Vol. 261, No. 1. Issue of January 5 pp.

66-70 1986

THE JOURNAL
OF BIOLOGICAL
CHEMISTRY
Q 1986 by The American Soeiety of Biological Chemists,Inc.

Prmted in

ti.^.^.

Selective Oxidation and Reduction of Methionine Residues

in Peptides
and Proteins by Oxygen Exchangebetween Sulfoxide and Sulfide*
(Received for publication, November 7,1984)

Yoram ShechterS
From the Department of Hormone Research, The Weizmann Institute of Science, Rehovot76100, Israel

Treatmentofamino acids, peptides,andproteins

T also oxidizes tryptophanyl residues (2). Although the latter
with aqueous solution of dimethyl sulfoxide (MezSO) reaction is usually eliminated at neutral or slightly alkaline
and hydrochloric acid (HCl) resulted in the oxidation pH, a particular tryptophan,if located at acidic microenvironof methionine to methionine sulfoxide. In addition to ment within the protein three-dimensional structure, may be
methionine, SH groups are also oxidized, but this re- oxidized at neutral pH aswell.
action proceedsafter a lag period of 2 h. Other amino
Another unsolved problem is the lack of mild conditions
acids are not modified by aqueous
MezSO/HCl.The for the conversion of methionine sulfoxide back to methioreaction is strongly pH-dependent. Optimal conditions nine. This can currentlybe achieved either by acid hydrolysis
are 1.0 M HCl, 0.1 M MezSO, at 22 O C . The reaction at high temperature (13) or by applying high concentrations
exhibits pseudo-first orderkinetics with KOb= 0.23 f of reducing agents such as 8-mercaptoethanol(14). The latter
0.015 M min at 22 C. Incubation of methionine
sulfoxide with dimethyl sulfide and HCl resulted in process, however, is extremely slow (K = 0.14 M h-) and
the conversion of methionine
sulfoxide to methionine. is certainly inapplicable to proteins having disulfide bonds.
The present study examines the potential of oxygen exThis reactionis fast (tH= 4 min at room temperature)
change between sulfoxides and sulfides, which at present is
andquantitativeat
relatively anhydrouscondition
(Le. at HzO:concentrated HC1:dimethylsulfide ratio of of limited use (15-19), for the selective oxidation and reduc2:20: 1). Quantitative conversions of methioninesulf- tion of methionine residues in peptides and proteins. The use
oxide back to methionine are obtained in peptides andof the volatile substances, Me2S0,2Me2S, as well as the use
proteins as well, with no observable other side reac- of HC1, for the nucleophilic assistance of C1- in promoting SO bond breaking to form the intermediate halosulfonium
tions in amino acids and proteins. The wide applications of this selective oxidation and reduction of me- cation > + S - C l (20) makes these oxidation-reduction procthionine residues are demonstrated and discussed. esses selective and applicable for methionine modification.
EXPERIMENTAL PROCEDURES

Materials-Bovine a-lactalbumin, ribonuclease, insulin, adrenoConditions and reagents which are highly selective in the corticotropic hormone (ACTH), methionine sulfoxide, 5,5-dithiobismodification of certain amino acids in peptides and proteins 8-nitrobenzoic acid), and chloramine-T were purchased from Sigma.
are useful for structure-function studies. Unfortunately, most Standard calibration mixture of amino acids type I (which contains
reagents are not selective and modify more than one func- 17 common amino acids) was a product of Spinko-Beckman. N tional group in proteins. Exceptions are several alkylating Acetyltryptophan was prepared by acetylating tryptophans with
anhydride/water/pyridine (1:1:0.8, v/v) for 1 h at 0 C.
agents which uniquely alkylate cysteinyl residues (summa- acetic
Methods-Amino acid analyses were performed with a Durrum
rized in Ref. 1). In a previous study we have demonstrated AminoAcid Analyzer ModelD-502. Hydrolysis of proteins were
that mild oxidizing agentssuch as chloramine-T and N- carried out in evacuated tubes with 6 N HCl at 105 C for 22 h. The
chlorosuccinimide, at neutral orslightly alkaline pH, will only derivative, 1-MetO-a-lactalbumin, was prepared by allowing a-lacoxidize methionine residues to methionine sulfoxide, in non- talbumin (20 mg in 10 ml of 0.1 M Tris-HC1 buffer, pH 8.4) to react
SH containing proteins (2). These mild chlorinating agents with 10 M excess of chloramine-T for 1 h at room temperature. The
solution was then extensively dialyzed and lyophilized. In this
were by far more suitable to this purpose since other previ- protein
derivative, the single methionyl residue of a-lactalbumin of bovine
ously used conditions and reagents such as periodate (3, 4), milk is oxidized to methionine sulfoxide. Reduced a-lactalbumin was
hydrazoic acid (5, 6), iodination (7, 8), photooxidation (9), prepared by dissolving a-lactalbumin (15 mg) in 1.0 ml of 0.1 M Trismild brominating agents (IO), and hydrogen peroxide (11,12) HC1 buffer, pH 7.4, 3 M urea, 12 mM dithiothreitol. The reaction
were less selective and modify other oxidizable groups in proceeded at room temperature for 30 min. The protein was then
proteins aswell. Chloramine-T itself is four to five times more dialyzed in the cold against 1M HCl, 0.1 mM dithiothreitol for 24 h.
Determination of methionine sulfoxide in the presence of methionine
reactive in oxidizing cysteinyl residues as compared to its was performed according to Ref. 2. Briefly, the protein sample was
affinity toward methionyl residues (2). Therefore, this oxidiz- subjected first to treatment with cyanogen bromide where methionine
ing agent cannot
be applied for modifying methionines in SH- was quantitatively converted to homoserine and itslactone (21), while
containing proteins. Furthermore, at acidic pH, chloramine- methionine sulfoxide remained intact. The latter is determined as
methionine after subsequent acid hydrolysis (2). Free SH groups of
* This study was supported by a grant from the Israel Academy of glutathionine were quantitatively determined according to Ellman
Sciences and Humanities. The costa of publication of this article were (22) using 1 mM 5,5-dithiobis-(2-nitrobenzoicacid) in 0.3 M Trisdefrayed in part by the payment of page charges. This article must HCl, pH 7.4, molar extinction, 412 nm = 13,600, was used. Biological
therefore be hereby marked advertisement in accordance with 18
Y. Shechter, unpublished observation.
U.S.C. Section 1734 solelyto indicate this fact.
The abbreviations used are: Me2S0, dimethyl sulfoxide; Me&,
$ Incumbent of the Charles H. Hollenberg Professorial Chair in
Metabolic and Diabetes Research established by the friends and dimethyl sulfide; ACTH, adrenocorticotropic hormone; MetO, methionine sulfoxide.
associates of Dr. Charles H. Hollenberg of Toronto, Canada.

66

67

Oxidation and Reduction of Methionine

activity of ACTH and insulin were determined by lipolysis (23) and
lipogenesis (24), respectively. Rat fat cells were prepared according
to Rodbell (25).
RESULTS

Conversion of Methionine to Methionine Sulfoxide by Me2S0

under Different Experimental Conditions-Table I summarizes 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, however, occurred a t 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 inaqueous 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, methionine was completely oxidized to methionine sulfoxide. Other
amino acids were recovered quantitatively (Table 11). There
wereno detectablequantities of cysteic acid, methionine
sulfone, or any other
ninhydrine-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).MezSOwas then added to a final concentration of 1M and
the spectrum was monitored again after 2 hat room temperature. No change in the spectrum (otherthan a small decrease
due to dilution) is detected, indicating that the tryptophanyl
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 theabsorbance
at 280 nm asexpected 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 SH groups are oxidized by aqueous
Me2SO/HC1, reduced glutathionine (3.6 mM) was incubated
for several hours, either in H20, 10% NJV-dimethylformamide, 1.2 M HCl, or at two different concentrations of Me2SO/
HC1. Aliquots were withdrawn a t intervals to determine SH
TABLEI
Oxidation of methionine to methionine sulfoxide at different
experimental conditions
The reaction mixture contained 0.2 mM methionine and 0.2 M
Me2S0in themedium specified in the Table. The reaction was carried
out for 2 h at room temperature.
Methionine
sulfoxide"

Medium

% of total

100
Hz0
0.1 M NaHC03, pH 8.5
100
0.01 M HCl
92
0.1 M HCl
30
1 M HCl
0
1 M Acetic acid
100
5 M Acetic acid
80
a Determined by automatic amino acid analyses.

0
0
8
70
100
0
20

TABLEI1
Recovery of amino acids after incubation with Me2SO-HC1
The amino acid mixture (0.5 pmollml of each) was made 2 M in
HC1 and 1 M in Me2S0. The reaction was carriedout for 3 h at 25 "C
before an aliquot was evaporated and its content analyzed.
Recoverf

Amino acid

Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Half-cystine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Cysteic acidb
Methionine
100 sulfoxide'
Methionine sulfone'
Alanine was taken as 100%.
An oxidation product of cystine or cysteine.
Oxidation products of methionine.

100
99
100
104
98
102
100
99
97
0
100
103
98
101
103
102
99
0
0

Wavelenghhml

FIG. 1. Spectrophotometric monitoring of N-acetyltryptophan treated with Me2SO/HC1 or chloramine-T. The spectrum
of N-acetyltryptophan (final concentration 0.35 mM in 2 M HCl) was
MelSO was then added to a final concentration of
monitored (-).
1 M and the spectrum was remonitored after 2 h of incubation at
22 "C (----). Chloramine-T (20 molar excess) was finally added and
the spectrum was remonitored (. . . . .).

content. As shown in Fig. 2, no detectable oxidation is observed at the first 2 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 that methionine can be modified selectively in the
presence of free SH groups.
Rate and Extentof 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 sulfoxide 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

68

Oxidation and Reduction of Methionine

I

2.6H DHF

TABLE
I11
Oxidation of methionyl residues in peptidesand proteins by aqueous
MezSO/HC1
Substance oxidized

Methionine
sulfoxide
%

0.L
3.6M HCI

vl

2
3
L
Time (hours)

FIG.2. 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-nitrobenzoicacid) in 0.3 M Tris-HC1 buffer, pH
7.4. The absorbance at 412 nm was then recorded.

Free SH
% initial

value

Methionine
100
Methionyl-valine
100
Methionyl-aspartic acid
100
Methionyl-phenylalanine
100
Methionyl-phenylalanylglycine
100
Adrenocorticotropic hormone
100
(ACTH)
Glucagon
100
Ribonuclease A
25
a-Lactalbumin
100
Reduced a-lactalbumin
lood
loo
a Extent of oxidation in the methionyl peptides and proteins was
determined by the cyanogen bromide method described under EXperimental 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 Experimental Procedures was oxidized for 2 h at 22 C in aqueous Me2S0
(0.1 M), HCl (1M).
e Samples were withdrawn for SH determination after 1- and 2-h
intervals.

72
.-

12

16

20

2L

Time Iminutesl

FIG.3. Rate of conversion of methionine to methionine sulfoxide 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 determined.

?Li2lLJ

by Me2SO/HC1-Table I11 demonstrates the extentof methionine oxidation of various peptides, polypeptides, and pro20
4
8
12
16 20 24 28 32 0.5M
l.OM
36MHCI
q S40
teins. Quantitative oxidations were obtained in methionylcontaining di- and tripeptides. Also, the single methionyl
Time (minutes)
residues of ACTH, glucagon, and a-lactalbumin of bovine
FIG.4. Conversion of methionine sulfoxide to methionine
milk were quantitatively oxidized. In bovine pancreatic ribonuclease, 25% of the methionyl residues were oxidized (Table by MepS/HCl. The reaction mixture contained 0.089 mM methionine
sulfoxide (MetO) and theindicated concentrations of dimethyl sulfide
111). Since in thisprotein only 1 out of the 4 methionyl (Me2S)and HCl. At intervals, 70-pl aliquots were withdrawn, diluted
residues is exposed to modification by other oxidizing agents 10-fold with cold HzO, lyophilized, and their Met and MetO content
(2), it seems likely that aqueous Me2SO/HC1 discriminates was analyzed.
between buried and exposed methionines in large polypeptides
as well.
In order to examine whether methionine can be modified
TABLE
IV
in thepresence of cysteinyl residues, the four disulfide bonds
Reduction of 1-Met0-a-lactalbumin undervarious conditions
of a-lactalbumin were reduced and the random polypeptide
Reduction of methionine
Conditions applied
was subjected to Me2SO/HC1 for 2 h. The single methionyl
sulfoxide in a-lactalbumin
residue was quantitatively modified with no observable reducm o l MetOlmol a-lactalbumin
tion in free SH groups within the first 2 h (Table 111).
1 M Me2S in Hz0
0.97
Reduction of Methionine Sulfoxide by Me2S/HC1-TreatHC1,
M MezS
0.05
10.7 M 0.5
ment of methionine sulfoxide with dimethyl sulfide (Me2S)
6 M0.5
HC1,
M Me2S
0.03
HC1,
M Me2S
0.03
4 M0.5
and high concentrations of hydrochloric acid resulted in the
2 M0.5
HC1,
M Me&
0.44
rapid conversion of methionine sulfoxide back to methionine
0.5 M HCl, 0.5 M MezS
0.69
(Fig. 4). The raection proceeds readily at 10.7 M HC1 and the
The protein derivative (2 mg) was incubated for 2 h at 22 C in
rate decreases as theH 2 0proportion in the reaction medium
increases. Thus, at 4.4-10.7 M HC1, the reaction proceeds to 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
completion, while at lower concentrations of HC1 (i.e. 1.0 M), cyanogen bromide (50 molar excess) was added. The reaction prothe extentof the reduction is retarded tremendously (Fig. 4). ceeded for 24 h at room temperature. The samples were then evapoReduction of 1-Meto-a-Lactalbumin by Me2S/HC1-Table
rated and acid hydrolyzed. MetO is converted to methionine during
IV demonstrates that the reduction of a MetO residue in a acid hydrolysis in 78% yield. Values were corrected according to this.

Oxidation and Reduction of Methionine

69

proteinisnotobtained
by Me& in the absence of HCl. biological interest after oxidation of their methionyl residues.
Quantitative reductions, however, were obtained at 4-10.7 M
HCl and were incomplete at lower HCl concentrations (Table
DISCUSSION
IV). This is essentially the same pattern of reduction as was
obtained with the free Met0 residue (Fig. 4).
The present study shows that Me2S0, Me2& and HCl can
Specificity of the Reduction by Me,S/HCl Inactivation and be efficiently used for the selective oxidation and reduction
Reactivation of ACTH-As shown in Table V, under the of methionyl residues in peptides and proteins. The reactions
conditions used for achieving quantitative conversion of me- involved and conditions applicable to peptides and proteins
thionine sulfoxide to methionine ( i e . at 10.7 M HC1,0.5 M are summarized in Scheme 1. Oxygen exchange between
Me2S for 2 h at room temperature), none of the standard Me2S0and methionine is acid-dependent (Table I). The
mixture of amino acids is modified. All the 17 amino acids chloride anion is effective in promoting the exchange. High
were recovered in quantitative yield. The same reaction con- concentrations of HCl, however, are not required and the
ditions, when applied to insulin did not cause any irreversible reaction proceeds readily to completion at 0.5-1.0 M HCl
denaturation of the hormone (not shown). As shown previ- (Table I, Fig. 3). Similarly, the reaction is completed at low
ously, treatment of ACTH with aqueous Me2S (0.1 M ) / H C ~ concentrations of Me2S0 ( i x . at 0.01 M Me2S0, Fig. 3),
(1 M ) for 3 h at 22 "C leads to oxidation of methionine to therefore eliminating the possibility of protein denaturation
methionine sulfoxide (Table 111). The modified derivative due to high concentrations of an organic solvent. The oxygen
retained about4% of the native hormonal activity (Table VI). exchange between M e 8 0 and methionine can be considered
Treatment of the oxidized protein with 10.3 M HC1,0.3 M specific to thisamino acid residue, since other side chains are
not modified (Fig. 1, Table 11),with the exception of cysteinyl
Me2S for 15 min at 37 "C resulted in nearly full reactivation
groups (Fig. 2). The latter, however, are oxidized very slowly
of the oxidized derivative (Table VI). It, therefore, seems that
and the reaction is initiated only after a lag period of 2 h at
Me2S/HC1 can be used to reduce and reactivate proteins of room temperature (Fig. 2). This issue also agrees with the
TABLE
V
amino acids after incubation with Me2S/HC1

Recovery of
The amino acid mixture (0.217 pmol/ml of each amino acid) was
incubated for 2 h at room temperature in 10.4 M HCl, 1M Me2S. The
sample was then evaporated and an aliquot of its content analyzed.
Alanine was taken as 100%.
acid
Amino
Recovery
~

~~~~

Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Half-cystine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine

100
104
97
100
98
107

103
97
96
100
97
100
103
101
96
96
108

TABLE
VI
Biological activity ofoxidized and reduced ACTH
Relative
Derivative
EDrn'
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 halfmaximal effect in lipolysis.
Determined by lipolysis in rat adipocytes according to Ref. 23.
'Oxidation was performed at 1 M HCl, 0.1 M Me2S0 for 2 h at
22 "C.
An aliquot of the oxidized ACTH was evaporated and reduced in
10.3 M HC1, 0.3 M Me2S for 15 min at 37 "C. The sample was then
loaded on a Sephadex G-10column (20 X 1cm), equilibrated and run
M HCl, 0.1 M NaCl. The proteinfractions werepooled.
with
ACTH concentration was determined by hydrolyzing an aliquot and
determined its amino acid content.

'

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

of the SHgroup of penicillamine by Me2S0 at37 "C is grossly
retarded at 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 arereadily 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 chloramineT (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 proceeds to completion (Fig. 4, Table IV).This reaction, however,
will be completed only at high concentrations of HC1 (i.e. 410.7 M), since an increased proportion of H 2 0 in the medium
is unfavorable for the reaction to proceed in the right direction. 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-"CO-

-NH-Ui-COI

Fh

F
S
I

cn,

0s
.:CH,
0.5MHCI,O.IMMe$30

22 OC,30-180min

-NH-CH-CO-

cn,

I
a 3

-NH-CH-COI

tY
7
S=O

F"
cn,
s.0

S;cY

7"

CH,
4.0-K17MHCl.a3"~~S

7
S

22 OC,30-180min

cy

SCHEME1. Oxidation and reduction of methionyl residues

by MezSO and MezS. Optimal conditions for the reactions.

Oxidation and Reduction of Methionine

70

the oxygen exchange between sulfoxidesand sulfides of macromolecules under hydrous or semianhydrous conditions and
(b) to determine the biological significanceof certain methionines in macromolecules of interest and the relation O f the
three-dimensional structure of the macromoleculeto therate
of methionine oxidation by Me2SO/HC1. The reduction procedure may also be useful for radioactively iodinated hermones and proteins, since oxidation of methionines to methionine sulfoxide is an undesirable side reaction which occurs
readily during iodination due to the presence of chloramineT (2) or hydrogen peroxide(28-30).
Acknowledgments-The skillful technical assistance ofB. Zarmi
and the careful reading of the manuscript bY Dr. Cynthia Webb are
gratefully acknowledged.
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