J. Biol. Chem. 1986 Shechter 66 70

8/9/2019 J. Biol. Chem. 1986 Shechter 66 70

1/5


2/5

Oxidation and Reduction of Methionine

67

activity of ACTH and insulin were determined by lipolysis (23) and

to Rodbell (25).

lipogenesis (24 ), respectively. Rat fat c ells were prepared according

RESULTS

Conversion of Methionine to Methionine Sulfoxide by Me2S0

under Different Experimental Conditions-Table I summa-

rizes varying conditions for the oxidation of methionine to

methionine sulfoxide by Me2S0. The reaction was found to

be strongly pH dependent. No oxygen transfer was observed

in an aqueous solution of 0.1 M sodium bicarbonate, pH 8.5,

and only slight reaction in 0.01

M

HC1 (Table

I .

The reaction

occurs readily, however, at 0.1-1

M

HC1. Acetic acid was a

poor substitute for hydrochloric acid. Some reactions, how-

ever, occurred at high concentrations (5 M ) of acetic acid

(Table I . The chloride anion, however, seems to be by far

more efficient in the acid-promoted S-0 bond breaking that

is required to oxidize methionine by Me2S0 in queous acidic

solutions.

Selectivity of Me2SO/HCl toward Methionine Residues-A

mixture of amino acids were treated with Me2SO/HC1 (Table

11 .

As shown in the Table, after exposure of the amino acids

to

1

M

Me2S0, 2

M

HC1 for 3 h at room temperature, methi-

onine was completely oxidized to methionine sulfoxide. Other

amino acids were recovered quantitatively (Table

11 .

There

wereno detectablequantities of cysteic acid, methionine

sulfone, or any otherinhydrine-positive derivatives of amino

acids. To examine whether tryptophan residues are modified

byMe2SO/HC1 the ultraviolet absorption spectrum of

N -

acetyltryptophan (0.35 mM in

2

M HCl) was monitored (Fig.

1).MezSO was then added to a final concentration of 1M and

the spectrum was monitored again after 2 hat room temper-

ature. No change in the spectrum (other han a small decrease

due to dilution) is detected, indicating that the ryptophanyl

moiety has remained unmodified. As an additional control,

about 20 eq of chloramine-T were added to the quartz cell

(Fig.

1).

This resulted in amarked decrease in the bsorbance

at 280 nm as xpected from the conversion of the tryptophanyl

moiety to 2-oxyindolealanine (26). Thus, aqueous Me2SO/

HC1 at room temperature is selective toward the oxidation of

methionine among the common amino acids side chains. This

selectivity is even preserved at elevated temperatures (i.e. at

50 and 100 C) as demonstrated in the study of Lipton and

Bodwell (19).

To examine whether free S H groups are oxidized by aqueous

Me2SO/HC1, reduced glutathionine (3.6 mM) was incubated

for several hours, either in H2 0, 10% NJV-dimethylformam-

ide, 1.2

M

HCl, or a t two different concentrations of Me2SO/

HC1. Aliquots were withdrawn a t intervals to determine SH

TABLE

Oxidation of methionine to methionine

sulfoxide

at different

experimental conditions

Th e reaction mixture contained 0.2 mM m ethionine and 0.2 M

Me2 S0 n themedium specified in the T able. The reaction was carried

out for

2

h at room temperature.

Medium

Methionine

sulfoxide

% of to ta l

Hz0

100 0

0.1 M NaH C03, pH 8.5 100 0

0.01

M

HCl 92

8

0.1 M HCl 30 70

1

M

HCl

0

100

1 M Acetic acid

100

0

5 M Acetic acid

80 20

a Determined by autom atic amino acid analyses.

TABLE1

Recov ery of am ino acids after incubation with Me2SO-HC1

The amino acid mixture (0.5 pmollml of each) was made 2

M

in

HC1 and 1 M n M e2S 0. The reaction was carriedout for 3 h at 25 C

before an aliquot was evaporated and its content analyzed.

Amino acid Recoverf

%

Aspartic acid 100

Threonine 99

Serine 100

Glutamic acid 104

Proline 98

Glycine 102

Alanine 100

Half-cystine 99

Valine 97

Methionine

0

Isoleucine 100

Leucine 103

Tyrosine 98

Phenylalanine 101

Lysine 103

Histidine 102

Arginine 99

Cysteic acidb 0

Methionine sulfoxide'00

Methionine sulfone' 0

Alanine was taken as 100% .

An o xidation product of cystine or cysteine.

Oxidation products of methionine.

Wavelenghhml

FIG. 1.

Spectrophotometric monitoring

of

N-acetyltrypto-

phan

treated with

Me2SO/HC1 or

chloramine-T.

The spectrum

of N-acetyltryptophan (final concentration 0.35 mM in 2

M

HCl) was

monitored

-).

MelSO was then added to a final concen tration of

1 M and the spectrum was remonitored after 2 h of incubation at

22 C

----).

Chloramine-T (20 m olar excess) was finally added and

the spectrum was remonitored

.

).

content.

As

shown in Fig.

2,

no detectable oxidation is ob-

served at the first2 h. Slow oxidation is obtained thereafter

(Fig. 2). As the oxidation of methionine residues by Me2SO/

HC1 are completed within shorter periods (next paragraph),

it implies tha t methionine can be modified selectively in the

presence of free SH groups.

Rate and Extent f the Reaction-When an excess of Me2S0

is applied over methionine (25-250

M

excess) in a fixed

concentration of HCl(1

M),

the rate of methionine oxidation

represents pseudo-first order rate (Fig. 3).Rateconstant

observed was calculated to be 0.23 f 0.015 M s at 22 C.

Methionine was quantitatively converted to methionine sulf-

oxide within 30 min (Fig. 3). Quantitative conversions were

still evident at 10 M excess of Me2S0 over methionine. Fifty

per cent conversion was obtained at about 4 M excess of

Me2S0 over methionine (not shown).

Oxidation

of

Methionyl Residues in Peptides

and

Proteins


3/5

68


I

I

2.6H

DHF 1

TABLE

Oxidation of methionyl

residues

in peptidesand proteins by aqueous

MezSO/HC1

0 L

vl

m

3.6M

HCI

1 2 3 L 5 6

Time (hours)

FIG.

. Oxidation of glutathione with time

by

MezSO/HCl.

Reduced glutathione (final concentration 3.6 mM) was incubated at

room temperature under the conditions specified in the figure. At

intervals, 25-p1 aliquots were withdrawn and added to solutions of 1

mM 5,5-dithiobis- 2-nitrobenzoic cid) in 0.3 M Tris-HC1 buffer, pH

7.4. The absorbance at 412 nm was then recorded.

L 8 12 16 20 2L

Time Iminutesl

FIG.

.

Rateof conversion of methionine to methionine sulf-

oxide by MeaSO/HCl. The reaction mixture contained 0 4 mM

methionine and the indicated molar concentrations of Me2S0 and

HCl.At intervals, 30-p1 aliquots were withdrawn, neutralized by

NH20H, lyophilized, and their Met and MetO content was deter-

mined.

by Me2SO/HC1-Table

I11

demonstrates the extent

of

methi-

onine oxidation of various peptides, polypeptides, and pro-

teins. Quantitative oxidations were obtained in methionyl-

containing di- and tripeptides. Also, the single methionyl

residues of ACTH, glucagon, and a-lactalbumin

of

bovine

milk were quantitatively oxidized. In bovine pancreatic ribo-

nuclease,

25%of

the methionyl residues were oxidized (Table

111).

Since in thisprotein only

1

out

of

the

4

methionyl

residues is exposed to modification by other oxidizing agents

2), it seems likely that aqueous Me2SO/HC1 discriminates

between buried and exposed methionines in large polypeptides

as well.

In order to examine whether methionine can be modified

in thepresence of cysteinyl residues, the four disulfide bonds

of

a-lactalbumin were reduced and the random polypeptide

was subjected to Me2SO/HC1 for 2 h. The single methionyl

residue was quantitatively modified with no observable reduc-

tion in free SH groups within the first 2 h (Table 111).

Reduction of Methionine Sulfoxide by Me2S/HC1-Treat-

ment

of

methionine sulfoxide with dimethyl sulfide (Me2S)

and high concentrations of hydrochloric acid resulted in the

rapid conversion

of

methionine sulfoxide back to methionine

(Fig.

4).

The raection proceeds readily a t 10.7 M HC1 and the

rate decreases as theH 2 0proportion in the reaction medium

increases. Thus, at 4.4-10.7 M HC1, the reaction proceeds to

completion, while a t lower concentrations of HC1 (i.e. 1.0 M),

the extentof the reduction is retarded tremendously (Fig. 4).

Reduction of

1

Meto-a-Lactalbumin

by

Me2S/HC1-Table

IV

demonstrates that the reduction

of

a MetO residue in a

Substance oxidized

Methionine

Free SH

sulfoxide

Methionine

Methionyl-valine

Methionyl-aspartic acid

Methionyl-phenylalanine

Methionyl-phenylalanylglycine

Adrenocorticotropic hormone

Glucagon

Ribonuclease

A

a-Lactalbumin

Reduced a-lactalbumin

(ACTH)

%

100

100

100

100

100

100

100

25

100

lood loo

% initial

value

a

Extent of oxidation in the methionyl peptides and proteins was

determined by the cyanogen bromide method described under EX-

perimental Procedures.

One residue (out of 4) was modified.

The single methionyl residue of a-lactalbumin was quantitatively

modified.

Reduced a-lactalbumin (prepared as described under Experi-

mental Procedures was oxidized for 2 h at 22 C n aqueous Me2S0

(0.1 M), HCl 1M).

e Samples were withdrawn for SH determination after

1-

and 2-h

intervals.

72

W

.

X

?Li2lL

0

4

8

12 16

20

24

28

32

0.5MOM

6

M q S

CI

40

Time (minutes)

FIG. . Conversion of methionine sulfoxide to methionine

by MepS/HCl.

The reaction mixture contained 0.089 mM methionine

sulfoxide (MetO) and thendicated concentrations of dimethyl sulfide

(Me2S)and HCl. At intervals, 70-pl aliquots were withdrawn, diluted

10-fold with cold HzO, lyophilized, and their Met and MetO content

was analyzed.

TABLE

V

Reduction

of

1-Met0-a-lactalbum in under arious conditions

Conditions applied

Reduction

of

methionine

sulfoxide in a-l actalbumin

m o MetOlmol a lactalbumin

1M

Me2S in Hz0 0.97

10.7

M

HC1,.5

M

MezS 0.05

6 M HC1,.5

M

Me2S 0.03

4

M

HC1,.5

M

Me2S 0.03

2 M

HC1,.5

M

Me& 0.44

0.5 M HCl, 0.5 M MezS 0.69

The protein derivative (2 mg) was incubated for 2 h at 22 C in

0.25 ml of the medium specified in the Table. The tubes were then

evaporated

to

dryness, dissolved in 0.3 ml of 70% formic acid, and

cyanogen bromide (50 molar excess) was added. The reaction pro-

ceeded for 24 h at room temperature. The samples were then evapo-

rated and acid hydrolyzed. MetO is converted to methionine during

acid hydrolysis in 78% yield. Values were corrected according to this.


4/5

Oxidation and Reduction of Methionine 69

protein snotobtained by Me& in he absence of HCl.

Quantitative reductions, however, were obtained at 4-10.7

M

HCl and were incomplete at lower HCl concentrations (Table

IV).This is essentially the same pat tern of reduction as was

obtained with the free Met 0 residue (Fig.

4).

Specifici ty of the Redu ction by Me,S/HCl Inactivation and

Reactivation of ACTH-As shown in Table

V,

under the

conditions used for achieving quantitative conversion of me-

thionine sulfoxide to methionine i e . at 10.7 M HC1,0.5 M

Me2S for

2

h a t room temperature), none of the standard

mixture of amino acids is modified. All the 17 amino acids

were recovered in quantitative yield. The same reaction con-

ditions, when applied to insulin did not cause any irreversible

denaturation of the hormone (not shown). As shown previ-

ously, treatment of ACTH with aqueous Me2S 0.1M)/HC~

1 M )

for 3 h at 22 C leads to oxidation of methionine to

methionine sulfoxide (Table 111). The modified derivative

retained about4% of the native hormonal activity (Table VI).

Treatment of the oxidized protein with 10.3 M HC1,0.3 M

Me2S for 15 min a t 37 C resulted in nearly full reactivation

of the oxidized derivative (Table VI).

It,

therefore, seems that

Me2S/HC1 can be used to reduce and reactivate proteins of

TABLE

Recovery

of

amino acids after incubation with

Me2S/HC1

The amino acid mixture (0.217 pmol/ml of each amino acid) was

incubated for 2 h at room temperature in 10.4M HCl, 1M Me2S. The

sample was then evaporated and an aliquot of its content analyzed.

Alanine was taken as 100%.

~ ~ ~~~~

Aminocid

Recovery

%

Aspartic acid 100

Threonine 104

Serine 97

Glutamic acid 100

Proline 98

Glycine

107

Alanine 103

Half-cystine 97

Valine 96

Methionine 100

Isoleucine 97

Leucine 100

Tyrosine 103

Phenylalanine 101

Lysine 96

Histidine 96

Arginine 108

TABLE

I

Biological activity of oxidized and reduced ACTH

Derivative EDrn'

Relative

bioactivitg

ng ml-

ACTH 2.7 100

Oxidized ACTH' 68

4

Oxidized and reduced 4.9 55

ACTHd

The ACTH or modified ACTH concentration that produces half-

maximal effect in lipolysis.

etermined by lipolysis in rat adipocytes according to Ref. 23.

22 C.

'Oxidation was performed at 1 M HCl, 0.1 M Me2S0 for 2 h at

An aliquot of the oxidized ACTH was evaporated and reduced in

10.3 M HC1, 0.3 M Me2S for 15 min a t 37 C. The sample was then

loaded on a Sephadex G-10 olumn (20 X 1cm), equilibrated and run

with

M

HCl, 0.1 M NaCl. The protein ractions were ooled.

ACTH concentration was determined by hydrolyzing an aliquot and

determined its amino acid content.

biological interest after oxidation of their methionyl residues.

DISCUSSION

The present study shows that Me2S0, Me2& and HCl can

be efficiently used for the selective oxidation and reduction

of methionyl residues in peptides and proteins. The reactions

involved and conditions applicable to peptides and proteins

are summarized in Scheme

1.

Oxygen exchange between

Me2S0and methionine is acid-dependent (Table I). The

chloride anion is effective in promoting the exchange. High

concentrations of HCl, however, are not required and the

reaction proceeds readily to completion at 0.5-1.0 M HCl

(Table I, Fig. 3). Similarly, the reaction is completed at low

concentrations of Me2S0 i x . at 0.01 M Me2S0, Fig. 3),

therefore eliminating the possibility of protein denaturation

due to high concentrations of an organic solvent. The oxygen

exchange between M e 8 0 and methionine can be considered

specific to this mino acid residue, since other side chains are

not modified (Fig. 1, Table 11),with the exception of cysteinyl

groups (Fig.

2).

The latter , however, are oxidized very slowly

and the reaction is initiated only after a lag period of

2

h at

room temperature (Fig.

2).

This issue also agrees with the

work of Snow et al.(27) who demonstrated that the xidation

of the SH roup of penicillamine by Me2S0 at7 C is grossly

retarded a t low pH values i.e.at pH 1-2, Ref. 27).Methionine,

methionyl containing di- and tripeptides, as well as exposed

methionyl residues of proteins are eadily oxidized by Me2SO/

HCl (Table 111). Methionines, which are buried within the

three-dimensional core of the protein molecule (such as the3

methionyl residues of ribonuclease A), do not seem to be

modified either by Me2SO/HC1 (Table 111)or by chloramine-

T

(Ref. 2). The methionyl residue of reduced a-lactalbumin

was quantitatively modified within

2

h with no concomitant

oxidation of the cysteinyl residue of the polypeptide chain

(Table 111).

Using Me2S and HCl, the opposite reaction, namely the

reduction of methionine sulfoxide to methionine,

also

pro-

ceeds to completion (Fig.

4,

Table IV).This reaction, however,

will be completed only a t high concentrations of HC1 i . e .4-

10.7

M ),

since an increased proportion of

H 2 0

in the medium

is unfavorable for the reaction to proceed in the right direc-

tion. A concentration range of 0.3-0.5 M Me2S was found to

be sufficient to obtain a complete conversion

of

methionine

sulfoxide to methionine. This was found to be valid for both

the free amino acid (Fig.

4)

or for methionine residues of a

protein molecule (Tables IV and VI).

The present study has several potential applications in

chemical and biological sciences. Examples are a ) to study

-NH-Ui-CO-

NH- CO-

I

Fh 0s

F

CH,

cn

S

0.5MHCI,O.IMMe$30 s.0

cn

22

OC,30-180min

a 3

-NH-CH-CO- -NH-CH-CO-

I I

t

ScY 7

CH,

S=O 4.0-K17MHCl.a3 ~~S S

I

I

cn

22

OC,30-180min

SCHEME

.

Oxidation and reduction

of

methionyl residues

cy

by MezSO and MezS.Optimal conditions for the reactions.


5/5

70


the oxygen exchange between sulfoxides and sulfides of mac-

10. Burstein,

Y.,

and Patchornik, A. (1972) Biochemistry 1 1 ,4 6 4 1 -

romolecules under hydrous or semianhydrous conditions and

4650

b ) to

determine the biological significance of certain methi-

11.

Hachimori,

Y.,

Horinishi, H., Kurihara, K., and Shibata, K.

onines in macromolecules of interest and the relation

O f

the

12. Toennies, G., and Callan, T. p. (1939) J . B ~ Lh m . 1 2 9 , 4 8 1 -

three-dimensional structure of the macromolecule o the ate

490

of methionine oxidation by Me2SO/HC1. The reduction pro-

13. Morihara, K. 1964) Bull. Chem. SOC. pn 37,1781-1784

cedure may also be useful for radioactively iodinated her-

14.

Tashjian,

A.

H., Jr., Ontjes, D. A., and Munson, p.

L. (1964)

mones and proteins, since oxidation of methionines to methi-

15.

Bodwell,

F.

G.,

a;ld

Pitt,

B.

M.

(1955)

J

Am

Chem.

sot,

,7,

onine sulfoxide is an undesirable side reaction which occurs

572-577

readily during iodination due to the presence of chloramine-

16.

Searles,

S.,

and Hays, H. R.

(1958) J.

Org.

Chem. 23,2028-2029

T

2)

or hydrogen peroxide

28-30).

17.

Hull,

C.

M., and Bargar,

T.

W.

(1975)

J Org.

Chem. 40 , 3152-

18.

Ruffato,

V.,

and Miotti,

V. (1978)

Gazz.

Ch im ica ZtaZ. 1 0 8 , 9 1-

and th e careful reading of the manu script bY Dr. Cynthia Webb are

19.

Lipton, S. H., an d Bodwell, C. E.

(1976)

J

Agric.

ood

C h m .

gratefully acknowledged.

2 4 ,2 6 -3 1

20.

Bovio,

A,.

and Miotti. V.

(1978)

J .

Chem. Soc.

Perkin Trans. I .

(1964) Biochim. Bwphys. Acta 9 3 .3 4 6 -3 6 0

Biochemistry 3 1175-1182

3154

Acknowledgments-The skillful technic al assistance of B. Zarmi

96

REFERENCES

1. Means, G. E., an d Feeney, R. E. (1971) Chemical Modifications of

2.

Shechter, Y., urstein, Y., nd Patchornik, A.

(1975) Biochem-

3.

Clamp,

J.

R., nd Hough, L.

(1965) Biochem.

J

9 4 , 1 7 - 2 4

4.

Knowles,J. R.

1965) Biochem.

J 95,

180-190

5.

Whitehead, J. K., and Bently, H. R.

(1952)

J.

Chem. SOC.

Part I1

6. Brill,

A.

S. and Weinryb, I.

(1967) Biochemistry 6,3528-3535

7.

Koshland, M. E. ngleberger,

F.

M., and Gaddone, S. M.

(1963)

8.

Filmer, D. L., nd Koshland, D. E., Jr .

(1964) Biochem. Biophys.

9.

Spikes,

J.

D., and Straight, R.

(1967)

Annu. Rev. Phys. Chem.

Proteins, Holden-Day Inc., San Francisco,

CA

istry 14,4497-4503

1527-1535

J. Biol

Chem. 238,1349-1352

Res. Commun.

17,189-195

18,409-436

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

, .

2 , i n - 1 7 7

Gross, E.

(1967)

Methods

Enzymol. 11,238-255

Ellm an, G. L.

(1959) Arch. Biochem . Biophys. 8 2 , 7 6 7 7

Shechter, Y. (1982) Endocrinology 110,1579-1583

Moody, A. J., Stan,M. A., Stan, M., and Gliemann, J.

(1974)

Rodbell, M. (1964)

J . Biol.

Chem. 2 3 9 ,3 7 5 -3 8 0

Patchornik,

A.,

Lawson, W. B., Gross, E., and W itkop, B.

(1960)

Snow,

J. T.,

Finley,

J.

W., and Friedman, M. 1975) Biochem.

Neumann, N. P., Moore, S., and Stein, W. H. (1962) Biochemistry

Koshland. D. E.. trumever. D. N.. an d Rov. W.

J.

(1962)

B m k -

Horm. Metab.

Res.

6 , 12-16

J .

Am. Chem.

Soc.

82,5923-5927

Bwphys. Res. Commun.

64,441-447

1,68-75

haven Symp. B ~ Z

5 , - 1 0 i - m

Chem. 238, PC3134-PC3136

_ .

. .

Schachter, H., Halliday, K. A., and Dixon, G. H. (1963) J Biol.

J. Biol. Chem. 1986 Shechter 66 70

Documents