Chemical modification of methionine residues in azurin

BIOCHEMICAL

Vol. 88, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 661-667

May 28, 1979

CHEMICAL MODIFICATION Richard Department

Received

H. L. Marks

of Biochemistry, Greenville,

April

OF METHIONINE

RESIDUES IN AZURIN

and R. David

Miller

East Carolina University, North Carolina 27834

School

of Medicine

12,1979

Summary. Azurin from Pseudomonas aeruginosa has been treated with bromoacetate at low pH to alkylate methionine residues. Two classes of methionine side chains are observed as a result of these reactions - four of the six methionines are reactive at pH 4, whereas all six are reactive at pH 3.2. The product containing four alkylated methionines maintains a significant portion of the blue color and spectroscopic characteristics of the native protein. The product which has been fully modified at the methionine residues, on the other hand, has lost all blue color and appears to be largely in a random coil form. The recent azurin

from

spatially

tribute

chains, ligand

groups blue

data

within

the

azurin

has not

importance tein,

(1).

to the metal. protein,

(7).

protein

been totally

molecule. focus

binding

groups

a series This

report

on the modification

and perhaps a cysteine

importance

on the

(2-6),

constitutes

of the

a preliminary side

the

Cu

x-ray

specific

transfer

chemical

of methionine

may confor

groups

activity

delineate

and reactivity

of site-specific

sulfur

from

to further

structure

is

sphere

electron

In an effort

residue

and two histi-

evidence

coordination

and the

of

its

has been postulated the

analysis

a methionine

previous

A similar

defined.

that

In addition

however,

in the copper

structure

protein,

plastocyanin,

As yet,

we have undertaken

that

has suggested

atom in this

groups

of some of these

the azurin studies

ligand

three-dimensional

as has been suggested'from

atom in another diffraction

the

aeruginosa

to the copper

one of the

side

concerning

Pseudomonas

close

atom is dine

report

of the

of the pro-

modifications account chains

to

of our

by bromoace-

tate. MATERIALS AND METHODS. Azurin was prepared from isolated cells of Pseudomonas aeruginosa as previously described (8). The apoprotein was prepared by dialyzing a solution of azurin against a cyanide solution at pH 10 (9). For composition analysis, protein samples were hydrolyzed in 6N HCl, dried, and resuspended in pH 2.2 citrate buffer before being applied to a Beckman 1198 amino acid analyzer. The extent of modification of methionine residues was analyzed 0006-291X/79/100661-07$01.00/0 661

Copyright All rrghts

@ I979

by Academic Press. Inc. in any form reserved.

of reproduction

Vol. 88, No. 2, 1979

BIOCHEMICAL

by subjecting the modified protein amount of unmodified methionine as was quantitatively analyzed either or as the sulfocysteine derivative

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

to peroxidation (10) and measuring the methionine sulfone (10,ll). Total cysteine as cysteic acid following peroxidation (10) following the method of Inglis and Liu (12).

Carboxymethylation of the protein was carried out under different condiBoth bromoacetate and iodoacetamide were used as alkylating agents, tions. the concentrations of reagent and time of reaction were varied, the pH of the In a typical medium was varied, and both holo- and apoprotein were modified. reaction, the protein was dissolved in a buffer solution at the appropriate pH, a small volume of a concentrated solution of the modifying reagent was added to give the desired concentration, and the pH was readjusted if necessary. The reaction mixture was left in the dark for the desired length of time at room temperature (23'C), excess reagent was removed either by gel filtration or dialysis, and the modified protein solution was lyophilized and

stored at -2O’C. RESULTS. alkylating

Although

agents,

bromoacetate, tively

results

position resistant

of the form

which

have is

lack

basis

24 hr,

side

the

dine

recovered

nine

sulfone

same conditions

all

chains

which

the

there

appears

toward

hand, using

is markedly is

essentially

other

cysteine

under

of the

under

to the copper

toward

reaction

out

here. but

the cysteine

of this

conditions

On the

that

apoprotein

Furthermore,

I).

have been with

reported

in the denatured

to be reactive.

(Table

far

iodoacetamide,

reported

expected

values

with

to be bound

even

histidine

thus

of the results

previously

of reactivity

pH 7 and 8 for

performed

have been used as

Qualita-

the reaction

complete.

thought

to alkylation, the

and iodoacetamide

reactions

the

and less

and Brown

112,

bromoacetate

have been obtained

to be slower

Ambler

firmed

most

and these

similar

appears

both

if the

diminished.

seen following of the methionine

atom

(0.16M) would

to be little

if

at these reaction

the amount

In addition,

We have con-

group

bromoacetate

only

of the sample;

is carboxymethylated

at be

any higher

is

pH

carried

of free

a trace this

at

is

bromoacetate

the alkylation

peroxidation

(2,3),

(2).

sulfhydryl

apoprotein,

residue

histi-

of methiosuggests

that

in the apoprotein

at

pH 7. In spite and histidine methionine

of the fact

that

in the holoprotein, side

chains

alkylation the

does reactions

have been carried

662

out

not seem to occur directed

at cysteine

specifically

at low pH to ensure

toward lack

of

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Amino

Acid

Following

Composition

of

Modification

Acid

Aspartic Threonine Serine Glutamic Proline Glycine Alanine Cysteine Valine Methionine

' d

1:.: 3:8 10.8 8.6

1;:; 5.9

3 9.9 3.5

11:2 3.2 10.6 7.3 2.9c 9.2 1.6

Es 1:7

Sulfoned(5.8) 4.3 9.6 2.0

t-i 9:9 2.0

4.1 trace 6.7 2.2

4.3 0.4 9.5 2.2

E 9:6 2.1

35-i 10:6 1.2

5.8 1:.; 1:1

6.2

:*i 10:7 1.3

:*: 10:5 1.2

;:i 10.3 4.1 10.9 8.Ob

E 10:5

hydrolysis.

1::;

No corrections

as

the

sulfocysteine

Measured

as

the

cysteic

Measured

on an equivalent

acid

conditions

consis

s of a O.lM

nction

losses

following

after

of

serine

and

threonine.

and sulfhydryl sodium

peroxidation.

peroxidation.

group.

formate

The standard

buffer

can be made between

carried

out

of copper

protein

retains

gradually

fades

copper(I1)

salts so it

for

set

of reaction

at pH 3.2 or 4.0,

0.16M

24 hr at room temperature.

reactions

color,

made

derivative

sample

imidazole

dist

were

b

derivative.

of the

for

Apoazurin reacted with BrAc, pH7

Protein

16.6 9.2 9.1 10.1 4.0 10.9 8.4 2.5b 9.7 3.0

17.2

reactivity

blue

of

15.7 8.9

16.3

Measured

A clear

Molecule

16.9 ii*:

Acid

bromoacetate,

Bromoacetatea

Azurin reacted with BrAc, pH4.0

Acid

a 24 hr

Samples

Azurin reacted with BrAc, pH3.2

Histidine Phenylalanine Lysine Arginine

b

per

Azurin reacted with BrAc, pH7

Azurin unmodified

Methionine Isoleucine Leucine Tyrosine

Azurin

with

Residues Amino

I

until

at pH 3.2 its

after

or potassium seems that

and 4.0.

blue about

color,

whereas is fails

reduction

663

results

At pH 4.0

12 hr it

ferricyanide neither

the

of the alkylation

after

24 hr the

at pH 3.2,

no longer

the

apparent.

to regenerate nor reversible

solution

blue

color

Addition any further

removal

of the

of

Vol. 88, No. 2, 1979

Cu(I1)

atom is

pH 3.2 for color

BIOCHEMICAL

responsible

there

of the azurin and subjected

is

with

bromoacetate a decline

decoloration

occurs.

amount

losses

observed

two pH values

analysis

(Table

I).

the

the

structure

characteristic

the protein of azurin,

decreased

lost;

UV region

that

has reacted

consistently

the

loss

low pH where

of cysteine

No losses

of histidine

values

of unfraction-

The far

formation

as might

be expected

from

time. sharp

UV region

alkylated

exhibits

of most

In spite the

amino

acid

show that

at 280 nm (or

CD spectrum

of the fact characteris-

at 280 nm has in the absorbance

a combination

structure still

of this

non-

of the fine

color

a decrease

band at 290 nm, though

664

blue

at 625 nm to that

Some of the fine

of the

the

samples

is devoid

represents

of several

at 625 nm is virtually

and holoazurin.

this

(pH 8.3)

at pH 7.

protein

the absorbance

in the absorbance

the

samples

of the modified

apo-

electrophoresis

show the

of the absorbance Whether

gel

mixtures

at pH 4 still

at this

the average

of the spectrum

of both

30%.

in particular

defined.

clear

at pH 3.2,

does the

Analytical

apoazurin

at pH 3.2,

the ratio

not clear

the

analyses

at 625 nm or an increase is

were

that

with

at pH 4.0 than

I represent product.

for

alkylated

by about

cysteine

per molecule.

heterogeneity,

alkylated

while

is

We have also

however,

of the product

severe

existent,

severe case,

alkylated

spectral

protein

less

in Table

This

is most

of analyzable

It

of methionine

pH values.

of one residue

focusing

Preliminary

well

at the

at

pH value.

of the

components.

analyses,

is

left

reaction

in the amount

In neither

given

and isoelectric minor

being

at either

mixtures

acid

levels

to the equivalent

The analyses

two)

of azurin

show any comparable

two different

in the

the

tic

Solutions do not

alkylation

difference at the

alkylations,

that

of color.

of bromoacetate

to amino

a significant

observed

for

loss

loss.

hydrolyzed

ated

the

24 hr in the absence

Products

are

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the

of the UV region

present, sample

is

shows a

less

Vol.

88,

No.

decrease the

2, 1979

in the negative

sample

random

BIOCHEMICAL

alkylated

coil

extremum

Neither

which

bears

the absorbance

per(I1)

protein,

nor

related

to the

secondary

hand,

by changing

the

which

an average

of four

retains

to a large

product protein,

The reaction is

reactivity

from

agent.

Spectral

change

little

spectral

but in

suggests

a totally

residues

of azurin

residues

in the

appears

increased

therefore,

that

pH is decreased

groups.

Cu(I1) atom is totally in a nonspecific

color displaced

is

the from

chains

4.0 to 3.2

toward

there

the reat 625 nm of

two extra that

pH 4 and is

in

increased

unchanged

of the

at pH 3.2 methio-

the fluores-

essentially

non-

is a conformational

4 which

bromoacetate

As a result

or both of these groups, the integrity blue

allow

residues

the absorbance

below

This

of the native

that

observations

below

thioether

characteristic

our

in

color.

appear

that

is other

modified.

methionine

reactivity

with

viously

unreactive

blue

side

to decrease

It seems, as the

is

pH is decreased

suggest

which

is obtained

low pH, and is essentially

to correlate

at 308 nm begins

protein

as the

cop-

a product chains

would

of these

of azurin to very

it

of the

characteristics its

pro-

On the

and accessible

accessibility

the

side

maintains

native

is present.

reaction,

Thus

to the

in the CD spectrum,

the spectral

of pH (14).

at pH 3 (15).

but

(13),

is characteristic

trough

bromoacetate

hand,

resemblance

methionine

qualitatively

is maintained

chains

six

but does

bound,

azurin

of the methionine

alkylation

directly

the

COMMUNlCATlONS

spectrum

of the molecule,

Cu(I1) chromophore

and the

of the

at 625 nm, which

degree

titrations

emission

existent

six

of the

between

On the other

cence

of all

pH of the

an increased

the protein

side

region

220 nm negative

of two methionine

(3,15).

this

structure

independent

results

nine

the

and at least

proteins

RESEARCH

structure.

to a product

tein.

BIOPHYSICAL

at 220 nm seen in native

at pH 3.2,

DISCUSSION. Modification leads

AND

does not affect

to react

with

the

two pre-

of the modification

of one

of the Cu(I1) environment is destroyed, lost. from

way.

665

We cannot the protein

say at this or whether

point it

whether

remains

Vol. 88, No. 2, 1979

It

is tempting

information lution

the

121 is

may provide

surface

atom derivatives.

121 and perhaps

13) would

to affect

and may well

also

44 is

indeed

involved

group

might

well

change in

render

"shielding" the

currently

product

results

from

test

these

methionines

109,

methionines

of the

it

13 is not

nearby

have been modified.

the

at position

48

44.

of this

should

accessible

the principal

at pH 4 so that It

If Met

Met 121)

and purify

reaction

13 or

Lowering

modification

(including

Pt

methionine

methionine

Cu environment, area

toward

56 and either

tryptophan

to isolate

bromoacetate

suggested

to be unreactive.

site

attempting

Methi-

reactivity 64,

(#44)

solvent.

at pH 4, whereas

of the the

is

to be

Another

of methionine of its

Cu binding

the

and it

no evidence

be expected

at 31 reso-

Cu atom.

Cu atom from

The orientation

bromoacetate

structural

64 and 56) appear

the Cu atom,

the reactivity

We are

which

near

the

the environment

to solvent.

determine

"shield"

give

with

(#109,

the

of azurin

and not near the

Thus,

to react

44 (or

appears

that

(1)

with

analysis

residues

to the metal.

Adman et al.

results

diffraction

but may help to be very

be expected

pH to 3.2

x-ray

these

of the molecule

a ligand

in the heavy

to correlate the

postulated

although

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the methionine

the surface

may be near

44 might

from

Three

at or near

given,

to try

derived

(1).

onine

8lOCHEMlCAL

then

we can

be possible

to

hypotheses.

ACKNOWLEDGEMENT.

This

work

was supported

by grant

GM-23935

from

NIH.

REFERENCES 1.

2. 3. 4. 5. 6. 7.

R. E., Sieker, L. C., and Jensen, L. H. (1978) Adman, E. T., Stenkamp, J. Mol. Biol. 123, 35-47. Ambler, R. P., and Brown, L. H. (1967) Biochem. J. E, 784-825. Finazzi-Agro, A., Rotilio, G., Avigliano, L., Guerrleri, P. Boffi, V., and Mondovi, B. (1970) Biochemistry 2, 2009-2014. Hill, H. A. O., Leer, J. C., Smith, B. C., Storm, C. B., and Ambler, R. P. (1976) Biochem. Biophys. Res. Commun. lo, 331-338. Proc. Nat. Acad. Sci., Solomon, E. I ., Hare, J. W., and Gray, H. B. (1976) USA, 73, 1389-1393. Ugurb?i, K., Norton, R. S., Allerhand, A., and Bersohn, R. (1977) Biochemistry 16, 886-894. Colman, P. M., Freeman, H. C., Guss, J. M., Murata, M., Norris, V. A., Ramshaw, J. A. M., and Venkatappa, M. P. (1978) Nature 272, 319-324.

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Vol. 88, No. 2, 1979

8. 9.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Marks, R. H. L., and Miller, R. D. (1979) Arch. Biochem. Biophys., in press. Yamanaka, T., Kijimoto, S., and Okunuki, K. (1963) J. Biochem. (Tokyo) 53, 256-259. firs, C. H. W. (1956) J. Biol. Chem. 219, 611-621. Vithayathil, P. J., and Richards, F. M. (1960) J. Biol. Chem. 235, 23432351. Inglis, A. S. and Liu, T. Y. (1970) J. Biol. Chem. 245, 112-116. Tang, S. W., Colemen, J. E., and Myer, Y. P. (1968) J. Biol. Chem. 243, 4286-4297. Gurd, F. R. N. (1972) Methods in Enzymology 25, 424-438. Marks, R. H. L., and Miller, R. D. (1979) Abstracts, XI International Congress of Biochemistry, Toronto.

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