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A spectrophotometric assay for iodide oxidation by thyroid peroxidase
ANALYTICAL
4, 341-345
BIOCHEMISTRY
A Spectrophotometric by
(1962)
Assay for Iodide Thyroid Peroxidasel
NICHOLAS
Oxidation
M. ALEXANDER
From the Radioisotope Service, Veterans Administration Hospital, West Haven, Connecticut, and the Department of Biochemistry, Yale University, New Haven, Connecticut Received
March
23, 1962
Manometry is useful, but inexpedient, for measuring thyroid peroxidase activity (1) and recently the enzyme was assayed by a colorimet.ric guaiacol method (2). This report describes a rapid, sensitive spectrophotometric assay with the nat.ural substrate, iodide ion, rather than an artificial hydrogen donor. Peroxidase catalyzes the formation of I, as depicted in Eq. (1) and periodide formation is then instantaneous (Eq. 2), when there is an iodide
2 KI
+ H?Oz
-
I?+L)KOH
%
KII
peroxidasr
IzfKI
(2)
excess of iodide substrate. Nearly all of the enzymically generated I, is bound as I,- because the equilibrium constant for &)/(I-) (I,) = 714 at 25” and the hydrolysis of I, or polyiodide formation is negligible (3, 4). Periodide can be spectrophotometrically determined at either of its absorption peaks 353 (5-7) or 287.5 rnp and the yield of I,- is directly proportional to peroxidase concentration in reaction mixtures containing approximate amounts of H,O, and enzyme. METHODS
Absorbancy
of I,- in Buffered
Solutions
Solutions containing varying amounts of periodide ion were prepared by adding aliquots (10 to 400 ~1) of 0.001 M I2 in ethanol to 1 ml of 0.133 M KI, 1.66 ml of 0.3 M, pH 7.4 sodium phosphate when indicated, and made up to 10 ml with water. The final concentrations of I,- were 90.4% of the added I? as calculated from the equilibrium (I-) (13)/(13-) ‘This investigation Public Health Service.
was
supported
in
part 341
by
a grant
from
the
United
States
342
ALEXANDER
= 0.0014M (3). The optical density of each solution was determined in Beckman cuvettes with a l-cm light path at room temperature (24-26’) using blank solutions that contained everything but Sodine. Preparation
of Digitonin
Solubilized
Thyroid
Mitochondria
Pig thyroid homogenate was prepared by homogenizing 10 gm of tissue with 40 ml of 1.1% KC1 in a Waring Blendor for 1 min. The homogenat,e was centrifuged for 15 min at 1000 X g and particles in the supernatant fluid that sedimented at 15,000 X g in 30 min were collected and are referred to as mitochondria. One ml of 1% digitonin in O.O2M, pH 7.4 phosphate was added for every 7.5 mg of mitochondrial protein and the suspension was mixed with the aid of a Potter-Elvehjem homogenizer. After 20 min at 3” with occasional stirring, the suspension was centrifuged at 40,000 X g for 20 min and the brown supernatant solution was collected. It contained 4 mg protein/ml and could be stored several weeks in the freezer without loss of peroxidase activity. One per cent digit,onin in buffer was brought into solution with the aid of heat and cooled before use. Protein determinations were carried out by the method of Lowry et al. (8). Procedure
for the Peroxidase
Assay
The assay for iodide oxidation by peroxidase is performed in the following manner (cf. 9) : The reaction mixture contains 40 pmoles KI, 150 pmoles pH 7.4, 0.3 M sodium phosphate, and an appropriate amount of enzyme in a total volume of 3 ml in a l-cm Beckman cuvette. The optical density at 287.5 or 353 rnEn.is set at zero and 10 ,ul of 0.08M H,O, added to the end of a stirring rod is dipped into the solution with a swirling motion. Optical density readings are recorded every 10 or 15 sec. Correction for the spontaneous oxidation of iodide by H,O,, which amounts to about 2 mpmoles IS-/ml in 60 set, is obtained by performing the assay in the absence of enzyme. Periodide production in m~moles/ml is calculated by multiplying t,he optical density by 10” and dividing by the molar extinction coefficient. RESULTS
24ND DISCUSSION
Molar Extinction. Coefficients for I,It is clear from Fig. 1 that I,- absorption obeys Beer’s law at either 287.5 or 353 rnp and that pH 7.4, 0.05 M phosphate buffer, unlike strong acid (6)) has no effect on the absorbancy of I-,. Free I? (
IODIDE
PEROXIDASE
ASSAY
343
thirty-two measurements in Fig. 1 are 34,690 (a = 21461) at 287.5 rnp and 22,900 (U = ~~855) at 353 mp. These values agree quit,e well (within ‘2 to 4%) with the data of others (5-7) although one paper (10) reports
0 FIG. 1. Absorption obtained iq 0.05 M, buffer ( x ).
4
8
12 16 20 24 28 32 36 nylmoles 13 per ml.
of varying concentrations pH 7.4 sodium phosphate
of periodide ion at 353 and 287.5 ma (solid circles) and in the absence of
extinctions for I,- that are 15% higher. In any event, I,- can be determined with about a 4% coefficient of variation. Kinetics
of Pig Thyroid Peroxidase ;Ictivity
In reaction mixtures containing appropriate concentrations of iodide, H,Oz, and enzyme, I,- formation is zero order for a convenient interval of time and the formation of I,- is proportional to the concentration of iodide peroxidase as shown in Fig. 2 [consult Chance (11) for a general discussion of the desired conditions for peroxidase assays]. For the first 40 set the rate of I,- formation is linear and dependent on enzyme con-
344
ALEXANDER
Seconds FIG. 2. Rate of periodide formabion with varying amounts of iodide peroxidase. Curves (A), (B), and (C) were obtained with digitonin solubilized pig thyroid mitochondria containing 200, 150, and 100 gg of protein, respectively. The points were from duplicate determinations at 287.5 mp and at room temperature (25”).
centration as seen with three different amounts of a digitonin extract from pig thyroid mitochondria. The rates decrease after 40 set and curve (C), which contains lesser amounts of enzyme, reaches a plateau sooner than (A) or (B), probably because iodide peroxidase is inactivated by H202, a phenomenon typical of other peroxidases (11). Identical results are obtained at 353 my. The amount of I, actually formed is 1.1 times greater because 9.5% of the generated I, is not bound as I,. Obvious alternatives are to measure the time required to obtain an arbitrarily chosen optical density or to use a recorder attached to the spectrophotometer. The assay is performed at 353 rn,u if materials that strongly absorb in the ultraviolet are present in the reaction mixture and, although the enzyme preparation absorbs at 287.5 rnp, it does not interfere with the assay at this wavelength because the optical density can be set at zero prior to adding the H,O,. All enzymic activity is destroyed by immersing the enzyme in a boiling water bath for 5 min. When supplied with H,O, (12, 13) digitonin solubilized mitochondria incorporate F-
1ODIDE
PEROXIDASE
345
ASSAY
iodide into tyrosine in amounts that correlate with the peroxidase activity assessed by this spectrophotometric method. Whole thyroid extracts cannot be assayed by this method because they contain considerable quantities of catalase and materials that react with I?. Catalase does not oxidize iodide and inhibits thyroid peroxidase by decomposing H,O,. However, crude lacrimal and submaxillary gland extracts that readily peroxidize iodide can be assayed by this method. Consistent with an earlier investigation (12), hematin increases the peroxidase activity of dialyzed (against 0.02 M, pH 7.4 phosphate) digitonin extra& by 40%. Other fractions that are obtained by t.reating the dialyzed extract with butanol show an absolute requirement for hematin. Detailed studies on hematin activation of various thyroid preparations are being carried out and will be reported later. This method offers the obvious advantages of speed and sensitivity. It is at, least 1000 times more sensitive than the standard manometric assay and can be performed in 2 min. For these reasons, it should be adequately suited for studies directed toward the purification and isolation of thyroid peroxidase. STJMMARY
A rapid, sensitive spectrophotometric assay for iodide oxidation by thyroid peroxidase is described. The method depends upon the measurement of periodide at either of its absorption peaks, 353 rnp or 287.5 mp, and is adequately suited for enzyme purification studies. ACKNOWLEDGMENT The
technical
assistance
of Betty
J. Corcoran
is gratefully
arknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
ALEXANDER, N. M., J. Biol. Chem. 234, 1530 (1959). HOSOYA, T., AND UI, N., Nature 192, 659 (1961). JONES, G., AND KAPLAN, B. B., J. Am. Chem. Sot. 50, 1845 (1928). DAVIS, M., AND GWYNNE, E., J. Am. Chem. Sot. 74, 2748 (1952). CUSTER, J. J., AND NATELSON, S., Anal. Chem. 21, 1005 (1949). PATRICK, W. A., AND WAGNER, H. B., And. Chem. 21, 1279 (1949). OVENSTON, T. C. J., AND REES, W. T., Analyst 75, 204 (1950). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RAND,~LL, R. J.. J. &o[. 193, 265 (1951). MAEHLY, A. C., ill “Methods of Biochemical Analysis” (D. Gliek, ed.), p. 386. Interscience, New York, 1954. AWTRY, A. D., AND CONNICK, R. E.. J. Am. Chem. Sot. ‘73, 1842 (1951). CHANCE, B., in “Methods of Biochemical Analysis” (D. Glick, ed.), p. 408. Int~erseience, New York, 1954. ALEXANDER, N. M., AND CORCORAM, B. J., J. Biol. Chem. 237, 243 (1962). SERIF, G., AND KIRKWOOD, S., J. Biol. Chem. 233, 109 (1958).
Chem.
Vol.
I,
Vol.
I,
4, 341-345
BIOCHEMISTRY
A Spectrophotometric by
(1962)
Assay for Iodide Thyroid Peroxidasel
NICHOLAS
Oxidation
M. ALEXANDER
From the Radioisotope Service, Veterans Administration Hospital, West Haven, Connecticut, and the Department of Biochemistry, Yale University, New Haven, Connecticut Received
March
23, 1962
Manometry is useful, but inexpedient, for measuring thyroid peroxidase activity (1) and recently the enzyme was assayed by a colorimet.ric guaiacol method (2). This report describes a rapid, sensitive spectrophotometric assay with the nat.ural substrate, iodide ion, rather than an artificial hydrogen donor. Peroxidase catalyzes the formation of I, as depicted in Eq. (1) and periodide formation is then instantaneous (Eq. 2), when there is an iodide
2 KI
+ H?Oz
-
I?+L)KOH
%
KII
peroxidasr
IzfKI
(2)
excess of iodide substrate. Nearly all of the enzymically generated I, is bound as I,- because the equilibrium constant for &)/(I-) (I,) = 714 at 25” and the hydrolysis of I, or polyiodide formation is negligible (3, 4). Periodide can be spectrophotometrically determined at either of its absorption peaks 353 (5-7) or 287.5 rnp and the yield of I,- is directly proportional to peroxidase concentration in reaction mixtures containing approximate amounts of H,O, and enzyme. METHODS
Absorbancy
of I,- in Buffered
Solutions
Solutions containing varying amounts of periodide ion were prepared by adding aliquots (10 to 400 ~1) of 0.001 M I2 in ethanol to 1 ml of 0.133 M KI, 1.66 ml of 0.3 M, pH 7.4 sodium phosphate when indicated, and made up to 10 ml with water. The final concentrations of I,- were 90.4% of the added I? as calculated from the equilibrium (I-) (13)/(13-) ‘This investigation Public Health Service.
was
supported
in
part 341
by
a grant
from
the
United
States
342
ALEXANDER
= 0.0014M (3). The optical density of each solution was determined in Beckman cuvettes with a l-cm light path at room temperature (24-26’) using blank solutions that contained everything but Sodine. Preparation
of Digitonin
Solubilized
Thyroid
Mitochondria
Pig thyroid homogenate was prepared by homogenizing 10 gm of tissue with 40 ml of 1.1% KC1 in a Waring Blendor for 1 min. The homogenat,e was centrifuged for 15 min at 1000 X g and particles in the supernatant fluid that sedimented at 15,000 X g in 30 min were collected and are referred to as mitochondria. One ml of 1% digitonin in O.O2M, pH 7.4 phosphate was added for every 7.5 mg of mitochondrial protein and the suspension was mixed with the aid of a Potter-Elvehjem homogenizer. After 20 min at 3” with occasional stirring, the suspension was centrifuged at 40,000 X g for 20 min and the brown supernatant solution was collected. It contained 4 mg protein/ml and could be stored several weeks in the freezer without loss of peroxidase activity. One per cent digit,onin in buffer was brought into solution with the aid of heat and cooled before use. Protein determinations were carried out by the method of Lowry et al. (8). Procedure
for the Peroxidase
Assay
The assay for iodide oxidation by peroxidase is performed in the following manner (cf. 9) : The reaction mixture contains 40 pmoles KI, 150 pmoles pH 7.4, 0.3 M sodium phosphate, and an appropriate amount of enzyme in a total volume of 3 ml in a l-cm Beckman cuvette. The optical density at 287.5 or 353 rnEn.is set at zero and 10 ,ul of 0.08M H,O, added to the end of a stirring rod is dipped into the solution with a swirling motion. Optical density readings are recorded every 10 or 15 sec. Correction for the spontaneous oxidation of iodide by H,O,, which amounts to about 2 mpmoles IS-/ml in 60 set, is obtained by performing the assay in the absence of enzyme. Periodide production in m~moles/ml is calculated by multiplying t,he optical density by 10” and dividing by the molar extinction coefficient. RESULTS
24ND DISCUSSION
Molar Extinction. Coefficients for I,It is clear from Fig. 1 that I,- absorption obeys Beer’s law at either 287.5 or 353 rnp and that pH 7.4, 0.05 M phosphate buffer, unlike strong acid (6)) has no effect on the absorbancy of I-,. Free I? (
IODIDE
PEROXIDASE
ASSAY
343
thirty-two measurements in Fig. 1 are 34,690 (a = 21461) at 287.5 rnp and 22,900 (U = ~~855) at 353 mp. These values agree quit,e well (within ‘2 to 4%) with the data of others (5-7) although one paper (10) reports
0 FIG. 1. Absorption obtained iq 0.05 M, buffer ( x ).
4
8
12 16 20 24 28 32 36 nylmoles 13 per ml.
of varying concentrations pH 7.4 sodium phosphate
of periodide ion at 353 and 287.5 ma (solid circles) and in the absence of
extinctions for I,- that are 15% higher. In any event, I,- can be determined with about a 4% coefficient of variation. Kinetics
of Pig Thyroid Peroxidase ;Ictivity
In reaction mixtures containing appropriate concentrations of iodide, H,Oz, and enzyme, I,- formation is zero order for a convenient interval of time and the formation of I,- is proportional to the concentration of iodide peroxidase as shown in Fig. 2 [consult Chance (11) for a general discussion of the desired conditions for peroxidase assays]. For the first 40 set the rate of I,- formation is linear and dependent on enzyme con-
344
ALEXANDER
Seconds FIG. 2. Rate of periodide formabion with varying amounts of iodide peroxidase. Curves (A), (B), and (C) were obtained with digitonin solubilized pig thyroid mitochondria containing 200, 150, and 100 gg of protein, respectively. The points were from duplicate determinations at 287.5 mp and at room temperature (25”).
centration as seen with three different amounts of a digitonin extract from pig thyroid mitochondria. The rates decrease after 40 set and curve (C), which contains lesser amounts of enzyme, reaches a plateau sooner than (A) or (B), probably because iodide peroxidase is inactivated by H202, a phenomenon typical of other peroxidases (11). Identical results are obtained at 353 my. The amount of I, actually formed is 1.1 times greater because 9.5% of the generated I, is not bound as I,. Obvious alternatives are to measure the time required to obtain an arbitrarily chosen optical density or to use a recorder attached to the spectrophotometer. The assay is performed at 353 rn,u if materials that strongly absorb in the ultraviolet are present in the reaction mixture and, although the enzyme preparation absorbs at 287.5 rnp, it does not interfere with the assay at this wavelength because the optical density can be set at zero prior to adding the H,O,. All enzymic activity is destroyed by immersing the enzyme in a boiling water bath for 5 min. When supplied with H,O, (12, 13) digitonin solubilized mitochondria incorporate F-
1ODIDE
PEROXIDASE
345
ASSAY
iodide into tyrosine in amounts that correlate with the peroxidase activity assessed by this spectrophotometric method. Whole thyroid extracts cannot be assayed by this method because they contain considerable quantities of catalase and materials that react with I?. Catalase does not oxidize iodide and inhibits thyroid peroxidase by decomposing H,O,. However, crude lacrimal and submaxillary gland extracts that readily peroxidize iodide can be assayed by this method. Consistent with an earlier investigation (12), hematin increases the peroxidase activity of dialyzed (against 0.02 M, pH 7.4 phosphate) digitonin extra& by 40%. Other fractions that are obtained by t.reating the dialyzed extract with butanol show an absolute requirement for hematin. Detailed studies on hematin activation of various thyroid preparations are being carried out and will be reported later. This method offers the obvious advantages of speed and sensitivity. It is at, least 1000 times more sensitive than the standard manometric assay and can be performed in 2 min. For these reasons, it should be adequately suited for studies directed toward the purification and isolation of thyroid peroxidase. STJMMARY
A rapid, sensitive spectrophotometric assay for iodide oxidation by thyroid peroxidase is described. The method depends upon the measurement of periodide at either of its absorption peaks, 353 rnp or 287.5 mp, and is adequately suited for enzyme purification studies. ACKNOWLEDGMENT The
technical
assistance
of Betty
J. Corcoran
is gratefully
arknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
ALEXANDER, N. M., J. Biol. Chem. 234, 1530 (1959). HOSOYA, T., AND UI, N., Nature 192, 659 (1961). JONES, G., AND KAPLAN, B. B., J. Am. Chem. Sot. 50, 1845 (1928). DAVIS, M., AND GWYNNE, E., J. Am. Chem. Sot. 74, 2748 (1952). CUSTER, J. J., AND NATELSON, S., Anal. Chem. 21, 1005 (1949). PATRICK, W. A., AND WAGNER, H. B., And. Chem. 21, 1279 (1949). OVENSTON, T. C. J., AND REES, W. T., Analyst 75, 204 (1950). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RAND,~LL, R. J.. J. &o[. 193, 265 (1951). MAEHLY, A. C., ill “Methods of Biochemical Analysis” (D. Gliek, ed.), p. 386. Interscience, New York, 1954. AWTRY, A. D., AND CONNICK, R. E.. J. Am. Chem. Sot. ‘73, 1842 (1951). CHANCE, B., in “Methods of Biochemical Analysis” (D. Glick, ed.), p. 408. Int~erseience, New York, 1954. ALEXANDER, N. M., AND CORCORAM, B. J., J. Biol. Chem. 237, 243 (1962). SERIF, G., AND KIRKWOOD, S., J. Biol. Chem. 233, 109 (1958).
Chem.
Vol.
I,
Vol.
I,