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Determination of spectral properties of aqueous I2 and I3− and the equilibrium constant
.~N.~LT'TIc.\I,
RIOCHl?MISTRY
Determination and
42,
t0.5201
(1!)71)
of Spectral I::- and
the
R~~ccivcd
Properties Equilibrium
Sovfmlwr
of Aqueous
12
Constant
10, 1970
.A number of reports liar-e :tpl)~aretl conceruing the spectral properties of aqueous solutions of I, anal I:, (-6). Howvcr, there has not been total agreement among investigators on these prolwrtics. Pul~lislwcl values for the extinction coeffiricwt. of 1::. vary as much as 20%‘. Rrccnt investigations of iodide metnbolism in biological systems and of thyroxine biosynthesis in particular have pointc~l up the ncwl for accurate values, ilot only for the spectral prolwrties, hut also for the eyuilihrium constant involving I-, I,, and I:,-. The present inr-cst,igation was unclertakcn to rc-evaluate these con&ants.
Lactoperoxidase was isolatccl and purified as pwviously outlined (7,8), and the concentration cktermincd by 412 nm nl~sorbancc, using a millimolnr extinction coefficient of 114 (9). Horseratlish lwroxidase (,HRP) , type VII-L, was obtained from Sigma Ckmical Co. The concentration of HRP was determined by 403 nm absorbance, using a millimolar extinction cocfficicnt of 91 (10). Reagent-grade rlicmicals were employed. Sodium thiosulfatc was standarclizcd against RIO, il I). Peroxide concentration was mcasurcd at 230 nm using a molar extinction coefficient, of 72.4 (12). All experiment s were performed at 24°C and in 0.001 31 H,SO,, to minimize hydrolysis of I, (131. A$ Zeiss P1IQ II dr~tcrlllinations and a spcctrophotometer was employc(l for alworl)ancr Gary model 14 rccordiiig s~,cctrol~llotolli~~t(~r wa. q usc~cl to ol,tnin slwctrn. cstinction Iodide could be dcterminr~tl at 226 11111,using :L millimolar coefficient of 12.3 (4). An Orion model 94-53 iodidcs electrode connected to :t Radiometer PHRI 22r millivolt mctcr ww used for iodide concentration measurement.
07 0.6 5 7 T
_
o5t-
IA
1
zw- 0.4 H" 0.3
1
0.2 01 0 300
Frc;. 1. Visible (~ll:d!~ze(l oxidation
I:,-
400
L.-. 450 X,nm
500
550
600
and ultraviol(,t slwctrunl of I?. I, \V:W oht:rined t)y of known aolrllions of I :~nd rl~cortl(~tl :is inclic:rtt~ti
P:xtinction
I?
350
4fiO :&50 :;:io :;;i:;
460
TARI,I:, Coefiicients
0.70 0. o:~:i 21 !) 2"'. 1 0.87
I for I? and I:,
11 746 O.I)IS
(4) (4)
26.4
(4);
2%.!+
3.1
(1);
21 .O (6’)
0.975
(4);
1.16
(5,) (1')
I)~~roxitlnsc~in the tc~sl.
35
30
25 ‘E 7% 20 H E
IO
275
I+;.
in t,hr
2. text.
Spc‘c.trurll
of
300
I, -. Iinoum
350
400
450 h,nm
concentrations
500 of
550 I.,-
600 w’re
oht:~ined
outlined
198
Solving equation respectively :
MOHRISON,
RAYSE,
AKiU
MICHAELS
A&i,, = “1.9.r + 0.035jj
0))
.-1go = 0.S7.C + 0.701/
Cc)
(1)) for x and then substituting
.r =
&(I
(c) gives,
- o.ox5y ‘1.9
A &cj(, -
!/ =
into equation
Cd)
0.0397Aa~" 0.699
(e>
From tlic I:,- conccutration and tlrc knowi atl(lition of iodide, equilihriurn iodide could be tletmnined. In Fig. 3 are 1)lotted the results of three .4
.3
‘,“I-”
.2
I-,mM FIG. 3. Iodirw-triioclide cqlds K”.,, CL+ outlined
in
equilibrium thy test.
constant.
The
slope
of the
lint.
0.768
m!W’.
different esl~eriineiits. The iiiillimolar ratio of 1:: /I, is plotted against millirnolar iodide concentrations at equilihriuln. The slolw of the line, an arcrage of 0.768 m%f-’ (76’8 Jf--‘), is A,,. Triiotlide contamination of an I, solution can give large errors in determination of I, concentration from 460 nm nbsorbancc readings. Fig. 4 illustrates this point. To a solution of iodine (0.22 1nX) were added five successive increments of 0.1 inM iodide. Large increases in the 350 nm absorbance :Irc’ noted, with little change at 460 nm. The similarity of I, and I::- millirnolar extinction coefficients at 460 nm may also be noted in Table 1. Figure 5 illustrates that LP and HRP will oxidize only lilnite(1 amounts of iodide quantitatively. The total iodide in solution is plotted versus I, produwl by t,hesc enzymes in the presence of excess peroxide. HRP catalyzes the oxitlatiotl to T, quantitatively ul) to a concentration of
c
c
L
350
400
450
500
550
600
h,nm FIG. 4. Effect of iodide on (-1 were added five successiw added : (. . .-. . . ) 0.1 IllM, ((-- -) 0.5 mill.
iodine qwcttwn. increments of -
-)
0.3
nm.
To n 0.22 m:ll iocline solution 0.1 mM iodide wrh. Total iodide (-.-)
0.3
Illlll,
(.
.)
0.4
n1.v.
1 .O mM I? while IA’ (Fig. 5B) will produce only 0.5 111M I, or somcvhat lower (0.35 mM) when starting solutions are ecluilibrium mixtures rather than pure iodide. One explanation for these results is that I, inhibits the initial rate of iodide oxidation by both IX and HR.P. Thr inhibition is much more pronounced in the CRW of LP. anal probably represents product inhibition.
Table 1 compares the extinction coefficient s obtwinvd in the present study and previously presented value:: in the literature. Although the values arc in reasonable :lgrcement, the 460 nm value of I, is 67:’ lower than previously reported. The published 353 nm rxtinction roefficients for I,- vary more than 20%. The values obtained in the present study are close to t,he more recently published data (5,61. Our values for Ia- are given at, 350 ant1 353 nm. Most literature ~alucs are given at 353 nm. the absorption maximum. It can be seen in Fig. 2 that, the 353 mn extinction is lees than l? higher than that at 350 nm.
200
MORRISON,
Ah-11
MICHAELS
.
-
dE s 5 h
BATSE,
.
0.5 -
I
/
3
.
.f -
/ I,
0
I
1 1.0
0
, I
I
I 2.0 Atoms, mM
1
I
I 3.0
/ I,
0.50
-
l -.‘7 ‘.
/
I
0 0
0.5 Total I Atoms.mM
I
I.0
FIG. 5. (-1) HRP-catalyzed iodine produc+ion. ‘To c,nrh indicated concentration of iodidt? RCW addrd fined concentrations of 3.3 mXf H,O, and 0.515 HRP. (B) 12’~catalyzed iodine production. Under identical conditions to (A) 0.133 pM I,P was cmploycd ( .-.). Starting solutions containing ecluilihrium mixtures of I:,, 12, :mtl I- ww also used (X-X).
pM
The equilibrium of equation (a) has long been of interest to chemists. A number of investigators (13, 16-18) have reported similar values for the equilibrium constant, when the experiments wcrc performed in dilute acid, which suppresses I, hydrolysis. The value obtained by Davies and Cwynne (16) with much more cumbersome techniques is in very good agreement with that obtained in the present study. Catalysis of iodide oxidation is frequently employed as an assay for peroxidases. Detection of triiodide at 353 nm is commonly used because of the sensitivity of the measurement.. Dilutions of pure I, in 0.001 M H2S0, obey Beer’s Law at 460 nm. However, as would be expected on the basis of the equilibrium constant, dilutions of I:,- in 0.001 M H.SOJ or in pH 7.4 phosphate buffer are linear with absorbance at 350 nm only in cases in which the iodide
concentration is wfficiently high. Contrary to the recent claim of Taurog ( 19), the deviation from Beer’:: IAX at lower concentrations of ioditlc can he completely attributed to the iodine-triioclidc equilihriunl.
Xt low pH \-dues mcl in tlw wwenc(L of txcc’s.c: lwrosicle, lactoperoxidasc ant1 horseradish perosi(l:wc n-ill catalyze the stoicliiornctric oxidation of iodide to iotline. Scithcr enzyme osicliecs ioclincs to iotlntc. Thcsc peroxidascs can he uwl, tlierc~forc. to l)rq)are j)ure I, solutions. This has maclc possible a r(w+cwmwt, of the extinction coefficients of aqueous I, arid I:,-. an(l of the iotliilc-triioditl~~ c~c~uilihriim coiist:~nt. Millimolar extinction coefficients for I:+- at 350 ~11 ant1 460 nnl arc 21.O and 0.87, rcspcctiwly. I\Iillinlolar extinction cweffirients for I, at the smw wavelengths are 0.035 nncl 0.70. Ak value of 768 W’ n-as ohtaincd for the iodine-triioclidc cquilihrium constant at 24°C.
7.
MORRISON.
M..
ASU
S.
ROMB~LTTS.
W.
4..
HU,TQUISP.
D.
B..
.I. Biol.
SCHROEDER.
\T-.
I\..
ANI)
Chcrn.
MORRISON.
238, M..
2847 (1963). Biochemisfc~/
6, 2965
(196i). M.. HA.MII,TON. H. B., .\IVI STOTZ. E.. J. Lsiol. Chrn2. 228, 767 (1957). B. C., HOLLIES-SIEDIX. A. G.. ASI) STARK. R. P.. “Pcrosi&sc.” 13uttcraorths. Ilondon. 1964. DYER. J. R.. Methods Biockrm. Acrl. 3, 111 (1956). Geortc~c, P.. Biochern. J. 54, 267 (1953). BRAY. IV. C.. .\ND MACKAY. G. M. J.. .I. Avl. (Ihrri~ f+)r. 32, 1207 (1910). BAYSIC, C:. S.. AND MORRISON. M.. A rcic. Bidwrn. Hiqh!p., in press (1971). Rw. (‘ornrn IOI. 32, i70 TIIOMAS. J. A.. .4%1) H.WEH. I,. P.. Riochen~. Biophys. (19688). D.wres. M., AND Gw~N?;E. E:.. J. A/n. C’hcm. Sot. 74, 274s (1952). JONES. G., AXI) KAPLAN. B. I%.. J. Am. Chwu. Sot. 50, 1845 (192s). ,JOSICS. G.. AND HARTX~~~S~N. l/l, I,.. J. Am. Clrrnr. %)c. 37, 752 (1015). T.A~-INK:. A.. An-h. Rioch~m. Binphys. 139, 212 (1970).
9. MORRIROS. 10.
11. 12.
13. 14. 15.
16. 17. 18. 19.
SAUNDERS.
RIOCHl?MISTRY
Determination and
42,
t0.5201
(1!)71)
of Spectral I::- and
the
R~~ccivcd
Properties Equilibrium
Sovfmlwr
of Aqueous
12
Constant
10, 1970
.A number of reports liar-e :tpl)~aretl conceruing the spectral properties of aqueous solutions of I, anal I:, (-6). Howvcr, there has not been total agreement among investigators on these prolwrtics. Pul~lislwcl values for the extinction coeffiricwt. of 1::. vary as much as 20%‘. Rrccnt investigations of iodide metnbolism in biological systems and of thyroxine biosynthesis in particular have pointc~l up the ncwl for accurate values, ilot only for the spectral prolwrties, hut also for the eyuilihrium constant involving I-, I,, and I:,-. The present inr-cst,igation was unclertakcn to rc-evaluate these con&ants.
Lactoperoxidase was isolatccl and purified as pwviously outlined (7,8), and the concentration cktermincd by 412 nm nl~sorbancc, using a millimolnr extinction coefficient of 114 (9). Horseratlish lwroxidase (,HRP) , type VII-L, was obtained from Sigma Ckmical Co. The concentration of HRP was determined by 403 nm absorbance, using a millimolar extinction cocfficicnt of 91 (10). Reagent-grade rlicmicals were employed. Sodium thiosulfatc was standarclizcd against RIO, il I). Peroxide concentration was mcasurcd at 230 nm using a molar extinction coefficient, of 72.4 (12). All experiment s were performed at 24°C and in 0.001 31 H,SO,, to minimize hydrolysis of I, (131. A$ Zeiss P1IQ II dr~tcrlllinations and a spcctrophotometer was employc(l for alworl)ancr Gary model 14 rccordiiig s~,cctrol~llotolli~~t(~r wa. q usc~cl to ol,tnin slwctrn. cstinction Iodide could be dcterminr~tl at 226 11111,using :L millimolar coefficient of 12.3 (4). An Orion model 94-53 iodidcs electrode connected to :t Radiometer PHRI 22r millivolt mctcr ww used for iodide concentration measurement.
07 0.6 5 7 T
_
o5t-
IA
1
zw- 0.4 H" 0.3
1
0.2 01 0 300
Frc;. 1. Visible (~ll:d!~ze(l oxidation
I:,-
400
L.-. 450 X,nm
500
550
600
and ultraviol(,t slwctrunl of I?. I, \V:W oht:rined t)y of known aolrllions of I :~nd rl~cortl(~tl :is inclic:rtt~ti
P:xtinction
I?
350
4fiO :&50 :;:io :;;i:;
460
TARI,I:, Coefiicients
0.70 0. o:~:i 21 !) 2"'. 1 0.87
I for I? and I:,
11 746 O.I)IS
(4) (4)
26.4
(4);
2%.!+
3.1
(1);
21 .O (6’)
0.975
(4);
1.16
(5,) (1')
I)~~roxitlnsc~in the tc~sl.
35
30
25 ‘E 7% 20 H E
IO
275
I+;.
in t,hr
2. text.
Spc‘c.trurll
of
300
I, -. Iinoum
350
400
450 h,nm
concentrations
500 of
550 I.,-
600 w’re
oht:~ined
outlined
198
Solving equation respectively :
MOHRISON,
RAYSE,
AKiU
MICHAELS
A&i,, = “1.9.r + 0.035jj
0))
.-1go = 0.S7.C + 0.701/
Cc)
(1)) for x and then substituting
.r =
&(I
(c) gives,
- o.ox5y ‘1.9
A &cj(, -
!/ =
into equation
Cd)
0.0397Aa~" 0.699
(e>
From tlic I:,- conccutration and tlrc knowi atl(lition of iodide, equilihriurn iodide could be tletmnined. In Fig. 3 are 1)lotted the results of three .4
.3
‘,“I-”
.2
I-,mM FIG. 3. Iodirw-triioclide cqlds K”.,, CL+ outlined
in
equilibrium thy test.
constant.
The
slope
of the
lint.
0.768
m!W’.
different esl~eriineiits. The iiiillimolar ratio of 1:: /I, is plotted against millirnolar iodide concentrations at equilihriuln. The slolw of the line, an arcrage of 0.768 m%f-’ (76’8 Jf--‘), is A,,. Triiotlide contamination of an I, solution can give large errors in determination of I, concentration from 460 nm nbsorbancc readings. Fig. 4 illustrates this point. To a solution of iodine (0.22 1nX) were added five successive increments of 0.1 inM iodide. Large increases in the 350 nm absorbance :Irc’ noted, with little change at 460 nm. The similarity of I, and I::- millirnolar extinction coefficients at 460 nm may also be noted in Table 1. Figure 5 illustrates that LP and HRP will oxidize only lilnite(1 amounts of iodide quantitatively. The total iodide in solution is plotted versus I, produwl by t,hesc enzymes in the presence of excess peroxide. HRP catalyzes the oxitlatiotl to T, quantitatively ul) to a concentration of
c
c
L
350
400
450
500
550
600
h,nm FIG. 4. Effect of iodide on (-1 were added five successiw added : (. . .-. . . ) 0.1 IllM, ((-- -) 0.5 mill.
iodine qwcttwn. increments of -
-)
0.3
nm.
To n 0.22 m:ll iocline solution 0.1 mM iodide wrh. Total iodide (-.-)
0.3
Illlll,
(.
.)
0.4
n1.v.
1 .O mM I? while IA’ (Fig. 5B) will produce only 0.5 111M I, or somcvhat lower (0.35 mM) when starting solutions are ecluilibrium mixtures rather than pure iodide. One explanation for these results is that I, inhibits the initial rate of iodide oxidation by both IX and HR.P. Thr inhibition is much more pronounced in the CRW of LP. anal probably represents product inhibition.
Table 1 compares the extinction coefficient s obtwinvd in the present study and previously presented value:: in the literature. Although the values arc in reasonable :lgrcement, the 460 nm value of I, is 67:’ lower than previously reported. The published 353 nm rxtinction roefficients for I,- vary more than 20%. The values obtained in the present study are close to t,he more recently published data (5,61. Our values for Ia- are given at, 350 ant1 353 nm. Most literature ~alucs are given at 353 nm. the absorption maximum. It can be seen in Fig. 2 that, the 353 mn extinction is lees than l? higher than that at 350 nm.
200
MORRISON,
Ah-11
MICHAELS
.
-
dE s 5 h
BATSE,
.
0.5 -
I
/
3
.
.f -
/ I,
0
I
1 1.0
0
, I
I
I 2.0 Atoms, mM
1
I
I 3.0
/ I,
0.50
-
l -.‘7 ‘.
/
I
0 0
0.5 Total I Atoms.mM
I
I.0
FIG. 5. (-1) HRP-catalyzed iodine produc+ion. ‘To c,nrh indicated concentration of iodidt? RCW addrd fined concentrations of 3.3 mXf H,O, and 0.515 HRP. (B) 12’~catalyzed iodine production. Under identical conditions to (A) 0.133 pM I,P was cmploycd ( .-.). Starting solutions containing ecluilihrium mixtures of I:,, 12, :mtl I- ww also used (X-X).
pM
The equilibrium of equation (a) has long been of interest to chemists. A number of investigators (13, 16-18) have reported similar values for the equilibrium constant, when the experiments wcrc performed in dilute acid, which suppresses I, hydrolysis. The value obtained by Davies and Cwynne (16) with much more cumbersome techniques is in very good agreement with that obtained in the present study. Catalysis of iodide oxidation is frequently employed as an assay for peroxidases. Detection of triiodide at 353 nm is commonly used because of the sensitivity of the measurement.. Dilutions of pure I, in 0.001 M H2S0, obey Beer’s Law at 460 nm. However, as would be expected on the basis of the equilibrium constant, dilutions of I:,- in 0.001 M H.SOJ or in pH 7.4 phosphate buffer are linear with absorbance at 350 nm only in cases in which the iodide
concentration is wfficiently high. Contrary to the recent claim of Taurog ( 19), the deviation from Beer’:: IAX at lower concentrations of ioditlc can he completely attributed to the iodine-triioclidc equilihriunl.
Xt low pH \-dues mcl in tlw wwenc(L of txcc’s.c: lwrosicle, lactoperoxidasc ant1 horseradish perosi(l:wc n-ill catalyze the stoicliiornctric oxidation of iodide to iotline. Scithcr enzyme osicliecs ioclincs to iotlntc. Thcsc peroxidascs can he uwl, tlierc~forc. to l)rq)are j)ure I, solutions. This has maclc possible a r(w+cwmwt, of the extinction coefficients of aqueous I, arid I:,-. an(l of the iotliilc-triioditl~~ c~c~uilihriim coiist:~nt. Millimolar extinction coefficients for I:+- at 350 ~11 ant1 460 nnl arc 21.O and 0.87, rcspcctiwly. I\Iillinlolar extinction cweffirients for I, at the smw wavelengths are 0.035 nncl 0.70. Ak value of 768 W’ n-as ohtaincd for the iodine-triioclidc cquilihrium constant at 24°C.
7.
MORRISON.
M..
ASU
S.
ROMB~LTTS.
W.
4..
HU,TQUISP.
D.
B..
.I. Biol.
SCHROEDER.
\T-.
I\..
ANI)
Chcrn.
MORRISON.
238, M..
2847 (1963). Biochemisfc~/
6, 2965
(196i). M.. HA.MII,TON. H. B., .\IVI STOTZ. E.. J. Lsiol. Chrn2. 228, 767 (1957). B. C., HOLLIES-SIEDIX. A. G.. ASI) STARK. R. P.. “Pcrosi&sc.” 13uttcraorths. Ilondon. 1964. DYER. J. R.. Methods Biockrm. Acrl. 3, 111 (1956). Geortc~c, P.. Biochern. J. 54, 267 (1953). BRAY. IV. C.. .\ND MACKAY. G. M. J.. .I. Avl. (Ihrri~ f+)r. 32, 1207 (1910). BAYSIC, C:. S.. AND MORRISON. M.. A rcic. Bidwrn. Hiqh!p., in press (1971). Rw. (‘ornrn IOI. 32, i70 TIIOMAS. J. A.. .4%1) H.WEH. I,. P.. Riochen~. Biophys. (19688). D.wres. M., AND Gw~N?;E. E:.. J. A/n. C’hcm. Sot. 74, 274s (1952). JONES. G., AXI) KAPLAN. B. I%.. J. Am. Chwu. Sot. 50, 1845 (192s). ,JOSICS. G.. AND HARTX~~~S~N. l/l, I,.. J. Am. Clrrnr. %)c. 37, 752 (1015). T.A~-INK:. A.. An-h. Rioch~m. Binphys. 139, 212 (1970).
9. MORRIROS. 10.
11. 12.
13. 14. 15.
16. 17. 18. 19.
SAUNDERS.