Flavin binding by bacterial luciferase: Affinity chromatography of bacterial luciferase

Vol. 57, No.4,

1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FLAVIN BINDING BY BACTERIAL AFFINITY

CHROMATOGRAPHY OF BACTERIAL

$

C. A. Waters+, J. R. Murphy

The Biological

Laboratories,

Cambridge,

Received

LUCIFERASE:

March

LUCIFERASE

, and J. W. Hastings

Harvard

Massachusetts

University,

02138

11,1974

Immobilized FMN covalently attached to Sepharose-6Bhexanoate binds bacterial luciferase. Immobilized flavin is also effective in its reduced form as a substrate in the light emitting reaction catalyzed by luciferase. Bioluminescence and aldehyde

oxygen

as substrates

(1,2,3).

is much lower constant

substrate

that

of the

with

binds

M-l)

bound

is

attachment luciferase.

in the

form

enough

utilizes

oxidation

of oxidized

reduced high

luciferase

function

affinity

FMN would

of Mosbach

FMN by covalent it

in a mixed the

than

of the procedure

by bacterial

Although

(K = 4 x lo3

chromatography

that

catalyzed

to suggest

be successful.

and co-workers

(7),

reduced

form it

luciferase

its

association

that

affinity

Using we have

to a Sepharose-GB-hexanoate In its

by molecular

FMN for

(4,5,6),

a modification immobilized

matrix is

FMNH2

effective

and found as a

reaction.

MATERIALS: Sepharose 6B was purchased as the pre-swollen gel from Pharmacia Fine Chemicals. FMN, riboflavin and cytochrome c were obtained from Sigma, dicyclohexylcarbodiimide and 6-aminohexanoic acid from Aldrich, bovine serum albmin cyanogen bromide (CNBr) from Eastman, crystallized (BSA) from Miles Laboratories and ovalbumin from Worthington. 5'Phosphopentyl flavin, Li salt, was a gift of Drs. D.B. McCormick and M.N. Kazarinoff. The two luciferases used in these studies, designated as Pf and MAV, were isolated frcun two species of luminous bacteria assigned by Reichelt and Baumann (8) to Photobaeterim fischeri (Pf, ATCC 7744) and Beneckea harveyi (MAV, Strain 392).

t

Fellow, National Institutes of Health ++ Predoctoral Department of Microbiology and Molecular Present Address: Harvard Medical School, Boston, Mass.

Copyright 0 I974 by Academic Press, Inc. All rights of reproduction in rrny form reserved.

1152

Genetics,

Vol. 57, No. 4, 1974

BIOCHEMICAL

METHODS AND RESULTS: --washed

with

0.1 M

in the

same buffer,

initiated

by the

formamide,

NaHCO

borate

stirred

for

500-ml

aside

preservative,

to 4o and adjusted

to pH 11.

was maintained

was filtered

activated

buffer,

pH 8.5,

18 hours

(v/v).

distilled

with

resuspended Activation

After

cold

was resuspended containing

at 4O.

The gel

After

followed

was

30 minutes

0.2 M sodium

was then

40 ml of 0.2 M

washed

pH 8.5,

by extensive the gel

A solution

was added

and the

acid

sequentially

and with

0.1 M NaHC03, 0.01

washing

with

was resuspended

q FMN and transferred

flask.

Erlenmeyer

24 ml pyridine

0.4

in

2.6 g 6-aminohexanoic

buffer,

filtration

H20 containing

shielded

at pH 11.

and washed

gel

0.5 M NaCl and H20,

pyridine

6B was

of 3 q CNBr in 6 ml N,N'-dimethyl-

each of 0.2 M sodilmn borate

HCl,

in

the

addition

slurry

of 30 ml of Sepharose

pH 0.5.

The washed, sodium

volume

to remove

dropwise

and the

buffer,

3

cooled

at 4 O, the material borate

A settled

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

M

80% aqueous in 6 ml

to a 250-ml

liqht-

of 10 q dicyclohexyl-carbodiimide slurry

gently

agitated

at 37O for

4 days. In order

to remove byproducts

was sequentially 95% ethanol,

washed H20,

with

and unbound

95% ethanol,

10q3 M HCl,

cold

and stored absorbance

of the acid

indicated

that

gel

was stable

for

hydrolysates

2.4 pmoles at least

Riboflavin-Sepharose procedure, gel

with

utilizing

but

only

about

the hexacarbon FMN.

EMN derivative a 5'-phosphopentyl

Attempts in which group

H20, warm

(45')

1-butanol,

0.1 M NaHCO 0.5 M NaCl and H20 3'

in 0.2% NaN 3 at 4 0 in. the

gels

FMN, the FMN-Sepharose

dark of the

of FMN were

until

used.

The 450 nm

flavin-substituted bound

per

Sepharose

ml of gel.

The

one year. conjugates

were

prepared

0.1 pmoles

were

bound

spacer

alone

were

the

link

unsuccessful.

1153

side It

chain

is possible

Control

the steps

5'-phosphopentyl

5'-phosphoribityl

same

per ml of gel.

was made by omitting

to covalently the

using

is

flavin,

an

replaced

by

that

the

Vol. 57, No. 4, 1974

coupling

BIOCHEMICAL

reaction

esterification

between

group

function

as an effective

mixed

side

using

This

equilibrated

was applied

and the

to baseline.

Bound

of either

column

by the method

Luciferase activity initiating

using

the

reduced by the

the

together injection

reaction

Luciferase

activity

is

standard

of Hastings

of FMN.

the

A),

sample

the A2So returned the

was determined

in

concentration

pattern using

with

a Cary Model

at O" with cell

a Radiometer

extracts

were

by Gunsalus-Miguel decanal

in which

with

and Weber

In an experiment

to as buffer The protein

A until

catalytically

Ill),

expressed

elution

cm)

(9).

with

Both MAV and Pf luciferases illustrates

x 2.6

M phosphate,

NaCl.

was measured

by the

of aldehyde

in

The FMN-Sepharose

by increasing

at 21' 2 2' with

a vial

(0.9

referred

buffer

as described

assay in

of a

yield

(0.02

without

ml)

of Lowry

"dithionite"

may

lower

colons

concentrations

was measured

group

same buffer. (1.2

was purified

the

(DTT),

was eluted

Protein

terminal

Michelson,

of 18 ml/b.

with

Conductivity

meter.

the

(A.M.

2 M NaCl in buffer

fraction

15 spectrophotometer.

either

rate

same buffer

luciferase

The A20o of each

determined

at 4O in

developed

NaCl or FMN in the

conductivity

out

with

in the

with

the production

for

5 x 10 -4 M dithiothreitol

pH 7, containing and then

through

intermediate

of 2 ml at a flow

by washing

group

same treatment.

was carried

a bed volume

was regenerated

agent

may account

by the

involves

The FMN phosphate

anhydride

riboflavin

Chromatography

hydroxyl

spacer.

acylating

ccmnnunication).

immobilizing

Sepharose-hexanoate

chain

of the hexanoate

phosphate-carboxylate

personal

It's

FMN and the

of a ribityl

carboxyl

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

addition excess

as the reduced

the

flavin

of solid oxygen

in quantassec

et al.

(10).

aldehyde, FMWH2 (lo),

and luciferase dithionite,

to initiate -IL (qssec-'

or are

followed the

1, using

reaction. the

(12). bind

to FMN-Sepharose.

of MAV luciferase

using

the

elution

Pf enzyme the

1154

Fig.

la

a linear pattern

gradient was

BIOCHEMICAL

Vol. 57, No. 4, 1974

similar,

except

been

of the fact

estimated that

to their

interaction with

that

slightly

the

NaCl,

MAV).

the binding

to be considerably

indicate

eluted

the Pf eluted

to 3 x 10 -3 M FMN, for

Pf compared in view

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

affinity

This

of free

is

the FMN ligand.

as shown in Fig.

lb for

FMN for

M

somewhat

surprising

FMN to Pf luciferase

(2 to 20 times)

of luciferases

with

-3 (5 x 10

later

for

weaker

this

The bound

gel

(5). is

Pf enzyme.

This

not

enzymes With

has may

due solely can also

be

MAV, the

0.4

03

02 01 0

FMN-Sepharose chromatography of MAV luciferase using a gradient Figure la. of FMN for elution MAV luciferase (4 m ) in 0.25 ml of buffer A, having a to a column prespecific activity of 1.4 x 1014 q.sec- 4 =mg-1, was applied Pf luciferase. equilibrated with the same buffer and eLuted with FMN. lb. The sample (3 mg) in 0.3 ml of buffer A, having a specifz activity of 1.2 x 1014 qssec-l*mg-l, was applied to a column pre-equilibrated in the same buffer and eluted with a linear NaCl gradient as shown (dashed line). x) and luciferase activities (0) are Absorbances at 280 nm (dotted line, plotted for each fraction. Specific activity of a pool of fractions 21-30 was 1.5 x 1014 q*secml-mgwl. Center ordinate scale, applicable to solid lines in both panels, luciferase activity in units of q.sec-l*ml-1-1013 (one division equals one unit).

activity

eluted

slightly

earlier

luciferases

do not bind

FMN; of the

4 x 10 14 activity

column, buffer

in the NaCl gradient

appreciably

90% of the Pf activity

to the

units

than

did

Pf.

Sepharose-hexanoate

of each enzyme loaded

and 98% of the MAV activity

The

lacking onto

such a control

eluted

in the

wash. Luciferase

Purified

can also

MAV luciferase

be separated was mixed

from

with

a mixture

BSA, cytochrome

1155

of non-flavoproteins. c and ovalbumin,

Vol. 57, No. 4, 1974

applied

BIOCHEMICAL

to the FMN-Sepharose

proteins

came through

characteristic

Crude

cell

extracts

gel

Luciferase

qsec

9

12

-1

q*sec

, similar

volume

colon,

0.35

This

with

buffer, enzyme

colan.

pH 7, (specific

on a Sepharose-

to the hexacarbon

in the buffer

and eluted

this

on DEAE-Cellulose.

was chromatographed bind

material.

using

M phosphate A.

at its

of eluted

starting

achieved

of buffer

eluted

activity

batch-adsorbed

which

came through

luciferase

to the

was

with

*rng-l)

the

The non-flavo-

The specific

MAV were

to remove proteins

the FMN-Sepharose

-1

of luciferase

a large

activity

wash while

was eluted

against

of 8 x 10

and chrcmatographed.

gradient.

-1

of strain

activity

and dialyzed

hexanoate

14

purification

The luciferase

activity

in the

was 1 x 10

A partial

column

in the buffer

position

luciferase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

spacer.

wash and was applied

a linear

NaCl gradient

to (Fig.

1.l 0.5 04

:

6

12

16

40

Ci I:

0.3

x

‘.I /’ 9. ‘.: ,,,’

44

48

FRAC T/O/V

52

I’ /’

0.2

1

56

2).

&

12.0 9.6 6

u

k

-z. F ‘: 4.6 c, $1 $ 1: z 7.2

60

NUMBER

purified luciferase was applied to the FMN-Sepharose Figure 2. Partially Some contaminating proteins came through in the wash (tubes 8-12). column. as well as scnne other unidentified proteins, was eluted with NaCl. Luciferase,

The pooled Since

luciferase

highly

purified

had a specific

activity

MAV luciferase

has a specific

1.4 x 1014 q-set-l-mg-l, Sepharose

step

Immobilized dithionite,

is

the MAV luciferase

estimated

to be about

FMN, reduced

is effective

either

of 6 x 10

activity

13 qssec -1 *mg -1 .

activity after

with

in

luminescence

1156

the FMN-

40% pure.

catalytically

as a substrate

of about

the

H2 or with reaction,

Vol.

57, No,

BIOCHEMICAL

4, f 974

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

60

40

20

0

6

12

I6

24

30

0

3

6

9

12

TIME fSEC0ND.S~ Figure 3. The kinetics of the reaction, measured by the time course of the bioluminescence, are the same with immobilized FMNH2 (x1 as with soluble FMNH (o), both with dodecanal (left) and decanal (right). Curves for reac 6 ions with soluble and insoluble F$@IH are normalized to the maximum initial intensities in each case. Note t 8 at the turnover time is quite different with the different aldehydes. Apparent first order rate constants for decay of luminescence: decanal = 0.37 set"; dodecanal, 0.056 set-l. M&V luciferase, 250C.

in

spite

in

less

of the than

2% of that the

that

one second with

turnover

same as that unexpected

fact

free for

for

soluble

(13)

and also

time

substantially.

(13).

competitive

the chain

because

the reaction

length

that

with

is only

of immobilization. by insoluble decanal

substitutions

of the

of FMNH2 occurs

maximum rate

initiated

FMNH2, both

of the fact

autoxidation

The apparent

FMNH2, perhaps

time

in view

the

aldehyde

about But

FMNH2 is

the

and dodecanal

(Fig.

on the

molecule

(14),

flavin

may alter

31,

turnover

was supported in part by National Science ACKNOWLEDGMENTS: This research Foundation Grant GB 31977X. The authors wish to thank Drs. T. 0. Baldwin, S.-C. Tu, and K. C. Backman for their critical reading of this manuscript We are also indebted to Ann Gunsalus-Miguel and nmerous discussions. for her luciferase preparations. REFERENCES 1. 2.

Hastings, J.W. (1968) Annu. Rev. Biochem. 37, 597-630. Hastings, J-W., Ebexhard, A-,-Baldwin, T-O., Nicoli, M-Z., Cline, Nealson, K.H. (1973) in Chemiluminescence and BiolUninescence

1157

T.W.,

Vol. 57, No. 4, 1974

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(M.J. Cormier, D.M. Hercules, J. Lee eds.) pp. 369-380, Plenm Publishing Co., New York. G.A., Bogacki, I.G., and Meighen, E.A. (1973) Dunn, D.K., Michaliszyn, Biochem. 12, 4911-4918. Gibson, Q.H., Hastings, J.W., Weber, G., Duane, W., and Massa J. (1966) in --Flavins and Flavoproteins (E.C. Slater ed.) pp. 341-359, Elsevier Publishing Co., Amsterdam. Baldwin, T.O. (1974) Biochem. Biophys. Res. Comm., in press. Nicoli, M.Z. (1972) Ph.D. Thesis, Harvard University, Cambridge, Mass. Mosbach, K., Guilford, H., Ohlsson, R., and Scott, M. (1972) Biochem. J. 127, 625-631. Reichelt, J.L., and Baumann, P. (1973) Arch. Mikrobiol. 2, 283-330. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-276. Gunsalus-Miguel, A., Meighen, E.A., Nicoli, M-Z., Nealson, K-H., and Hastings, J.W. (1972) J. Biol. Chem. 247, 398-404. Meighen, E.A., and Hastings, J.W. (1971) J. Biol. them. 246, 7666-7674. Hastings, J.W., and Weber, G. (1963) J. Opt. Sot. Am. 53, 1410-1415. Gibson, Q.H., and Hastings, J.W. (1962) Biochem. J. 83, 368-377. J.W. (1969) J. Biol. Chem. 244, 2572-2576. Mitchell, G.W., and Hastings, Hastings, J.W., Spudich, J.A., and Malnic, G. (1963) J. BE. Chem. 238, 3100-3105.

1158