Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MOLECULAR WEIGHTS OF NITROGENASE

COMPONENTS

FROM AZOTOBACTER VINELANDII Ronald

H. Swisher,

Michael

Landt

and Francis

J.

Reithel

Department of Chemistry Universitv of Oregon Eugene, OEegon 97&O 3 Received

August

25,

1975

SIJMi4AHY. Meniscus depletion sedimentation equilibrium ultracentrifuge experiments were performed on purified MoFe and Fe proteins-of Azotobacter vinelandii. The MoFe protein was found to have a molecular weight of 245,000, using an experimentally confirmed partial specific volume of 0.73. The MoFe protein formed one band on sodium dodecyl sulfate gel electrophoresis and had a subunit molecular weight of 56,000. The subunit molecular weight from ultracentrifuge experiments in 8 Ill urea was 61,000. The molecular weight of the Fe protein was calculated to be 60,500 in meniscus depletion experiments. Similar experiments in 8 M urea solvent indicated a subunit molecular weight of 30,000. A subunit molecular weight of 33,000 was obtained from sodium dodecyl sulfate gel electrophoresis experiments. Nitrogenase

consists Neither

and an Fe protein. alone,

but

nature

of the

when they

and accurate ents

also

In genase filtration values for

the

'SDS:

are

physical

a recent

activity

complex

hindered

in --

from

and SDS1 gel

(1)

dodecyl

is

vivo

extreme

component molecular

A. vinelandii

for

The value

sulfate

fix

the for

nitrogen

restored.

is still

The

obscure,

separate

oxygen

compon-

sensitivity

proteins. weights

of the

were determined

nitro-

using

The molecular

electrophoresis.

were 64,000

MoFe protein.

the

will

of the

by the

of both

paper

components

sodium

combined

an MoFe protein

protein

characterization

instability

reported

component

nitrogenase

has been

and general

of two proteins,

Fe protein the

gel

weight

and 216,000

MoFe protein

is

sub-

Vol. 66, No. 4,1975

stantially They

BIOCHEMICAL

lower

reported

Archibald

than

a value

approach and SDS gel

of the

two component

quantitative

are

as modified

molecular

of both

molecular

weight

'?"ne Chervenka attained

method

rapidly

Reliable

molecular

depletion

sedimentation

Chervenka

(4)

if

on microweights

further

of each of

the

advantage

that

traditional

the

as well

component

can be obtained

the

equilibrium

to determine

NoFe and Fe proteins

has the

than

baseo

electron

to be obtained

(2,3).

to be pursued.

and data

concentrations

daltons

experiments,

need

by

weight

and coworkers

to 300,000

electrophoresis. proteins

RESEARCH COMMUNICATIONS

by Hardy

270,300

used meniscus

experiments

of

of

studies

de have

reported

to equilibrium

graphs

subunit

that

AND BIOPHYSICAL

as the

proteins.

equilibrium over

is

a wider

meniscus

range

depletion

experiment. WETHODS.

The component

Shah and Brill twice

(5)

and the

cation

except

last

brand).

the

ultracentrifuge

were prepared an Amicon of subunit

(pH 8.01,

for

with

0.25

(pH 7.0)

2.5

was used

purifi-

proteins containing

was used

double

sector

as the

dialysate

cells.

Samples

by ultrafiltration

a denaturing

in

For determination solvent

of

8 M urea,

inM EDTA and 50 mI\I potassium and runs

were conducted

at room

temperature. Sedimentation using

a Spinco

equilibrium model

experiments

E ultracentrlfuge

1477

of

and 50 mM sodium

device.

weights,

native

buffer

8MC microultrafiltration molecular

Fe protein

a solvent

Iii NaCl

method

was crystallized

of the

on the

ultracentrifugation

125 mM 2-mercaptoethanol, phosphate

step

This

by the

MoFe protein

experiments

(Xannox in

tile

at O-5'C

50 mN Tr5.s chloride

reference

that

All

anaerobically

dithionite

were purified

electrophoresis

was omitted.

were done

proteins

were carried using

12 mm double

out

Vol. 66, No. 4, 1975

sector

BIOCHEMICAL

synthetic

interference copic

boundary patterns

plates.

cells

with

sapphire

were recorded

Fringe

Shadowgraph

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

patterns

and processed

windows.

on Kodak

were measured

by computer

Rayleigh

type

II

with

a Nikon

programs

G spectros-

written

in

this

laboratory. SDS gel of the

electrophoresis

method

denatured

in

of Weber a solvent

was conducted and Osborn

(6).

consisting

of

ethanol,

0.2% EDTA and 0.33

disc

electrophoresis

gel

polyacrylamide Activity

gels

Aerograph

Porapak

(7)

RESULTS.

tation

since

runs

one sharp

band

phoresis

activity nanomoles

albumin

Kleiner

weight

and Chen (1)

regular the

Plots

value

c vs.

band

a

D20 replacing

method

was 0.73 r2 from

of Edelstein

polyproduced

electrodetermined

was 1450

specific acid

sedimen-

Specific

of Fe protein

amino

in

also

was 56,000.

the

homo-

during

weight

experimentally

run with

obtained

of In

from

the

peak

The partial

was verified

using

solvent,

with

was judged

molecular

excess

mg.

an ultracentrifuge

on a

biuret

SDS and subsequent

on SDS gels

calculated

the

The MoFe protein with

of ethylene/min

assay

fitted

using

7%

buffer.

reduction

symmetrical

The subunit

a large

with

as standard.

a single

of migration

were

Standard

cm tubes

(pH 9.2)

MoFe protein

on treatment

MoFe protein

acetate

acetylene

electrophoresis.

with

0.5

and moved as a single

on SDS gels.

on the basis

in

was measured

produced

velocity gel

(8);

serum

it

(pH 8.0).

1200 gas chromatograph

Protein

with

acrylamide

out

the

The recrystallized

geneous

the

Series

R column.

reaction

using

proteins

1% SDS, 1% 2-mercapto-

chloride

and 50 mM Tris

a modification

Component

was performed

was measured

Varian

M Tris

using

volume analysis

for of

by carrying 1~20 as the and Schachman

ml/gm. meniscus

1478

depletion

ultracentri-

Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1. Results obtained from meniscus depletion equilibrium The natural logarithm of MoFe sedimentation of IJIoFe protein. protein concentration in fringes is plotted versus the square of the radius. Rotor speed was 9057 rpm. Temperature was 0.9'C and run time has 15 hours. Solvent conditions were described in the text.

fuge

experiments

as shown in MoFe protein

on the

Fig. based

Sedimentation in

subunits

of the

obtained

on several

order

experiments the

NoFe protein.

and the

weight

such runs

to determine

unchanged

MoFe protein

The molecular

equilibrium

8 Pl urea

to remain

1.

recrystallized

(9).

molecular

calculated

were also molecular

plots

weight

for

was 245,000

volume

of In

c vs.

was determined

linear the

2 5,000.

performed

weight

The specific Linear

were

in

of the was assumed r2 were

to be 61,000

+1,000. The Fe protein the in with

Shah and Brill the

ultracentrifuge SDS and analysed

obtained

from

procedure

(5)

the

was found

and to yield by SDS gel

1479

second

DEAE column

in

to be homogeneous

a single

electrophoresis.

band

when treated The subunit

Vol. 66, No. 4, 1975

molecular In

BIOCHEMICAL

weight

c vs.

protein

r2 from were

molecular

acid the

weight

tl,ooo.

linear

plots

30,000

volume

protein

of In

of 0.72. from with

from

c vs. the

data

Fe protein,

most plot

concentration

of 0.72,

conducted

in

obtained

of

Fig.

2.

the

runs

also

a partial

of the

on titration

was 600 nanomoles

amino

to be gave

a molecular

assuming

Fe

we calculated

8 N urea

r2 and indicated

of

average

from

of several

activity

range

shown in

data

subunits,

on native

of the

calculated

and Chen (l),

Plots

of weight

is

volume,

The specific the

over

the

was 33,000.

experiments

A typical

Experiments

t 500 for

calculated

depletion

specific of Kleiner

molecular

60,500

of

versus

partial

analysis

on SDS gels

to be linear

measured. weight

the

meniscus

found

concentrations

Using

determined

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

weight specific

Fe protein, of the

of ethylene/min

MoFe mg.

weight distribution obtained from meniscus Figure 2. Molecular Weightdepletion equilibrium sedimentation of Fe protein. average molecular weight of the Fe protein is plotted versus The rotor speed was 18138 rpm, cell concentration in fringes. and run time was 11 hours. Solvent temperature was 0.25oc, conditions were described in the text. 1480

Vol. 66, No. 4,1975

BIOCHEMICAL

DISCUSSION. proteins

Previous from

SDS gel

electrophoresis, Gel

actually

and thus a firm

remove

all

We have

molecular

and to

would

qualification. values

determined

of the

partial

specific

this

paper

protein

using

nation which sued

in

partial

fraught this

of

with

be taken

the

SDS gel

upon published

(81,

precision.

but

depend

experimental

molecular

The values acid

verified

for

the only

experimental

difficulties,

the

used

composition

method

of

one

upon a knowledge

protein.

Direct volumes

with

absolute

this

content.

equilibrium

reliable

amino

has been

specific

from

of the

of the

sulfhydryl

to

electrophoresis

to be more

method

method

precautions

appear

by this

by this

electrophoresis

obtained

obtained

molecular

SDS gel

reduce

the

filtration,

accurate,

than

The values

D20 method digit

of the is

thus the

two significant

fully

component

ultracentrifuge

rather

that

volume

are based

The value

(1).

on gel

determinations

The validity

weight

in

of the

reasonably

foundation.

10%.

ultracentrifugation major

weight

precision

to be only

radius

requires

iron

the

been based

although

Stokes

proteins

found

method

the

bound

have

values

and non-equilibrium

thermodynamic

of iron-sulfur

weight

filtration,

measures

weight lack

molecular

A. vinelandii

experiments.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MoFe yields determi-

component is being

proteins, pur-

laboratory.

ACKNOWLEDGEIQQ~TS. R. S. was supported by USPHS Training Grant 00715. N. L. was supported by USPHS Training Grant 00444.

REFERENCES. 1. 2. 3.

Kleiner, D. and Chen, C. H. (1974) Arch. Microbial. 98, 93-100. Burns, R. C., Holstein, R. D. and Hardy, R. W. F. (1970) Biochem. Biophys. Res. Comm. 2, 90-99. Stasny, J. T., Burns, R. C., Korant, B. C. and Hardy, R. W. F. (1974) J. Cell. Bi01. 60, 311-316.

1481

Vol. 66, No. 4,1975

;: 6. 7.

a. 9.

BfOCHEMfCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Chervenka, C. H. (1970) Anal. Biochem. 34, 24-29. shah, v. K. and Brill, W. J. (1973) Biochem. Biophys. Acta 305, 445-454. Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 44064412. Gornall, A. G., Bardawill, C. J. and David, M. M. (1949) J. Biol. Chem. 177, 751-766. Edelstein, S. J. and Schachman, H. K. (1967) J. Mol. Chem. 242, 306-311. Reithel, F. J. and Sakura, J. D. (1963) J. Phys. Chem. 67, 2497-2498.

1482