ARCHIVES OF RIOCHEMISTRY AND RIOPHYSICS 122, 164-173 (1967)

Comparison of the Chemical Properties of Selenocysteine

and Selenocystine with Their Sulfur Analogs’

R. E. HUBER’J AND R. S. CRIDDLE

Department of Biochemistry and Biophysics, University of California, Davis, California 95616

Received May 24, 1967; accepted May 24, 1967

Some chemical properties of cystine and cysteine have been compared with those
of their selenium-containing analogs. Major differences were noted between their
t,itration curves. pK values of 2.01,5.24, and 9.96 were observed for the ionizations of
the carboxyl, selenohydryl, and amino groups, respectively, of selenocysteine. These
values are compared with a pK of 2.3 for the carboxyl group of cysteine and values in
the range of 8-10 for the ionization of the sulfhydryl and amino groups. Selenocysteine
is much more reactive with halo acid derivatives than is cysteine, and reacts readily
with iodoacetate even at pH values much below the pK of the selenohydryl group.
Selenocysteine has an apparent half-wave potential of a.212 V compared with 0.021
V for cysteine. It is unstable to acid hydrolysis, being completely decomposed by
heating at 110” in 6 N HCl. It is also more soluble in water than is cysteine.

Since sulfur and selenium are similar in
their chemical properties, and differ only
slightly in properties such as ionic radius,
electronegativities, covalent radius etc.,
several attempts have been made to replace
the sulfur needed for growth in biological
systems with selenium (l-6). Selenium has
been shown to be quite readily incorporated
into living systems to form selenomethionine,
selenocystathionine, Se-methyl-selenocys-
teine, selenothiamine, etc., but evidence
for the occurrence of selenocystine or seleno-
cysteine in biological systems is limited.
Peterson and Butler (2) have located trace
amounts of a free amino acid in plant
extracts which migrates with authentic
selenocystine in chromatography and elec-
trophoresis experiments. However, the ex-
istence of either the free amino acid or of
proteins containing this amino acid in other
organisms has not been well documented, in
spite of many attempts to demonstrate its
presence. Reports of selenocystine in proteins
are subject to considerable uncertainty due
i This work was supported in part by
Public Health Service grant GM 10017.
e Shell Predoctoral Fellow.
to the limited stability of the amino acid
under hydrolysis conditions.
Many enzymes which have been isolated
and studied have been termed sulfhydryl
proteins because of the extent to which their
biological activity is dependent upon the
cysteine moieties of such proteins. The bio-
logical activities of these proteins can readily
be destroyed by reaction with various
reagents such as heavy metals, iodoacetic
acid, p-chloromercuribenzoate, or others
which chemically combine with or modify
sulfhydryl groups. Substitution of seleno-
hydryl moieties for the sulfhydryl groups of
these proteins could therefore result in an
altered biological activity which should in
part be predictable from the chemical differ-
ences in reactivity of the two amino acids.
A study was therefore undertaken to
investigate those properties of selenocysteine
and selenocystine which would most directly
affect its synthesis, utilization, and detection
in biological systems and to observe any
marked chemical differences in the properties
U.S. of these amino acids as compared with those
of cysteine and cystine which might be re-
flected in proteins containing them.
164
 CHEMICAL STUDIES OF SELENOCYSTEINE 165

MATERIALS AND METHODS 2 X 10-4~ cysteine or selenocysteine at pH 1 and

the respective amino acids in the sample being
Reagents. Seleno-DL-cystine and nL-cystine
titrated. The maxima of the difference spec-
were purchased from the Sigma Chemical Com-
tral peaks in the range 235-245 rnb were used as a
pany of St. Louis, Missouri. Sodium borohydride
was purchased from Metal Hydrides Inc. The 5,5’- measure of the concentration of the ionized form
of the sulfhydryl, and the maximum at 243 rnp was
dithiobis-2.nitrobenzoic acid (DTNB) was pur-
used to determine the concentrations of the
chased from the Aldrich Chemical Co. Special
ionized selenohydryl group (7).
grade potassium bromide powder for infrared spec-
Reaction rates. The reaction rates of cysteine
troscopy, iodoacetic acid, and iodoacetamide were
and selenocysteine with halo acid derivatives (8)
purchased from Matheson, Coleman and Bell.
were followed by means of a Metrohm Ltd. pH Stat
cu.Chloroacetjamide was purchased from Eastman
using 0.1 N sodium hydroxide to maintain the pH
Organic Chemicals. Triton X-100 was purchased
during the reaction. The selenocystine and cystine
from Rohm and Haas Company. Two per cent so-
were again reduced with sodium borohydride and
dium amalgam was prepared in the laboratory.
Acid-base tifrations. Standard acid-base titra- were kept under nitrogen gas for these exper-
iments. The concentrations of iodoacet,at,e, chloro-
tions of the selenohydryl, sulfhydryl, carboxyl,
acetamide, iodoacetnmide, cysteine, and seleno-
and amino groups of the amino acids were carried
cysteine were 0.05 pi.
out using a Radiometer type PHhl 22r pH meter
Solubility. The solubilities of the oxidized
with a (iK2021C Radiometer electrode. Prior to
forms of t,he amino acids in neutral solution were
titration, selenocystine and cystine were reduced
determined by adding excess amino acids to form
by means of 2yo sodium amalgam (10) or by the use
saturated solutions and then stirring for several
of sodium borohydride (22). Concentrations of 4,2
and 1 mg of selenocysteine or cysteine per milliliter days at 25” and pII 7. After stirring, the solutions
were centrifuged at 35,000 g for 30 minutes at 25”.
of water were used for the titration. The titrants
The supernatant fluid was then filtered through
used were 0.1 N HCl and 0.1 N NaOH. Nitrogen
Whatman No. 1 filter paper. The concentrations
gas was bubbled through all solutions to remove
of selenocystine and cyst,ine remaining in solution
oxygen and through the titration medium to keep
were determined by mealls of the ninhydrin reac-
it anaerobic. ThepK valuesreported were obtained
tion by using solutions of leucine of known concen-
by extrapolation to zero amino acid concentration.
tration for standards (9).
Spectral titration. The spectral titrations were
Stability to HCl. Hydrolysis was carried ollt
carried out in a Cary model 14 spectrophotometer
anaerobically under those conditions normally
modified to follow addition of titrant by simulta-
used for hydrolysis of proteins for the purpose of
neous measurement of spectra and pH. Measure-
amino acid analysis (6 N HCl at a temperature of
ment of pH was made with a combination
110”). The concentrations of unhydrolyzed seleno-
Metrohm type E300 pH meter (expanded scale)
and Metrohm glass electrode inserted directly into cystine and cystine were determined at variolls
the 3.ml quart,z cuvette used for spectral measure- hydrolysis t,imes by reducing with 2% sodium
ments. The amino acids in solution were reduced amalgam (10) and the11 reacting with (DTNB), itl
0.1 M phosphate buffer at pI1 7.5. One rmole of
with sodium borohydride and kept anaerobic
DTNB was added per milliliter reaction mixture.
throughout the titration by bubbling nitrogen gas
The reaction took place essentially instanta-
through the samples. That samples remained re-
neously and the color formed was measured
duced lmder these conditions was determined from
the constant heights of the spectral peaks over pe- spectrally at 412 mp in a Zeiss spectrophotometer
riods of up to 30 minutes, which was longer than (11). The hydrolysis products were analyzed in :t
Phoenix Precision Instrument Co. amino acid ana-
was required to complete the experiment. The
nitrogen gas, q-hich was introduced through capil- lyzer, model K8OOOVG.
lary Tygon tubing into the cell during the Selenocysteic acid was prepared from seleno-
titration, also effected mixing of the sample with cystine by the performic acid oxidation method of
the titrant. The standard acid and base solutions Hirs (12), and was used to determine whether oxi-
used as t,itrant also had nitrogen passed through dation might yield a product stable to acid
them for 15 minutes before use. Titrant was intro- hydrolysis. Products obtained following a 20-hour
duced through capillary Teflon tubing connected hydrolysis in G N HCI were investigated in the
to an Agla micrometer syringe. The concentrations amino acid analyzer.
of amino acids used for the spectral titrations were Polarography. Polarography was performed
generally 0.02 M. with a Sargent Model XXI polarograph with
Titrations over a range of pH values were fol- a dropping-mercury electrode. The solutions con-
lowed by observing the difference spectra between tained 1 ml of 0.027, Triton X-100, 8 ml of 0.1 N
166 HUBER AND CRIDDLE

PH

FIG. 2. Spectrophotometric titration curve of

cysteine plotted from the data. of Fig 1.

from NH,+-R-S- to NH2--R-S-. Figure

2 shows a curve obtained by plotting the
percentage maximum absorbance against the
pH. This curve represents the SH titration
if the assumption is made that no difference
in extinction coefficient exists between these
b
two forms. It has previously been noted that
210 220 230 240 250 260
the sulfhydryl titration curve for cysteine
exhibits the complex form shown here (2).
These observations were interpreted to indi-
FIG. 1. Difference spectra for cysteine at var- cate a parallel dissociation of the amino
ious pH values. Cysteine concentration was
group and sulfhydryl group of cysteine over
2 X 10-4 M. A solution of 2 X W4 Mcysteine at pH
1.0 was used in the reference cell.
this pH range, such as is shown in the scheme
in Fig. 3. Thus, in this scheme it is proposed
KCl, and 4 ml of 0.004 M selenocystine or cystine
that as the amino group becomes unionized
under a nitrogen atmosphere. Apparent half-wave the ultraviolet absorption spectrum of the
potentials were estimated from the midpoints of dissociated sulfhydryl group is altered such
the curves. that the peak is shifted to longer wave-
lengths. However, De Deken et al. (13) inter-
RESULTS AND DISCUSSION pret the observed spectral titration curves
Titration curves. The observed difference and the shift in the wavelength of the absorp-
spectra for cysteine at various pH values are tion maximum with pH as being indicative
shown in Fig. 1. These results are similar to of intramolecular hydrogen bonding. They
those of Benesch and Benesch (7). The propose the formation of a hydrogen bonded
maxima of the absorption peaks shift to intermediate of the type shown in Fig. 4 and
longer wavelengths at higher pH values as its subsequent ionization to account for the
the predominant absorbing species changes observed spectral shift. Edsall (14), after
 CHEMICAL STUDIES OF SELENOCYSTEINE 167

s-

H,N’ -I---+

*-\ s-
SH /
+
w-
“3 I “ZN I

*\
1” /

FIG. 3. S’cheme proposed by Benesch and

Benesch (2) for:the dissociation of cysteine.

0.6

FIG. 4. Ionization scheme showing hydrogen

bonded intermediate proposed by De Deken et al.
(8) to account for the titration and spectral prop-
erties of cysteine.

consideration of general structural evidence

and the pH dependence of ultraviolet and
Raman spectra, has questioned the existence
of such an intermediate in appreciable con-
centration. Lindley (8) points out that the
failure of all methods employed so far to give
an unequivocal answer to the pattern of
ionization of cysteine is due to the fact that 0.0
220 230 240 250 260 270 280
none gives a measure solely of either of the w
intermediate ionic species. FIG. 5. Difference spectra for selenorysteine
The spectral titration curves of the seleno- at various pH values. Selenocysteine coucentra-
hydryl group at different pH values are quite tion was 2 X 10m4M. A solution of 2 X 1OW &I seleno-
different than the curves for cysteine. As can cysteine at pH 1.0 was used in t,he reference cell.
be seen in Fig. 5, the peak maxima are all at
a somelvhat longer wavelength than was the standard acid-base titration curves show
case for cysteine, and there is no shift in three distinct buffering regions with pK
wavelength of the maximum at higher pH values at 2.01, 5.24, and 9.96 for the
values. The molar absorbance is also greater carboxyl, selenohydryl, and amino groups,
for selenocysteine than cysteine. The spectral respectively. The dissociation of the carboxyl
titration curve, shown in Fig. 6 in conjunc- proton of selenocysteine occurs at a somc-
tion with a simple acid-base titration curve what lower pH value than the corresponding
of all the titratable groups, does not exhibit dissociation in cysteine, which has a pK of
the complex shape observed for the titration 2.30. Titration of selenocyst’eine therefore
of the sulfhydryl group of cysteine and the proceeds by the scheme shown in Fig. 7.
pK obtained from the spectral titration is The lon pK value of t,he selenohydryl
observed to be 5.24. This is much lower than group in relation to the sulfhydryl pK ‘is
that of the sulfhydryl group, and therefore consistent with what would be expected
the titration of the selenohydryl residue does based on the reported ionization constants of
not overlap the region of titration of the H2S and H,Se. Latimer and Hildebrand (15)
amino group to any significant extent. The list the ionization con&ants of H2S as 1.1 X
16s HUBER AND CRIDDLE

.60 -

PH
FIG. 6. Titration curves of selenocystine. X, Spectral titration curve plotted from the
data of Fig. 5. 0, Standard acid-base titration.

SeH SeH Se- se-

pK=Z.Ol pK=5.24 pK=9.96
I 1 - 1 -
HCH - HCH - HCH HCH

I - I I -
HOOC-CC-NH3+ -OOC-CC-NH3+ ‘OOC-CC-NH3+ -OOC-C-NH2
H H H H

FIG. 7. Scheme for the ionization of selenocysteine.

lo-’ and 1.0 X 1P6, while the correspond-
ing ionization constants of HzSe are listed
as 1.7 X 1O-4 and 1 X lo-lo.
If either the explanation proposed by De-
Deken et al. illustrated in Fig. 4 or that pro-
posed by Benesch and Benesch illustrated in
Fig. 3 holds true for cysteine and accounts
for the observed spectral shift to higher
wavelengths, one might expect a similar
scheme to also hold true for selenocysteine.
(Indeed, the selenium analog would be ex-
pected to have somewhat less strain in the
formation of t,he hydrogen bonded inter-
mediate and be more stable than its sulfur-
containing counterpart.) For selenocysteine
there should be no shift in the wavelength
from pH 5 to about 9. At about pH 9 the
amonium group will begin to loose its proton
and t,he hydrogen bonded intermediate
should begin to break up (13) with a subse-
quent shift to higher wavelengths, or alter-
natively, according to Benesch and Benesch
(7) the deionization of the ammonium group
should have the effect of shifting the wave-
length of absorption of the selenide group to
longer wavelengths. Consideration of the
curves of Fig. 5 indicates that no detectable
shift in absorption maximum occurs in solu-
 CHEMICAL STUDIES OF SELESOCYSTEINE 169

tion of pH values up to 12. It seems unlikely
that selenocysteine can exist as the hydrogen
bonded intermediate without demonstrating
the change in absorption spectrum upon
breaking that bond at higher pH. The fact
that no change in absorption was noted does
not allow selection of either of t,he models
for sulfhydryl ionization however.
Rates of reactim of sulfhytlryl and selen.o-
hydryl groups. Reactions of the general type
illustrated by E‘ig. S were follo\ved as a func-
tion of pH by using iodoacetate, iodoacet-
amide, and chloroacetamide. Since the
reaction as written results in the liberation
of prot’ons, a pa-stat may be used to observe
t’he course of the reaction and allo\\- deter-
mination of initial reaction rates. As t,he re-
action takes place with the unprotonated
selenide or sulfide ions, the rate of reaction
is dependent on pH. Thus at low pH
RSH - RS
\ the maximum rate with selenocysteine is 2.2
values, where the concentration of the
dissociated form is low:, the reaction is ex-
tremely slow, but at higher pH values the
concentration of the dissociated form in-
creases and the initial reaction rate increases
accordingly. The equation for the initial
reaction rate in the presence of excess (0.05~)
iodoacetate \vould then be
R = k(iodoacetate) (RF) = K(RS-).
K is a pseudo-first order rate constant which
includes t,he initial iodoacetat,c concentra-
ation. The initial rate R is dependent on
(RS-), which in turn is dependent upon
the pH.
l;igure 9 illustrates a typical family of
curves obtained by measuring the rate of
reaction of iodoacetate \vit’h cysteine as a
function of pH. The maximum pseudo first-
order rate constant for t’his react.ion \vas 1.0
X 1OP min+, while that for reaction with
+ H+ selenocysteinc was 2.2 X lo-” min-I. Thus

I
times greater than the rate for cgsteine. With
ICH$OOH iodoacetnmide the rate constant was 0.61
X lop3 for cystcine and 0.96 X 10-” for
selenocyst,eine, or a 1.6-fold increase in rate
RS-CH2-COOH lyith the selenium analog. The rate con-
stants for reaction with chloroacetamide
FIG. 8. Carboxymethylation reaction of sulf- \\-ere 0.20 X 1O-3 rniIl-l and 0.2s X 1O-3
hydryl group with iodoacetate. The rate of pro- mir+.‘, respectively. In this case t’he rates are
ton liberation in this t,ype reaction was followed
in a pH-stat t,o determine reaction rates of cysteine
much slower and the relative maximum rate
and selenocysteine with iodoacetate, iodoaccta- is only 1.4 times greater for selenocysteine.
mide, and chloroacetamide. In all cases the rate of reaction with seleno-

I I
0 I 2 3 4 5 6 7

Time (min 1
FIG. 9. Rates of reaction of iodoacetate with cysteine at the different pH values indicated.
170 HUBER
0 2 4 6 6 IO 12
PH
FIG. 10. Titration curves of cysteine and
selenocysteine obtained from different spectral
measurements and from measurement of reaction
rates. Ultraviolet spectral titrations for: cys-
teine, ----; selenocysteine, -v-s-.-.. Reaction rates
as a function of pH for: cysteine plus iodoacetate,
“““; selenocysteine plus iodoacetate, -; cys-
teine plus iodoacetamide, -. . .-. . .-; selenocys-
teine plus iodoacetamide ----; cysteine plus
chloroacetamide, . . . . .... ...*; selenocysteine
plus chloroacetamide, ~ p.
cysteine is greater than the corresponding
rate of reaction with cysteine.
Since the reaction rate depends upon the
extent of dissociation of the selenohydryl
sulfhydryl group, one can obtain titration
curves for the dissociation of these groups by
plotting initial reaction rate versus pH as is
shown in Fig. 10. The reaction rate titration
curves for cysteine are slightly displaced to
lower pH values than the titration curve
obtained by the spectral method. Also, it, is
observed that the curve has the form of a
simple titration curve, lacking the second
inflection point obtained with acid-base
titration. The pK for sulfhydryl ionization
obtained by this method is 8.25.
When the reaction of selenocysteine with
iodoacetate or iodoacetamide is studied, the
titration curve obtained from reaction rates
is displaced to much lower pH values than
are obtained with the spectral or standard
acid-base titration. A pK value of 2.0 is ob-
tainedcompared with the spectral pK value
of 5.24. In the case of reaction with chloro-
acetamide the titration curve obtained is
much nearer to that obtained spectrally al-
though still slightly lower. The reaction rate
titration curve for chloroacetamide reacting
with cysteine overlaps the curves for reaction
with iodoacetate and iodoacetamide.
AND CRIDDLE
The reaction pK values of selenocysteine
thus seem to be dependent upon the halo-
derivative used. The faster reacting iodo-
acetate and iodoacetamide react rapidly with
selenocysteine at considerably lower pH
values than does chloroacetamide. The
selenohydryl group reacts even in the undis-
sociated form with the iodo-derivatives,
probably because the selenium atom is a
good nucleophile and iodine is in addition a
14
good leaving group. In the case of chloro-
acetamide, where the chlorine atom is not as
readily removed by nucleophilic displace-
ment, the reaction may require the selenide
form of selenocysteine for reaction. Since
sulfur is probably not, as good a nucleophile
as is selenium, the sulfide ion is the reactive
form with each of the halo-derivatives.
The reaction rates of iodoacetate and iodo-
acetamide wivith selenocysteine are very pH
dependent and yield a curve which resembles
the titration curve of a carboxylate group
since this is the only species present titrating
in this pH range. Since the rate seems to be
the same with either iodoacetate or iodo-
acetamide, it is then suggested that the ioni-
or zation of the carboxyl group of the seleno-
cysteine may play a role in this reaction with
the carboxylate anion helping to displace the
proton from the selenohydryl moiety. The
reasons for the differences in pK values ob-
tained spectrally and those obtained by
reaction kinetics for reaction with cysteine
and for the reaction of selenocysteine with
chloroacetamide is not clear at, the present
time.
Solubilities. The solubilities of selenocys-
tine and cystine were compared at pH 7 and
25”. The data is shown in Table I along with
the summary of the chemical properties of the
two amino acids. On a molar basis seleno-
m,-cystine is about six times as soluble as
m,-cystine. The solubilities of selenocysteine
and cysteine were not compared since they
are both quite soluble, and it, is especially
difficult to keep them in a reduced form in
the process of determining their solubilities.
Stability to hydrolysis. An investigation of
the stability of the two amino acids in 6 N
HCl at 110” was undertaken, since determi-
nations of amino acids incorporated into
proteins are normally carried out after
 CHEMICAL STUDIES OF SELENOCYSTEINE 171

TABLE I
CHEMICAL PROPERTIES OF CYSTEINE AND SELENOCYSTEINE

Property Selenocysteine Cyst&e

PK 5.2 8-9
Reaction rate (iodoacetate) constant 2.2 X 10e3 min-1 1 .O X 1OP mini
Reaction rate (iodoacetamide) constant 0.96 X 1P min-i 0.61 X 10-S mine1
Reaction rate (chloroacetamide) 0.28 X 1tP min-i 0.20 X 19+ mine1
constant
Solubility 0.00235 M (Se-cystine) 0.00038 M (cystine)
Fraction left after 6 hours of 0.05 0.95
hydrolysis in 6 N HCl 110”
Apparent half-wave potentials -0.212 v 0.021 v

100%

75%
% of
original
collccntmtil

25%

HOURS
FIG. 11. Decrease in concentration of cysteine and selenocysteine as a function of time
hydrolysis in 6 N HCl at 110”.

hydrolysis under these conditions. While
selenocystine has not generally been found in
such hydrolyzates, there are several reports
claiming to have detected it (16, 17). Such a
hydrolysis tends to slowly decompose cystine
(18), and Peterson and Butler (2) have sug-
gested that acid hydrolysis might break
down selenocystine. In this study, the per-
centage of amino acid remaining after
heating with 6 N HCl for various periods of
time was determined. The results are shown
in Fig. 11. Essentially all of the selenocys-
tine was destroyed within the first 6 hours of
heating, but only a small fraction of the
cystine was destroyed.
When products of the acid hydrolysis of
selenocystine were investigated by using an
automatic amino acid analyzer, three major
172 HUBER AND
ninhydrin-reactive products were detected.
The first of these was ammonia. This prob-
ably is formed by hydrolysis of selenocystine
to dehydroalanine, which, in acid solut)ion,
is hydrolyzed to yield equal amount,s of pyru-
vate and ammonia. Pyruvate was established
as a hydrolysis product by reaction with
dinitrophenyl hydrazine (19). Ko spectrally
detectfable (20) dehydroalanine was found in
the reaction mixture after hydrolysis. Ap-
proximately 33 % of t’he initial selenocystine
can be recovered as pyruvate and ammonia.
The other two products are eluted in the
region between alanine and valine by using
the buffer system of Moore et al. (21). The
ident’ity of these two compounds has not yet
been established. Because they migrate in
this general area and because they react with
ninhydrin, it is evident that both the amino
group and the carboxyl group remain intact.
Another quite minor product is present
which, based on its migration, on the
analyzer, appears to be alanine. This
accounts for only about l-3% of the total
products. During hydrolysis of selenocystine,
the hydrolysis tube becomes coated with a
black precipitate, which indicates that
another breakdown product of selenocystine
hydrolysis is elemental selenium.
Cystine hydrolysis does not result in the
formation of either pyruvate or ammonia.
The only detectable compounds reacting
with ninhydrin after 20 hours of hydrolysis
of cystine are cystine and a compound which
elutes from the ion-exchange column quite
soon after cystine. The identity of the later
compound was not determined.
Selenocysteic acid is also unstable to acid
hydrolysis. After oxidation of selenocystine
by the method of Hirs (la), the major prod-
uct detectable on the amino acid analyzer
eluted from the column very near to the
region of elution of cysteic acid and was
probably selenocysteic acid. However, sev-
eral other compounds amounting to approxi-
mately 10 % of the total ninhydrin color were
also found, including small peaks in the area
of glycine and alanine. Acid hydrolysis (20
hours) completely destroyed the selenocys-
teic acid. The major products of the hydrol-
ysis were ammonia, pyruvate, and two peaks
eluting in the regions that glycine and
CRIDDLE
alanine usually elute from the analyzer
column. Several other minor peaks were
present, and the black precipitat’e of sele-
nium was again formed, The normally used
procedures for determining cysteine or cys-
t,ine in proteins are therefore completely in-
capable of detecting bhe presence of seleno-
cystine, and reports of selenocystine in acid
hydrolyzates of protein must be disregarded.
If selenocysteine and selenocystine are to be
det’ected in proteins, it will necessitate
hydrolysis by a procedure such as proteolyt,ic
digestion.
l’olarography. Polarographic studies of the
two compounds showed that selenocystine
has a lower apparent half-wave potential
than does cyst’ine (Table I). This fact is re-
flected by the observation that the reduced
selenocysteine is more readily oxidized to
selenocystine than is the case for cysteine
forming cystsine.
CONCLUSIONS
The observations on the chemical prop-
erties of selenocystine and selenocysteine as
compared with those of the corresponding
sulfur-cont,aining amino acids (summarized
in Table I) demonstrate that some major
differences exist in the properties of these
amino acids. The chemical differences are
sufficiently great that it seems unlikely that
the selenium analog of the amino acid could
replace cysteine or cystine in a protein mole-
cule without, marked alteration of the protein
structure and function. This should be partic-
ularly true for the class of “sulfhydryl pro-
teins” whose activity seems directly depen-
dent on the presence of a free cysteine side
chain. In the physiological pH range near
pH 7, the cysteine side chain exists predom-
inantly in the sulfhydryl form, while seleno-
cysteine is found almost exclusively as the
selenide ion. The pH dependence of activity
of a selenocysteine containing “sulfhydryl
protein” would therefore be expected to be
greatly changed. It is more likely, however,
that activity would be completely lost due
to the loss of the fine balance between charge
and structural orientation at the active site.
Since the chemical reactivities of the two
reduced amino acids are different, this would
also be reflected in the properties of proteins
 CHEMICAL STUDIES
containing them. For example, the kinetics
of enzyme reactions involving the acylation
of a sulfhydryl group as an intermediate
would be altered in the selenium-containing
protein. The inactivation of such an enzyme
by reactions with “sulfhydryl” reagents
would also be enhanced. A frequent cause of
activity loss in proteins is t’he oxidation of
sulfhydryl residues and the formation of di-
sulfide bonds. As the polarography experi-
ments indicate, this would be even a greater
greater problem with a selenohydryl group
since it is difficult to maintain it in a reduced
form. Diselenide bond formation or mixed
sulfur-selenide bonds could readily form to
inactivate the enzyme.
In view of these differences, it is not sur-
prising that there have been few reports of
the detection of selenocysteine in biological
systems. All reports of selenocysteine recov-
ered from acid hydrolysis of proteins must,
be discounted on the basis of instability of
this selenium amino acid.
Selenocystine may exist as a free amino
acid in certain organisms grown in the pres-
ence of selenium compounds, and the possi-
bilit#y still exists that some of this amino acid
may be incorporated into proteins. How-
ever, extreme care must be used in interpret-
ing chromatographic data indicating the
presence of this amino acid, since selenium-
containing products from acid hydrolysis of
prot’ein extracts have been observed to mi-
grate with and have been identified as seleno-
cystine.
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