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Polarography and zone electrophoresis of uranyl-L-methionine complexes
JOURXAL
OF
ELECTRO-AKXLYTICIL
POLAROGRXPHY AKiD URANYL-L-RIETHIONINE
TSA-TEH
LA1
Depm-imeni
of CJzemicaZ
XSD
September
(Received
CHEMISTKY
ZOI\;E ELECTROPHORESIS COMPLEXES
HEXG-SHOE Epzgzneevixg.
19th.
225
OF
CHEXG Cherzg
K?r~r 5 Urriuevsity,
Tainan.
Taiwan
(China)
1965)
IXTRODUCTION
studied
The formation of complexes between methionine and metal ions has been by various methods. PELLETIER~ has reported the formation of I : I-, I : 2- and
I :3-complexes between nickel and methionme, and determined then formation constants by Bjerrum’s method_ PERRIX~.~ measured the formation constants of I : Iferric- and -ferrous-methioninate complexes by potentiometric titration_ The I I I and I : a-cupric-methioninate complexes have been studied by several investigatorsJ=s. LI AND MXXXING~ deduced the binding site in the complexes of zinc and lead with methionine and measured their formation constants_ In the present x-ark, the complex of r-methionine with uranyl ion has been studied by polarography and zone electrophoresis_
EXPERIMENTAL.
The apparatus and procedures used in this work have been reported elsehaving a flow rate of where7. Polarograms were taken at 30 k 0.1~ using a capillary 2.140 mg/sec and a drop time of 4-39 set at 69.1 cm of mercury height (1~) in 0.5 M sodium perchlorate solution at -0-250 V us. S_C_E. Triton X-100 (o_ooz~/~) was used as a maximum suppressor and 0.2 M sodium perchlorate as supporting electrolyte. The preparation and analysis of the uranyl perchlorate solution used was described in an earlier papers_ A 2.00-M L-methionine solution was freshly prepared before use by dissolving 29.85 g of special grade (Nutritional Biochemicals Corporation) in deionized water and diluting to IOO ml. RESULTS
Nature
_4ND
DISCUSSION
of yeduction
The reversibility of the electrode reaction was deduced immediately from the value of E, -E, and a plot of log i/( id-i) was made for further confirmation. The (EP -E&value for all polarograms falls in the range 0.054-0_060 V and the log plot has an average reciprocal slope of o.ogS, in the pH range 0.8-4-7 and ligand-concentration range o_o&o.z M_ All these values are in good agreement with the theoretical J_ EZeclroanaZ
Chem..
12
(1966)
zz5-zzg
226
IAT
value
for a one-electron
temperature (which
reversible
coefficient
is less than
of the
+ I mV/O)
reduction.
half-wave
provides
The
value
potential
further
of
-
in the
evidence
0.23
T_ T., C,YESG
iX
mV,/degrce
temperature
for the
range
of the reversible
S.
6-45,”
character
of
the reaction_ solution ate and
A constant value for id,/h+=o_34S fo.ooq was obtained for the polarographic of 1.0 rnlM uranyl perchlorate, 0-06 Jf r-methionine, o-2 LW sodium perchloro_ooz~/, Triton X-100 at pH 4.~4 at a mercury height of 47 z-So.5 cm. The
temperature
coefficient
data indicate Effect
of PW
The
of the diffusion
that the electrode asvd .&and
effect
of
reaction
cm-rent
was found
is entirely
to be r.r5:4/degree
These
diffusion-controlled_
concemtratiolz
pH
on
the
polarographic
behaxiour
of
uran>-l-L-methionine
complex was studied over the pH range 0.5-4-7 by polarographing solutions of 1.0 O.OOZ~/~ Triton X-100 and rdkf uranyl perchlorate in 0.2 M sodium perchlorate, various concentration of r;methionine. The upper limit of pH was restricted to 4.7 by the precipitation of uranium- The greater the concentration is the pH value that can be reached before precipitation
0_06o
Fig.
I. Variation of E, vrith At_ I-0 mfll UO2(C104)2,
Fig_ 0.06
z_ Variation M Cm and
of 0.0020,<
of L-methionine.
pH and ligand concn.. C,: (0). o-zoo; ( 0). o-z _W NaC104 and O.OOZ~/~ Triton 3”roe.
polarographic characteristics Triton X-roe_
x-ith pH.
I o ml11
o-140;
the higher
(X).
UOz(C10~)z.
o.Ioo;
o-1
(A).
iW NaClOa.
It can be seen from Fig. I, that the four curves are divided into four sections_ In section I, alI the curves coincide with the horizontal line of the half-wave potential, -0-177 V. which is the first half-wave potential of the simple uranyl ion and it can therefore be inferred that the complex formation between uranyl ion and L-methionine does nottakeplaceinthisregionofpHvalueandligandconcentration_ due
In section II of Fig_ I, the half-wave potential is shifted to the negative side to the complex formation of uranyl ion with L-methionine. As the values9 of
pKcoon aud pK_2 ligand species for
for methionine are 2-28 and 9.21, respectively, the most probable complexing under these experimental conditions should be iso-
electric dipolar L-methionine ion, CHBS(CHZ)&H(NH~+)COO-. Hence, formula~of the complex may be expressed by UOaM?z+ where M denotes L-methionine ion ; the half-cell electrode reaction may be expressed as UOaMpz+se
=
the general the dipolar
UOzM,++(p-q)M
If the nq&naleoucentratiou~of
L-methionine
is denoted
by C,,,, and
KCOOH
by K,,
POLAROGRAPHY the half-wave
(E,).
OF URAXYL-L-31ETHIOXIXE
potential = (5)s
of the complex
+ 0.06
log K-
-00.06@--){log
227
COMPLEXES
is given
o.o6(p
-
aslo
4) log C,
[H’])}
I-,-log&+
(1)
where K is the ratio of dissociation constants of the oxidized and reduced complex ions. For the greater portion of this section, the pH-value is small enough to give an as compared to K,. Then eqn. (I) reduces to exceedingly large H+ concentration (E&c = const.-oo.06(p-9)
log C,-00.06(p-q)pH
(2)
This equation indicates that the half-wave potential of the complex should be a linear This was verified in this section. The slope function of pH when C, is kept constant. ion is consumed for the reduction of of the line is o.ozg, showin, = that one hydrogen two uranyl ions. However, as the pH-value approaches pk’, and Ka is appreciable as compared to the H+ concentration, the linear relation no longer holds. As the pH value exceeds p&Z, and the Hf concentration becomes negligibly small in comparison with I<,,
eqn.
(E,),
=
(r) then reduces const.
-
0.06Cp -
Hence, a region where the observed_ This corresponds electrode reaction_
q) log C,
(3)
pH is independent of the half-wave potential should be to section III, where no hydrogen ion is involved in the show a third inflexion is greater than 4.00, the curves slope of -o_o2g is obtained_ It indicates that one hydroxyl
When the pH-value and section IV with a mean ion is released
to
in the reduction
of two hydroxylated
pH
uranyl
ions.
C
I3 Z-40
pH 320
017
-Es
Fig.
3
Plots
of
Fig.
4_ l3ectrochromatograms
-E+
US. log
“5
E
S C E.(V)
C,.
pH:
uoy
( A),
Z-I-Z-~;
(
0).
of the uranyl--L-methionine
2-5-4-0;
(X).
complexes
pH 4
300
O-4-7.
pH.
at various
The effect of pH on diffusion current is shown in Fig_ 2. The variation of halfwave potential with pH for the same polarographic solution is also shown for comparison_ It can be seen that the diffusion current constant has a value of 1.52 at pHvalues lower than z_6S, and r-37 at pH-values greater than 2.72. The sudden drop of J_ Elecfroanal.
Chetn.,
12
(1966)
7-25-2zg
zzs
LA1
the diffusion current constant from potential becomes pH-independent, has occurred_
1.5~2 to 1-37 around pH reveals that a change
T.
T.,
CHEKG
2.70, where the half-wave of metal complex species
In order to study the ligand number of the complex, a plot of half-wave tial ‘us. the log of the ligand concentration is sho\vn in Fig. 3_ The slopes of L3E,/;I for the three pH regions, Z-I-2.5, z-5-+0 and 4.~4 7 are of the same -o.ogS, indicating that one more ligand is attached to U(V1) than to U(V) _ Zo?te
H_ 5.
potenlog C, value,
eEectro@wesis
In order to determine the formulae of the complexes, their electrical behaviour was studied by zone electrophoresis. A 7-0 x z-o-cm strip of Toyo No. 52 chromatography paper was cut into three pieces. The two end-pieces were moistened with 0.10 M L-methionine solution_ The centre piece was moistened with 1.0 mM uranyl perchlorate solution containing o-10 M L-methionine solution_ The three pieces had the same pH-value which was varied from 2.4 to 4-47 for each experiment. The three pieces were placed side by side on a glass plate. Platinum electrodes were laid across the ends of the strip and the assembly covered with another glass plate. A potential of 200 V d-c_ was applied for 6 min in each experim ent_ Potassium ferrocyanide was used as developer. The results are shown in Fig. 4_ A control paper, A, indicates the pattern of the chromatogram for zero-potential applied. The shaded areas show the final position of uranium. B, C and D indicate that the complex species at pH 3-40 is cationic, and that at pH 3.20 and 4-37, the complexes are of the same species, a neutral molecule. The results are in good agreement with the prediction made from the Id-pH curve in Fig. Z_ A chromatogram of simple uranyl ion, as a reference, was obtained by moistening the two end-pieces with a very dilute and the centre piece with 1.0 mM uranyl
solution of sodium perchlorate at pH 3.00, perchlorate solution at the same pH as the
end-pieces. The result is given in E, in which the zone has moved about twice as far as in B, toward the negative electrode. As the electrophoretic mobility of simple uranyl ion is essentially equal to that of its complex species, it was concluded that the complex species for B is unit-positively charged. Thus, taking into consideration that the uranyl ion usually shows a co-ordination number of four, the complex species of uranyl-_-methionine complexes were confirmed to be UOa(OH)l&+ at pH 2.40 and UOZ(OH)ZMZ at pH 3-30 and pH 4_37_ TABLE EVIDENCE
PH
I FOR
THE
No_ of H+ DE-
2-1~2.5
COhZPLEX
o-5
4--4-7
or
inuoZued (H+)
z-5-4-o E-5 (OH-)
SPECIES
P-_q I I I
AND
ELECTRODE
Charge U( VI) +I
cl 0
RE9CTIONS
of cornpzex
Complex
species
ut VI)
Ul V)
UOy(OH)Nxf UOz(OH)zMz UOz(OH)zMz
UOz(OH)BG UOz(OH)&(UO+(OH)xM-
Results based on the foregoing data and discussion and the electrode reactions are expressed as follows :
are s nmmarized
in Table
I,
POLAROGRAPHY
at pH
OF
UR%NYL-L-JIETHIOh-IKE
COMPLEXES
229
2.1-2.5,
2
at pH
UO,(OH)M,‘+H++z
e =
3 UO~(OH)M~+H~I++M
3_5-q-0, uOz(0H)zM~
at pH
+ e =
UO,(OH).M-+M
4_*4_7, 2 U02(OH)2M2+2
e =
(UO&(OH)a~I-+3
M+OH-
ICKSOWLEDGEMEKT
The
authors
thank
the
National
Council
on
Science
Development
for
their
support.
The uran3-1-r-methionine complex has been studied using polarography zone electrophoresis over the pH range o-S-4-7 and the ligand concentration with the formufae UOz(OH)Ma+ o.o~o.zo iW at 30 ko_r”. Two complexes UOI(OH)ZMZ, M denoting dipolar L-methionine ion, have been pH-values. The effects of pH and ligand concentration on the current-voltage curves are discussed and the electrode reactions
and range and
confirmed at various characteristics of the represented_
REFERENCES I z 3 4 5 6 7 8 g IO
S. D. D. C. N. N. T. T. 0. T.
J_ PELLETIER, JChim. Phys.. 57 (1960) 297. D_ F’ERRIN, J_ Chenz. SOG;., (1958) 31~5. D. PERRIN. ibid., (1959) zgoJ_ HAWKINS _XKD D. D. PERIUN. Imorg Chew., 1 (1963) 843. C_ LI AKD E. DOODY, /_ Am. Chenz. Sot , 74 (1951) qSq_ c. LI AXD R. -9. ~~A~~IXG. ibid., 77 (1955) 5~25. T- LAI AND S- J_ XVEY, /_ EZecl~ocJ~m. SOG , III (1964) 1183. T. LAI X-D T. L. CHANG. Amal. Chenr., 33 (1961) IIg3_ H. EMERSON, P. L. KIRK XXD C. L. A. SCHMID%-, J. Biol. Chem., gz (1931) T. LAI, S. X_ CHEN, B. C WXNG XYD C. C. HSIEH, dxaL_ CJzaenz., 35 (1963) J.
Electyoazal
CJLem.,
IZ
_t+g 1531. (1966)
zz5-zzg
OF
ELECTRO-AKXLYTICIL
POLAROGRXPHY AKiD URANYL-L-RIETHIONINE
TSA-TEH
LA1
Depm-imeni
of CJzemicaZ
XSD
September
(Received
CHEMISTKY
ZOI\;E ELECTROPHORESIS COMPLEXES
HEXG-SHOE Epzgzneevixg.
19th.
225
OF
CHEXG Cherzg
K?r~r 5 Urriuevsity,
Tainan.
Taiwan
(China)
1965)
IXTRODUCTION
studied
The formation of complexes between methionine and metal ions has been by various methods. PELLETIER~ has reported the formation of I : I-, I : 2- and
I :3-complexes between nickel and methionme, and determined then formation constants by Bjerrum’s method_ PERRIX~.~ measured the formation constants of I : Iferric- and -ferrous-methioninate complexes by potentiometric titration_ The I I I and I : a-cupric-methioninate complexes have been studied by several investigatorsJ=s. LI AND MXXXING~ deduced the binding site in the complexes of zinc and lead with methionine and measured their formation constants_ In the present x-ark, the complex of r-methionine with uranyl ion has been studied by polarography and zone electrophoresis_
EXPERIMENTAL.
The apparatus and procedures used in this work have been reported elsehaving a flow rate of where7. Polarograms were taken at 30 k 0.1~ using a capillary 2.140 mg/sec and a drop time of 4-39 set at 69.1 cm of mercury height (1~) in 0.5 M sodium perchlorate solution at -0-250 V us. S_C_E. Triton X-100 (o_ooz~/~) was used as a maximum suppressor and 0.2 M sodium perchlorate as supporting electrolyte. The preparation and analysis of the uranyl perchlorate solution used was described in an earlier papers_ A 2.00-M L-methionine solution was freshly prepared before use by dissolving 29.85 g of special grade (Nutritional Biochemicals Corporation) in deionized water and diluting to IOO ml. RESULTS
Nature
_4ND
DISCUSSION
of yeduction
The reversibility of the electrode reaction was deduced immediately from the value of E, -E, and a plot of log i/( id-i) was made for further confirmation. The (EP -E&value for all polarograms falls in the range 0.054-0_060 V and the log plot has an average reciprocal slope of o.ogS, in the pH range 0.8-4-7 and ligand-concentration range o_o&o.z M_ All these values are in good agreement with the theoretical J_ EZeclroanaZ
Chem..
12
(1966)
zz5-zzg
226
IAT
value
for a one-electron
temperature (which
reversible
coefficient
is less than
of the
+ I mV/O)
reduction.
half-wave
provides
The
value
potential
further
of
-
in the
evidence
0.23
T_ T., C,YESG
iX
mV,/degrce
temperature
for the
range
of the reversible
S.
6-45,”
character
of
the reaction_ solution ate and
A constant value for id,/h+=o_34S fo.ooq was obtained for the polarographic of 1.0 rnlM uranyl perchlorate, 0-06 Jf r-methionine, o-2 LW sodium perchloro_ooz~/, Triton X-100 at pH 4.~4 at a mercury height of 47 z-So.5 cm. The
temperature
coefficient
data indicate Effect
of PW
The
of the diffusion
that the electrode asvd .&and
effect
of
reaction
cm-rent
was found
is entirely
to be r.r5:4/degree
These
diffusion-controlled_
concemtratiolz
pH
on
the
polarographic
behaxiour
of
uran>-l-L-methionine
complex was studied over the pH range 0.5-4-7 by polarographing solutions of 1.0 O.OOZ~/~ Triton X-100 and rdkf uranyl perchlorate in 0.2 M sodium perchlorate, various concentration of r;methionine. The upper limit of pH was restricted to 4.7 by the precipitation of uranium- The greater the concentration is the pH value that can be reached before precipitation
0_06o
Fig.
I. Variation of E, vrith At_ I-0 mfll UO2(C104)2,
Fig_ 0.06
z_ Variation M Cm and
of 0.0020,<
of L-methionine.
pH and ligand concn.. C,: (0). o-zoo; ( 0). o-z _W NaC104 and O.OOZ~/~ Triton 3”roe.
polarographic characteristics Triton X-roe_
x-ith pH.
I o ml11
o-140;
the higher
(X).
UOz(C10~)z.
o.Ioo;
o-1
(A).
iW NaClOa.
It can be seen from Fig. I, that the four curves are divided into four sections_ In section I, alI the curves coincide with the horizontal line of the half-wave potential, -0-177 V. which is the first half-wave potential of the simple uranyl ion and it can therefore be inferred that the complex formation between uranyl ion and L-methionine does nottakeplaceinthisregionofpHvalueandligandconcentration_ due
In section II of Fig_ I, the half-wave potential is shifted to the negative side to the complex formation of uranyl ion with L-methionine. As the values9 of
pKcoon aud pK_2 ligand species for
for methionine are 2-28 and 9.21, respectively, the most probable complexing under these experimental conditions should be iso-
electric dipolar L-methionine ion, CHBS(CHZ)&H(NH~+)COO-. Hence, formula~of the complex may be expressed by UOaM?z+ where M denotes L-methionine ion ; the half-cell electrode reaction may be expressed as UOaMpz+se
=
the general the dipolar
UOzM,++(p-q)M
If the nq&naleoucentratiou~of
L-methionine
is denoted
by C,,,, and
KCOOH
by K,,
POLAROGRAPHY the half-wave
(E,).
OF URAXYL-L-31ETHIOXIXE
potential = (5)s
of the complex
+ 0.06
log K-
-00.06@--){log
227
COMPLEXES
is given
o.o6(p
-
aslo
4) log C,
[H’])}
I-,-log&+
(1)
where K is the ratio of dissociation constants of the oxidized and reduced complex ions. For the greater portion of this section, the pH-value is small enough to give an as compared to K,. Then eqn. (I) reduces to exceedingly large H+ concentration (E&c = const.-oo.06(p-9)
log C,-00.06(p-q)pH
(2)
This equation indicates that the half-wave potential of the complex should be a linear This was verified in this section. The slope function of pH when C, is kept constant. ion is consumed for the reduction of of the line is o.ozg, showin, = that one hydrogen two uranyl ions. However, as the pH-value approaches pk’, and Ka is appreciable as compared to the H+ concentration, the linear relation no longer holds. As the pH value exceeds p&Z, and the Hf concentration becomes negligibly small in comparison with I<,,
eqn.
(E,),
=
(r) then reduces const.
-
0.06Cp -
Hence, a region where the observed_ This corresponds electrode reaction_
q) log C,
(3)
pH is independent of the half-wave potential should be to section III, where no hydrogen ion is involved in the show a third inflexion is greater than 4.00, the curves slope of -o_o2g is obtained_ It indicates that one hydroxyl
When the pH-value and section IV with a mean ion is released
to
in the reduction
of two hydroxylated
pH
uranyl
ions.
C
I3 Z-40
pH 320
017
-Es
Fig.
3
Plots
of
Fig.
4_ l3ectrochromatograms
-E+
US. log
“5
E
S C E.(V)
C,.
pH:
uoy
( A),
Z-I-Z-~;
(
0).
of the uranyl--L-methionine
2-5-4-0;
(X).
complexes
pH 4
300
O-4-7.
pH.
at various
The effect of pH on diffusion current is shown in Fig_ 2. The variation of halfwave potential with pH for the same polarographic solution is also shown for comparison_ It can be seen that the diffusion current constant has a value of 1.52 at pHvalues lower than z_6S, and r-37 at pH-values greater than 2.72. The sudden drop of J_ Elecfroanal.
Chetn.,
12
(1966)
7-25-2zg
zzs
LA1
the diffusion current constant from potential becomes pH-independent, has occurred_
1.5~2 to 1-37 around pH reveals that a change
T.
T.,
CHEKG
2.70, where the half-wave of metal complex species
In order to study the ligand number of the complex, a plot of half-wave tial ‘us. the log of the ligand concentration is sho\vn in Fig. 3_ The slopes of L3E,/;I for the three pH regions, Z-I-2.5, z-5-+0 and 4.~4 7 are of the same -o.ogS, indicating that one more ligand is attached to U(V1) than to U(V) _ Zo?te
H_ 5.
potenlog C, value,
eEectro@wesis
In order to determine the formulae of the complexes, their electrical behaviour was studied by zone electrophoresis. A 7-0 x z-o-cm strip of Toyo No. 52 chromatography paper was cut into three pieces. The two end-pieces were moistened with 0.10 M L-methionine solution_ The centre piece was moistened with 1.0 mM uranyl perchlorate solution containing o-10 M L-methionine solution_ The three pieces had the same pH-value which was varied from 2.4 to 4-47 for each experiment. The three pieces were placed side by side on a glass plate. Platinum electrodes were laid across the ends of the strip and the assembly covered with another glass plate. A potential of 200 V d-c_ was applied for 6 min in each experim ent_ Potassium ferrocyanide was used as developer. The results are shown in Fig. 4_ A control paper, A, indicates the pattern of the chromatogram for zero-potential applied. The shaded areas show the final position of uranium. B, C and D indicate that the complex species at pH 3-40 is cationic, and that at pH 3.20 and 4-37, the complexes are of the same species, a neutral molecule. The results are in good agreement with the prediction made from the Id-pH curve in Fig. Z_ A chromatogram of simple uranyl ion, as a reference, was obtained by moistening the two end-pieces with a very dilute and the centre piece with 1.0 mM uranyl
solution of sodium perchlorate at pH 3.00, perchlorate solution at the same pH as the
end-pieces. The result is given in E, in which the zone has moved about twice as far as in B, toward the negative electrode. As the electrophoretic mobility of simple uranyl ion is essentially equal to that of its complex species, it was concluded that the complex species for B is unit-positively charged. Thus, taking into consideration that the uranyl ion usually shows a co-ordination number of four, the complex species of uranyl-_-methionine complexes were confirmed to be UOa(OH)l&+ at pH 2.40 and UOZ(OH)ZMZ at pH 3-30 and pH 4_37_ TABLE EVIDENCE
PH
I FOR
THE
No_ of H+ DE-
2-1~2.5
COhZPLEX
o-5
4--4-7
or
inuoZued (H+)
z-5-4-o E-5 (OH-)
SPECIES
P-_q I I I
AND
ELECTRODE
Charge U( VI) +I
cl 0
RE9CTIONS
of cornpzex
Complex
species
ut VI)
Ul V)
UOy(OH)Nxf UOz(OH)zMz UOz(OH)zMz
UOz(OH)BG UOz(OH)&(UO+(OH)xM-
Results based on the foregoing data and discussion and the electrode reactions are expressed as follows :
are s nmmarized
in Table
I,
POLAROGRAPHY
at pH
OF
UR%NYL-L-JIETHIOh-IKE
COMPLEXES
229
2.1-2.5,
2
at pH
UO,(OH)M,‘+H++z
e =
3 UO~(OH)M~+H~I++M
3_5-q-0, uOz(0H)zM~
at pH
+ e =
UO,(OH).M-+M
4_*4_7, 2 U02(OH)2M2+2
e =
(UO&(OH)a~I-+3
M+OH-
ICKSOWLEDGEMEKT
The
authors
thank
the
National
Council
on
Science
Development
for
their
support.
The uran3-1-r-methionine complex has been studied using polarography zone electrophoresis over the pH range o-S-4-7 and the ligand concentration with the formufae UOz(OH)Ma+ o.o~o.zo iW at 30 ko_r”. Two complexes UOI(OH)ZMZ, M denoting dipolar L-methionine ion, have been pH-values. The effects of pH and ligand concentration on the current-voltage curves are discussed and the electrode reactions
and range and
confirmed at various characteristics of the represented_
REFERENCES I z 3 4 5 6 7 8 g IO
S. D. D. C. N. N. T. T. 0. T.
J_ PELLETIER, JChim. Phys.. 57 (1960) 297. D_ F’ERRIN, J_ Chenz. SOG;., (1958) 31~5. D. PERRIN. ibid., (1959) zgoJ_ HAWKINS _XKD D. D. PERIUN. Imorg Chew., 1 (1963) 843. C_ LI AKD E. DOODY, /_ Am. Chenz. Sot , 74 (1951) qSq_ c. LI AXD R. -9. ~~A~~IXG. ibid., 77 (1955) 5~25. T- LAI AND S- J_ XVEY, /_ EZecl~ocJ~m. SOG , III (1964) 1183. T. LAI X-D T. L. CHANG. Amal. Chenr., 33 (1961) IIg3_ H. EMERSON, P. L. KIRK XXD C. L. A. SCHMID%-, J. Biol. Chem., gz (1931) T. LAI, S. X_ CHEN, B. C WXNG XYD C. C. HSIEH, dxaL_ CJzaenz., 35 (1963) J.
Electyoazal
CJLem.,
IZ
_t+g 1531. (1966)
zz5-zzg