluminescence therefore appears to be in the class of sensitized chemiluminescence with FMN acting as the specific sensitizing agent. FMNH2 reacts hyde

to form

with

aldehyde

mediates first

bacterial

long-lived

with after

molecule

with

luciferase

light

"MAV" type

luciferase

to which

absence

can at

later

Lee and Murphy

(2)

intermediates

to produce

reaction

in the

(1).

of luciferase, one molecule

which

(E) (3).

of aliphatic times

FMNH2 and oxygen of FMN (oxidized)

be reacted

identified

The intermediate

alde-

two interwhich

was a reduced

appeared luciferase

was bound:

E + 2FMNH2 + 02 -f Xl + FMN + H202.

(t%,

The intermediate

Xl took

1.3 min

form

5O C) to

up oxygen

in a psuedo-first

intermediate

order

reaction

the

fluorescence

X2:

x1 + 02 -f x2. The fact absorption better from Copyright All rights

the

that

X2 was a flavoprotein

properties

of the

characterize

this

mixture

by Sephadex

mixture

intermediate

0 I974 by Academic Pres.r, Inc. of reprodurtion in any jorm reserved.

(G-25)

was deduced (intermediate it

was decided

chromatography

119

from

X2 and FMN). to

separate

at O" C where

and

In order

to

the

free

FMN

the

longer

Vol. 58, No. 1, 1974

lifetime

of

found

the

however,

BIOCHEMICAL

intermediate that

intermediate

is

not

only

removed

The holoprotein

(t%,

14 min)

allows

free

FMN but

also

by this

(holo-X2)

AND BIOPHYSICAL

process,

RESEARCH COMMUNICATIONS

this most

thus

manipulation. of the

producing

can be reconstituted

FMN bound

It

was

to

the

an apoprotein

by addition

(ape-X2).

of FMN to the

apoprotein. Though

the

apo-X2

in the

holoprotein,

it

phatic

aldehyde

The light moved

yield

from

the

of flavin

is

sensitizing

preparation

contained

was observed

to the

apo-X2

is

reduced

not

apo-X2

than

the

preparation until

mixture.

greater

that

less

lOO%,

than

light

yield

was the

last

traces

Since

under

then

in this

5% the

amount

on addition

same as that

of

flavin

of

ali-

of holo-X2.

of FMN are vigorously

these

conditions

step

flavin

the must

re-

quantum

yield

be acting

as a

agent. EXPERIMENTAL

Luciferase were

measured

with

a Cary

made with ing

used

column

results

previously

cc,

of

protein were

15 to

G-25 eluted

in

essentially

mixed

the

by the

flavodoxin LeGall) quenched

Both

same.

Sephadex with

at a rate

of

column

where

column

all 3

and in-

components When

ml/min.

(2 cm x 30 cm) was

respectively

columns

were

APO-X2 was made by layerG-25

minutes

taken

measurements

luciferase

chromatographed

experiments,

constant

for

the

generation

of apo-Xp

was determined

by the

was prepared by extensive the

the

were

(QB)

completely FMN rather

and the

final

separated than

FMN from

FMNH2, was

proteins.

The equilibrium

concentration

a jacketed

2-3 and 9-11

the

or BSA in control

measured

of

(4).

(1 cm x 15 cm) or a longer

luciferase with

described

yields

spectra

Fluorescence

30 NM) into

was then

quantum

Absorption

spectrophotometer.

The mixture

the

(2,6).

(~80 PM, ICC) on top

FMNH2 (0.1

a short

and bioluminescence

described

14 recording an instrument

O" C.

purified

as previously

luciferase

jecting at

and FMN were

from dialysis

fluorescence

system

and addition amount

Desulfovibrio

added

2 M KBr

FMN in the 120

+ FMN z holo-X2)

back

of FMN.

of FMN fluorescence gigas

at pH 3.9, of

(ape-X2

flavodoxin (5).

manner

The holo-X2 quenched.

(a gift

The apoflavodoxin reported

was

(5).

from

ApoJ.

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

WAVELENGTH

RESEARCH COMMUNICATIONS

(nm)

Figure 1. Curve A, absorption spectrum of the chromatographed intermediate (apo-X2); B, apo-X2 (3.6 PM) and added FMN (2.8 uM); C, after warming this mixture; D, the holo-X2 estimated by subtracting the free FMN calculated from the binding constant.

Flavin tially

from

removed

irreversibly

contaminating

flavoproteins

by dialysis denatured

against at

in the

1 M KBr but

luciferase

at pH 7.0

used was par-

since

luciferase

is

low pH. RESULTS

Absorption Curve with

A, Figure

1, shows

FMNH2 immediately

absorptions would

can be seen

require

spectrum

after

that

if

result.

products,

is very

there

(2.8

uM, final

(3.6

uM) the

and free

with

absorption

amount

bound

little

is

to this

(curve

On warming,

carries

in this

added

luciferase

spectrum.

than

an absorption

absorption 20 x 10m3 cm-'

intermediates

to final

However,

when FMN

chromatographed

B) of a mixture

121

X2 present

measurable

greater all

reacted

No distinct

of intermediate

density

which

change

of

FMN, an easily

an optical

spectrum (2).

spectrum

on G-25 Sephadex.

the

On warming

concentration)

FMN results

though

contained

of a flavoprotein

at 445 nm would

absorption

chromatography

even

it

the

of spectrum

intermediate the

X 2 flavoprotein identical

to

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

InoLo-xzi (TOTAL

x,1

Figure 2. Scatchard plot of the binding to FMN to intermediate X2 as determined by addition of FMN to apo-X2. The amount of holo-X2 was estimated by quenching of FMN fluorescence.

that

of

from

fluorescence

(curve

FMN returns

(curve

C).

quenching

B.

This

reported

the

spectrum

constant

absorption

spectrum

of the

corresponds

free

calculated of bound

FMN

FMN contribution

to that

from

of intermediate

X2

(2).

The chromatography, bound

equilibrium

by substraction

absorption

before

the

(below),

D) can be computed

curve

Using

FMN forming

therefore,

apo-X2

from

results

which,

in the

by addition

removal

of both

of FMN, the

free

holo-X2

and

can be

reconstituted. Fluorescence After apo-X2

preparation

by the

less

responds present. FMN is

a detectable

warming,

to

by fluorescence

sensitive

absorption

less

10% of

than

In control carried

down the

vious

conclusion

results

are constant

(2)

the that

shown in Figure is

expected

observed

amount,

varies

based

from

on the

with

the

bound 2 in

the

run

amount

(BSA + FMN or luciferase

fluorescence the

PM) can be found

This

measurements, that

column

of FMN (0.1-0.3

measurements.

experiments

APO-X2 quenches

tion

amount

not

in the

detectable

to run

and cor-

of X 2 (2-3 !-Ml

+ FMN) no detectable

protein. of added

FMN.

This

confirms

FMN in X2 was nonfluorescent. form

to be 7 x lo5

122

of a Scatchard

M-l.

The somewhat

plot. greater

our preThese

.

The associathan

one

BIOCHEMICAL

Vol. 58, No. 1, 1974

AND BIOPHYSICAL

Quantum yields on reaction and the additions indicated.

TABLE.

of

the

apo-X2

RESEARCH COMMUNICATIONS

preparation

with

decanal

Addition

none

0.085

0.45

uM)

0.079

0.0052

FMN (28 PM)

0.093

0.00045

FMN (2.8

apoflavodoxin

(12.8

apoflavodoxin

(64 PM) none

uM)

C

apoflavodoxinC

(13 uM)

0.056

>1

0.056

>l

0.079

.1.2

0.036

>>l

X2 as equal to one half the added FMNH2 ( QB(FMNH2) = 0.03, a. Estimated reference 6). b. Estimated FMN by fluorescence of the flnal reaction mixture after warming. c. Prior dialysis of luciferase against KBr (2 M, O" C, 2 days).

to one binding reduction lead

of FMN by room

to a greater

amount

of

--Quantum

flavin

formation

necessary of

for

of intermediate

these

intermediate

by photo-

manipulations.

than

that

This

estimated

would

from

the

added.

Yields yield

apoprotein

1 mg/ml) is used

since

into

decanal

at

O" C, though

production

is greatly

with

respect

the quantum FMN does

measurements

solution

containing

of light yield

light

concentration

reduced

Quantum the

may be due to additional

not

yield affect

of apo-X2

were made by injecting

1 cc of buffer

solution

final

quantum

reduced.

of Xp is very light

nearly yield

yield

As seen

to FMN can be much greater

the

(phosphate,

(40 uM) at room temperature. the

the or the

123

This is

in the

than

the Table,

any of the

same as aldehyde rate

0.1

of

light

cc of

pH 7, BSA temperature same, the

the

quantum

reactants (0.05)

emission.

rate

while

(6). If

Added how-

Vol. 58, No. 1, 1974

ever,

BIOCHEMICAL

apoflavodoxin

not

completely.

first

dialyzed

is

against

yield

concentration

FMN on the

replace

the

light

yield

is

can be increased

48 hours, FMN in

FMN in

RESEARCH COMMUNICATIONS

which

if

results

role

but

luciferase in the

a contaminating

its

quenched

is

removal

of

flavoprotein.

in the

final

steps

This leading

to

(7).

The quantum its

of quenching

perhaps

material,

emission

aldehyde

1 M KBr for

can apparently

light

before

The amount

some fluorescent protein

added

AND BIOPHYSICAL

of X2 is probably

may be slightly

the

same as that

underestimated

of

due to the

aldehyde

but

again

photoreduction

of

column. DISCUSSION

These

results

intermediate

support

in the

composition

strongly

bound most

FMN (2).

dicated

by the

by the

quenching

The light decanal

are low

result

support

peroxide

from

the

it

the

established,

Sephadex simply

column

of

breaks

FMN is

is

the

to produce back

the

FMN, as in-

X2 absorption

spectrum

the

a recent

claim

little

doubt

or other,

be a specific

(a),

certainly

of apo-X2

the

probably

it

since

light bound

bound

are

and

reduced M.

two possible

roles

for

the

124

apo-X2

This

hydro-

flavin

comes from

to the protein

hydro-

a flavin

as a cation

in an enzymatic

in

to

breakdown.

emission

chromophore

with

flavin

of the

removal

to its

is

or lo-*

luciferase

lead

sensitizing

reaction

X2 concentration

that

that

the

of FMN until

the

intermediate would

of

concentration 1% of

protein is

the

yield

oxidation

aldehyde. There

down

not

which

by adding

characteristic

than

the

in some form

FMN must

of

much less

does not

molecule

of

and quantum

independent

there

on the

is reconstituted

intensity

constitutes

of

separated

long-lived

a flavoprotein

aldehyde

show that

rapidly

the

of FMN fluorescence.

peroxide

then

FMN is

is

aliphatic

results is

(2) that

reaction

of

The present

reappearance

levels,

Since

absence

to X2 and equilibrium

of the

conclusion

bioluminescence

In the

The holoprotein

apo-X2.

very

bacterial

E(H202)FMN.

to H202 and free

reason

our previous

the

complete

reaction

(9),

Vol. 58, No. 1, 1974

scheme.

The first

specific

sensitizer.

BIOCHEMICAL

is that

w

it

AND BIOPHYSICAL

reacts

directly

RESEARCH COMMUNICATIONS

with RCHOand the FMN is a

- X2 + RCHO+ apo - Xp - RCHO + FMN E + RCOOH+ hv

Alternatively can be described

the Xp may react with RCHOdirectly as a catalyst

as well

in which case the FMN

as a sensitizer.

aw - X2 + FMN = holo - X2 holo - X2 + RCHO-+ X2 - RCHO + E + RCOOH+ FMN + hv REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Hastings, J. W. and Gibson, Q. H., J. Biol. Chem. 238, 2537 (1963). Lee, J. and Murphy, C. L., Biochem. Biophys. Res. Comm. 53, 157 (1973). Hastings, J. W., Weber, K., Friedland, J., Eberhard, A.,%tchell, G. W., and Gunsalus, A., Biochemistry 8, 4681 (1969). Wampler, J., and DeSa, R., ApplFed Spectroscopy 6, 623 (1971). Mayhew, S. G., in "Flavins and Flavoproteins" Kahn, H., Ed., University Park Press, Baltimore, p. 185 (1971). Lee, J. and Murphy, C., in "Chemiluminescence and Bioluminescence" Cormier, M. J., Hercules, D. M., and Lee, J., Plenum Press, New York, p. 381 (1973). Lee, J., Murphy, C. L., Faini, G. J., and Baucom, T. L., in "Liquid Scintillation Counting: Recent Progress" Stanley, P. E., and Scoggins, B. A., Eds., Plenum Press, New York, in press (1974). Hastings, J. W., Balny, C., Le Peuch, C., and DOUZOU,P., Proc. Nat. Acad. Sci. (USA) 70, 3468 (1973). Eley, M., Lee, J., Lhoste, J.-M., Lee, C. Y., Cormier, M. J., and Hemmerich, P *, Biochemistry 2, 2902 (1970).

125