ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 1, April 15, pp. 244-257, 1979

Hog Thyroid

ALLEN

Peroxidase: Physical, Chemical, and Catalytic of the Highly Purified Enzyme’ B. RAWITCH,”

Properties

ALVIN TAUROG,tT’ STEVEN B. CHERNOFF,* MARTHA L. DORRIST

AND

*Department of Biochemistry, University oj’riansas Medical Cexfer, Kansas Cify, Kansus 66103, and +Department qf‘Pharmacology, Cniuersity of‘ Texas Health Science Center, Dallas, Texas 7.5235 Received September

12, 1978; revised November

30, 1978

Studies are reported on the purity and on the physical, chemical, and catalytic properties of a highly purified, stable, thyroid peroxidase (TPO). The enzyme was solubilized by treatment with deoxycholate and trypsin, and it was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. The final product, designated TPO VII, had a value for A,,,IA,,,, of 0.54, and its specific activity based on the guaiacol assay (794 pmol of guaiacol oxidized/min/mg) was considerably greater than that of any previously described TPO. Specific activity values based on other peroxidase-catalyzed reactions were also higher for TPO VII than for previous TPO preparations. Purity estimates for TPO VII, based on polyacrylamide disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels, ranged from 80 to 95%. The molecular weight, determined by sedimentation equilibrium, was 93,000. Results of sodium dodecyl sulfate-gel electrophoresis also indicated a molecular weight of approximately 90,000. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions indicated that TPO VII is composed of two peptide chains of unequal size, with the larger about 2.5.fold the size of the smaller. Carbohydrate analysis revealed that TPO is a glycoprotein containing about 10% by weight of carbohydrate. The predominant sugars were mannose and N-acetyl glucosamine. A significant amount of glucose was also found, along with small amounts of galactose, fucose, and xylose. The amino acid composition of TPO VII showed a high proline content, a predominance of arginine over lysine, and a ratio of [Asp] plus [Glu] to [Lys] plus [Arg] of over 2. Isoelectric focusing in polyacrylamide gels indicated an isoelectric pH of 5.75. In agreement with observations made on earlier preparations of TPO, heme spectral data showed significant differences between the pyridine hemochromogens of TPO VII and horseradish peroxidase, suggesting that the heme in TPO is notferriprotoporphyrin IX. Circular dichroism measurements indicated that approximately 40% of TPO VII involves 01helix or @ structure.

Thyroid peroxidase (TPO>”is a membrane- coupling of diiodotyrosine residues in thyrobound, hemoprotein enzyme that catalyzes globulin to form thyroxine (1,2). Because of both the iodination of thyroglobulin and the its particulate nature TPO has been difficult to purify, and although attempts have been ’ Aided by USPHS Grants AM-18896 (Dr. Rawitch) made to isolate the enzyme in its native form and AM-03612 (Dr. Taurog). (3-5), preparations with high specific activ2 Career research awardee, USPHS. Reprint reity have been isolated only after first releasquests should be addressed to Dr. Alvin Taurog, ing the enzyme from the membrane by proDepartment of Pharmacology, University of Texas teolysis (6-11). Although it is likely that the Health Center, Dallas, Tex. 75235. native enzyme is altered by this procedure, :I Abbreviations used: TPO, Thyroid peroxidase; the fragment that is isolated is nevertheless BSA, bovine serum albumin; PB, phosphate buffer; extremely active and undoubtedly carries MMI, methimazole; T,, thyroxine; SDS-PAGE, sodium the active site. dodecyl sulfate-polyacrylamide gel electrophoresis; A previous communication from this laboCD, circular dichroism; HRP, horseradish peroxidase; Tg, thyroglobulin; DIT, diiodotyrosine. ratory (8) described a purification scheme 0003-9861/79/050244-14$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

244

245

PROPERTIES OF THYROID PEROXIDASE

for hog TPO involving the sequential use of deoxycholate and trypsin for solubilization, followed by chromatography on DEAEcellulose and gel filtration through Bio-Gel P-100. A preparation withA,,,,lA,,, equal to 0.34 was isolated by this procedure. Subsequently the solubilization procedure was modified, and deoxycholate and trypsin were added simultaneously (Taurog and Dorris, unpublished). Two preparations, designated V and VI, were isolated using the latter procedure, withA4,,,lA280values of 0.40 and 0.38, respectively. These preparations were estimated to be 36-40% pure on the basis of heme content and an assumed molecular weight of 64,000 (12). Their catalytic activities based on iodination of goiter thyroglobulin and BSA were also reported (12). In the present communication we report studies with a more highly purified preparation of TPO (preparation VII), isolated by a procedure similar to that used for preparations V and VI, but including as a final purification step the use of a column of hydroxylapatite. TPO VII displayed a value of 0.54 for&lo~&so, a much higher ratio than those of most previous preparations. On the basis of the guaiacol assay it has the highest specific activity yet reported (794 pmol of guaiacol oxidized/min/mg). The most active thyroid peroxidase previously reported is the bovine TPO described by Alexander (lo), with A,,,IA,,, equal to 0.55 and a guaiacol specific activity of 460. However, in contrast to the marked instability of the Alexander preparation, TPO VII was exceedingly stable in frozen solution at -20°C. It was isolated in 1974 and it has shown no significant loss in enzymatic activity after storage in the freezer for more than 4 years. Also in contrast to the preparation described by Alexander (lo), preparation VII was isolated in sufficient quantity to permit measurement of several of its physical and chemical properties. Purity estimates based on disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels ranged from 80 to 95%, thus making TPO VII particularly suitable for studying the physical and chemical properties of the enzyme. In the present communication we present data on amino acid composition, content of various sugars, molecular weight by sedimentation equilib-

rium, heme spectrum, circular dichroism spectrum, and behavior on electrophoresis and isoelectric focusing in polyacrylamide gels. Data are also presented comparing the specific activity of TPO VII with previous TPO preparations, based on several different peroxidase assays. The trypsin-solubilized preparation of TPO studied here most likely represents a cleavage product from a larger membranebound precursor. However, since our solubilized TPO preparation appears to retain essentially all of the enzymatic activities of “native” TPO, and since, as shown in this study, it is at least 80% pure, we believe that the data reported herein reflect the physical and chemical properties of the active core of the thyroid peroxidase. METHODS AND MATERIALS

Solubilixation of Thyroid

and Purification Peroxidase

The procedure was similar to that described previously (8), but several important modifications were introduced. The major changes included the following: (i) The pellet suspension was treated initially with both trypsin and deoxycholate, instead of with deoxycholate alone, (ii) extraction of the acetone pellet was performed with a mixture of Triton X-100 and deoxycholate, instead of with deoxycholate plus trypsin, and (iii) chromatography on hydroxylapatite was used for the final purification step. Details of the enzyme purification procedure are presented in the miniprint supplement.4

Protein

Measurement

Unless otherwise stated protein was measured by the method of Lowry et al. (13).

Methods of Assaying

Enzyme Activity

During the purification procedure enzyme activity was assayed by the iodide oxidation method (14, 15). The assay was performed in 0.02 N KI at pH 7.4 in 0.04 M phosphate. The reaction was initiated with 10 ~1 of 0.1 M H,O, (final concentration 0.5 mM), and AA,,, was recorded in 15 s. A unit is defined as AA,,,lmin equal to 1.0. Three other assay procedures were also used to com4 The table and figure numbers in the miniprint supplement are preceded by the letter S, to distinguish them from the tables and figures in the main body of the paper. Enlarged copies of the material in the miniprint supplement will be provided on request.

246

RAWITCH ET AL.

pare the specific activity of TPO VII with previous TPO preparations. Guaiaco2 oxidation. This assay, a modification of that described by Hosoya et al. (15), was performed in a Model 124 Hitachi double-beam spectrophotometer with attached recorder. Both the sample and the reference cuvettes contained 3 ~1of TPO (100 yglml), 1.20 ml of 0.067 M phosphate buffer (PB), pH 7.0,0.2 ml of BSA (5 mg/ml in PB), and 0.7 ml of 0.1 M guaiacol in PB. The reaction was initiated in the sample cuvette by the addition of 10 ~1 of 66 mM freshly diluted H,O,, and AA,,, after 60 s was determined from the chart recording. A unit of activity was defined as AA,,,, = 1.0 in 60 s. Iodirzation assay. The incubation mixture contained 5 ~1 of TPO (100 pglml), 745 ~1 of 0.067 M phosphate buffer, pH 7.0, 100 pl of BSA (5 mg/ml in PB), and 100 ~1 of 0.1 mM la’1 in PB. The reaction was initiated at 25°C by the rapid addition of 50 ~1 of 2 mM H,O?. After 1 min the reaction was terminated by rapid addition of 10 ~1 of 0.5 M methimazole (MMI), and the reaction tubes were placed in an ice bath at 0°C. An aliquot (25 ~1)of the incubation mixture was delivered on a strip of Whatman 3MM paper, and the fraction of the total ‘:“I remaining at the origin after developing the chromatogram for 60 min in collidine-3 N NH,OH (3:l) was determined. The latter represents ‘“‘I incorporated into BSA (2). Incorporation of 1 pmol of iodide/min into BSA was defined as a unit of iodination activity. Coupling assay. Thyroid peroxidase catalyzes the coupling of DIT residues in the thyroglobulin to form T,, as previously reported (2). It was of interest to compare preparation VII with previous preparations based on this activity of the enzyme. Goiter thyroglobulin (0.04% I) was iodinated chemically with ““I:; to a level of 25 atoms of I per molecule as previously described (2). The solution of labeled protein was dialyzed thoroughly to remove inorganic ‘:‘lI. The assay system contained the following: 500 ~1 of 1.5 pM [‘3LI]thyroglobulin in PB, 5 ~1of TPO (100 pg/ml in PB), and 5 ~1of glucose (100 mg/ml in PB). The reaction was initiated at 37°C by addition of 5 ~1of glucose oxidase (100 kg/ml, 39 unitsimg) and it was terminated after 10 min by addition of 5 ~1of 0.5 M MM1 and cooling of the reaction tubes in an ice bath at 0°C. The thyroglobulin was digested with Pronase plus aminopeptidase (16), and the [‘“‘I]T, fraction was determined by paper chromatography in t-amyl alcohol-NH,OH. The increase in the percentage of [‘S’I]T, produced by incubation with the TPO (compared to a control incubated without TPO) was taken as a measure of coupling activity. A unit was defined as an increase of 1.0% in the [‘“‘IIT, fraction.

ing modifications. The gel buffer was made 0.5% in SDS. The TPO samples, already in phosphate buffer, were made 2% in SDS, 0.1% in @mercaptoethanol, and incubated at 60°C for 30 min. P-Mercaptoethanol was omitted in some samples to obtain SDS-gel patterns of the nonreduced enzyme. When SDS-PAGE was performed in tubes, the gel length was 10 cm. When SDSPAGE was performed in a slab, the dimensions were 10 by 7 cm with 3-mm thickness. The current applied to the slabs was 120 mA, the same current per unit of cross-sectional area as was used in the tube gels. Molecular weight standards included: phosphorylase A, lactoperoxidase, bovine serum albumin, chymotrypsinogen, and ribonuclease. Standard disc gel electrophoresis was performed using the method of Davis (18). Gels, 7.5%, were run in both tubes and slabs. TPO samples were brought to the correct pH by the addition of at least twofold the volume of 10x buffer. In some experiments disc gels were stained for activity by placing them in a solution of 0.005 M guaiacol in 0.02 M phosphate, pH 6.8, and then adding H,O, to a final concentration of 5 x lOmaM. In other gels run simultaneously the protein was stained with Coomassie blue. This enabled us to determine the correspondence between enzyme activity and protein. Isoelectric focusing was performed in tubular 5% acrylamide gels using ampholytes in the pH range of 4-7 and staining with bromophenol blue (19). Sulfuric acid, 0.01 N, was used at the anode and 0.01 N sodium hydroxide at the cathode.

Sedimentation

TPO, at a concentration of 0.7 mglml, was dialyzed against 0.1 M sodium phosphate buffer at pH 7.0 for 24 h. Dilutions from this dialyzed stock were made with the dialysate to concentrations of 0.3,0.2, and 0.15 mgi ml. Sedimentation equilibrium experiments were carried out according to the high speed method of Yphantis (20) in a Beckman Model E analytical ultracentrifuge with standard interference optics at a rotor speed of 21,740rpm. Photographic plates were taken after 24,48, and 72 h and analyzed using a Nikon microcomparator. Insignificant change was seen in the fringe distribution pattern after 48 h at equilibrium speed. Column heights were near 0.2 mm and a layer of FC-43 fluorocarbon was used to provide a uniform base for the liquid columns. The partial specific volume of TPO was calculated from the amino acid and carbohydrate compositions of the protein according to Schachman (21).

Amino Polyacrylamide and Isoelectric

Gel Electrophoresis Focusing

SDS-Polyacrylamide gel electrophoresis (SDSPAGE) was performed on 7.5% acrylamide gels using the method of Weber and Osborne (17) with the follow-

Equilibrium

Acid Analyses

Aliquots of a TPO solution of known concentration were taken for amino acid analysis. Hydrolysis was performed at 110°C in acid-washed glass hydrolysis tubes under vacuum using redistilled 5.7 N HCl. Hydrolysis time was 22 h. Following hydrolysis, the

247

PROPERTIES OF THYROID PEROXIDASE tops of the tubes were carefully opened and known amounts of pyridyl ethylcysteine and norleucine were added as internal recovery standards. The contents of the tubes were taken to dryness under reduced pressure on a shaking evaporator, and the dried hydrolpsates were dissolved in measured amounts of pH 2.2 sodium citrate buffer. Samples were analyzed by standard twocolumn methodology on a Beckman Model 121automatic amino acid analyzer equipped with a system AA computer-integrator and high sensitivity option. Short- and long-column results were normalized using the internal standards. Tryptophan was determined spectrophotometrically according to the method of Bredderman (22) after extensive dialysis against 6 M guanidine hydrochloride.

Carbohydrate

Analysis

Aliquots of a TPO solution of known concentration were taken for hydrolysis. Neutral sugars were determined according to the procedure of Kim et al. (23) after hydrolysis in 0.25 N H,SO, in the presence of Dowex 50 resin. Arabinose and inositol were added as internal standards. Following hydrolysis, H&SO,, free amino acids, amino sugars, and peptides were removed by passing the samples through a column (0.5 cm i.d.) consisting of0.5 cm of Dowex-50 x 2 (H+ form) layered over 5 cm of Dowex-I x 8 (formate form). After reduction and formation of alditol acetate derivatives, analyses were carried out on a Hewlett-Packard Model 58308 gas chromatograph using a %-in. x lo-ft column packed with Supelco SP2340. Concentrations of neutral sugars were calculated from peak areas by comparison with both known standards and internal standard. Amino sugar analyses were carried out on 4-h hydrolysates (lOO”C, 4 N HCl) employing the short column of the amino acid analyzer.

method of Yang and co-workers (25, 26) with model spectra generated using the known crystallographic structure of several proteins. The analyses were carried out using a BMD 073 Computer Program (Biomedical Computer Programs, University of California Press, 1973) and an IBM 370/145 computer.

Heme Spectra The characteristics of the heme moiety in TPO were examined using the pyridine hemochromogen procedure (8). Horseradish peroxidase (HRP) was used as a reference under each set of conditions. Absorption spectra were obtained on an Aminco-Chance doublebeam spectrophotometer for both the oxidized and dithionite reduced enzyme forms.

Fluorescence

Spectrum

The intrinsic fluorescence of TPO VII was examined using a Perkin-Elmer MPF 44A spectrofluorometer with a micro-processor based corrected spectral unit to give true or corrected excitation and emission spectra. Spectra were obtained in 0.05 M phosphate buffer at pH 7.0. RESULTS

Purity

qf Enzyme

The polyacrylamide disc gel electrophoresis pattern of TPO VII is illustrated in the upper panel of Fig. 1. Approximately 80% of the total stained protein appeared in a single band, designated II in the protein scan. The most significant minor component, designated III, appeared as a shoulder on the leading edge of component II. It accounted Circular Dichroism Measurements for about 15% of the total protein. Two addiCircular dichroism (CD) measurements were made tional very light-staining bands, designated with a JASCO J-20 automatic recording CD spectro- I and IV, comprised less than 5% of the total polarimeter. The thyroid peroxidase was examined at protein. Densitometry of unstained gels a known concentration (40 fig/ml) in 0.05 M sodium (Fig. 1, lower panel) indicated that the heme phosphate, pH 7.0. The instrument was calibrated with absorption peak corresponded to the major standard D-lo-camphorsulfonic acid (24). Mean residue protein band. In other experiments (results weight ellipticities, [H],,s,,., were calculated according not shown) enzyme activity in unstained to the equation: gels, localized by treatment of the gels with guaiacol plus H,02, also corresponded to the 0 scale factor’ MRW [%uw,= major protein component. Clearly, there1O.L .c fore, the major protein peak in Fig. 1 repreWhere 0 is the observed ellipticity in degrees, MRW sents active TPO. The relatively low levels is the mean residue weight calculated from amino acid of the minor components made it difficult to analysis (110 for TPO VII), L is the cell pathlength in determine if they also contained heme. Atcentimeters, C is the protein concentration in grams per milliliter, and the scale factor is in degrees per tempts to resolve this point by overloading the gels were not successful because the centimeter. Secondary structure of TPO was estimated by the minor component of greatest interest (III)

248

RAWITCH ET AL. Prote,n

scan

1

ProkIn

scan

I

Heme

Scan

I

TOP

FIG. 1. Densitometer scan of disc gel electrophoresis (7.5%) of TPO VII. The lower panel is a scan at 412 nm of the unstained gel to locate the heme band. The upper panel is a scan at 600 nm of the gel after staining in Coomassie brilliant blue. The anode is on the right. The sample load was 30 wg.

FIG. 2. Densitometer scan of SDS-polyacrylamide (7.5%) electrophoresis of nonreduced TPO VII. The major band corresponds to a molecular weight of 87,000. The small peak corresponds to a molecular weight of 60,000. The gel was stained in Coomassie brilliant blue prior to densitometer scanning at 600 nm. The anode is on the right. The sample load was 20 Fug.

observed at the same location. Careful examination of the gels after staining with migrated very near the major band, and bromophenol blue revealed that the TPO resolution was unsatisfactory at large sample band actually consisted of a series of three to loads. It is not clear from these results four subbands of very similar isoelectric whether component III represents an active points which were not well resolved in denform of TPO. However, this possibility was sitometry of the gel. The densitometer scans suggested by the results obtained with indicated that 86% of the bromophenol blueguaiacol staining, since it appeared that the guaiacol stain extended into the region of peak III. If component III does indeed correspond to an active form of TPO, then 95% of the total protein in TPO VII represents active enzyme. Figure 2 shows the SDS-polyacrylamide gel electrophoresis pattern for nonreduced z’ I ‘i TPO VII. Almost all of the stained protein ;I (93.6%) was present in a single peak, cor2 responding to a molecular weight slightly greater than 80,000. Presumably this peak includes components II and III of Fig. 1. TOP One minor component was observed, representing only about 4% of the total stained -r protein and corresponding to a molecular FIG. 3. Densitometer scan of gel isoelectric focusing weight of about 60,000. of TPO VII. Ampholytes were used to produce a pH When TPO VII was subjected to isoelec- gradient from 4 to 7. The pH gradient was determined tric focusing in 5% polyacrylamide gels con- by slicing duplicate blank gels into 3-mm sections and taining ampholytes from pH 4-7, virtually incubating each slice in 1 ml H,O for 3 h. The pH of all of the protein focused in a narrow region each of the resultant solutions was then determined. at pH 5.75 (Fig. 3). A heme band was also The sample load was 30 pg.

PROPERTIES

OF THYROID

PEROXIDASE

249

staining material was in the major TPOassociated bands (Fig. 3). Weight Determinations

Molecular

The molecular weight of TPO VII was determined both by sedimentation equilibrium and by SDS-polyacrylamide gel electrophoresis. Results of the sedimentation equilibrium study are shown in Fig. 4. The linearity of a typical plot of In C against the square of the radial distance is illustrated in Fig. 4A. Figure 4B shows the concentration dependence of the apparent weight-average molecular weight. The extrapolated molecular weight at infinite dilution was 93,000. This was based on a partial specific volume of 0.721, calculated from the amino acid and carbohydrate composition of TPO VII. SDS-polyacrylamide gel electrophoresis under reducing conditions (Fig. 5) revealed two major bands, designated B and C, with apparent molecular weights of 60,000 and

.’J

0 I/ I . 495 rz m

t"

24,000, respectively, and a single minor band, designated A, with an apparent molecular weight of 87,000. The staining intensities of the two major bands as judged from densitometry were consistent with equimolar amounts of the two species. Component A, comprising about 5% of the total stained protein, was assumed to represent undissociated TPO VII. Its molecular weight (87,000) was in reasonable agreement with that determined by sedimentation equilibrium (93,000) and was equal to the sum of the molecular weights of components B and C. Cott2posifiott

500

100

FIG. 5. Densitometer scan of SDS-polyacrylamide (7.5%;) electrophoresis of reduced TPO VII. A, B, and C correspond to molecular weights of 87,000, 60,000, and 24,000, respectively. The gel was stained in Coomassie brilliant blue prior to densitometer scanning at 600 nm. The anode is to the right. The sample load was 20 fig.

505

1I

(cm’) 1

FIG. 4. Sedimentation equilibrium results with TPOVII. (A) A representative plot of In [fringe concentration] versus radial distance squared. The line represents the linear regression fit of the data. The protein concentration was 0.3 mgiml in 0.1 M sodium phosphate, pH 7.0. Rotor speed was 21,740 rpm. Photograph was obtained after 72 h and analyzed on a Nikon microcomparator. (B) A plot of weight-average molecular weight versus protein concentration. The line is the linear regression fit to the data.

The amino acid and carbohydrate composition of TPO VII, shown in Table I, revealed the following notable features: (i) an unusually high proline content compared to most globular proteins, (ii) approximately 13 half-cystine residues per mole, (iii) 6 residues of methionine per mole, (iv) 13 residues of tryptophan per mole, and (v) a predominance of arginine (51 residues) over lysine (12 residues). Carbohydrate analysis of TPO (see Table I) revealed 8.6% recovered sugars, representing about 10% of the total recovered weight of the enzyme. While the predominant sugars were mannose (27 residues) and N-acetyl glucosamine (12 residues), significant amounts of glucose (8 residues) and galactose (2 residues) were detected along with small amounts of both

250

RAWITCH ET AL. TABLE I

Asp Thr Ser Glu Pro ‘JY Ala Cysl2 Val Met Ile Leu Tyr Trp Phe Lys His Ax Fucose Xylose Mannose Galactose Glucose N-acetylglucosamine Heme

Percentage by weight

Residues per mole”

7.31 2.54 1.81 9.37 7.24 3.73 5.64 1.48 4.04 0.82 2.22 8.17 2.05 2.60” 5.05 1.61 2.95 8.56 0.10 0.11 4.64 0.36 1.45

59 23 19 67 69 61 74 13 38 6 18 67 12 13 32 12 20 51 1 1 27 2.0 8

2.02 0.47

12 0.71’

dithionite-reduced forms of the hemochromogens. As reported previously with less pure preparations of TPO (81, significant differences were observed between the spectra of the two peroxidases. The cyband for the reduced pyridine hemochromogen of TPO reproducibly displayed a peak at 562 nm, significantly different from the peak at 557 nm for the protoferriheme known to be present in HRP. The ratio of absorbance of the TPO (Y peak to its Soret peak was greater than the corresponding ratio observed for HRP. Comparison of the Soret peaks of the oxidized pyridine hemochromogens also showed a major difference. The maximum for TPO occurred at 420 nm, while that for HRP was observed at 401 nm. Spectra of the acid-acetone-extracted hemes (results not shown) displayed differences similar to those described previously. These heme spectral studies with TPO VII confirm earlier findings (8), which suggest that the heme in TPO is not identical with the ferriprotoporphyrin IX of HRP. Circular

Dichroisrn

Spectruw

The circular dichroism spectrum, obtained for a solution of TPO in 0.05 M sodium phosTotal 86.34” 706 phate at pH 7.0, indicated a weak CD band in the visible-near-uv region corresponding U Rounded off to the nearest whole residue number to the heme moiety. An unremarkable far-uv based on a molecular weight of 93,000. h Determined spectrophotometrically (see text for CD spectrum was obtained, which was normalized and fitted with the parameters details). (I Based on A,,, of the pyridine hemochromogen, as- derived from the X-ray diffraction based suming a millimolar extinction of 32 (the millimolar ex- structures of known proteins (25, 26). The tinction coefficient of ferriprotoporphyrin IX at 557 computer analyses were consistent with 18% nm). This value is an estimate since the heme found in o(helix and 20% ,Bstructure in TPO. AnalyTPO appears to differ from ferriprotoporphyrin IX. ses based on the method of Greenfield and ‘I Weight recovery based on Lowry protein deterFasman (2’7) indicated 16% (Yhelix, in good mination. agreement with the computer data fit. fucose and xylose (about 1 residue of each). Sialic acid was not determined because of the limited amount of material available for analysis. Heme Spectral Studies

Figure 6 shows the pyridine hemochromogen spectrum of TPO VII compared with that of horseradish peroxidase (HRP). Results are shown for both the oxidized and

Fluorescence

Spectrum

The intrinsic fluorescence of TPO was characteristic of a tryptophan-containing protein with a corrected emission maximum near 338 nm and a small shoulder at 305 nm (indicating some emission from tyrosine). The corrected excitation spectrum maximum occurred at 282 nm. No fluorescence signal associated with the heme in TPO was detected.

251

PROPERTIES OF THYROID PEROXIDASE

400

420

440

460

480

500

520

540

560

580

600

620

640

660

NANOMETERS

FIG. 6. Comparison of pyridine-hemochromogen spectra of TPO VII (TPO) and horseradish peroxidase (HRP). The pyridine hemochromogens were formed by mixing 0.6 ml of pyridine, 1.0 ml of 0.3 N NaOH, and either 717 pg of TPO VII or 250 wg of horseradish peroxidase, in a total volume of 3 ml. The spectrum of the oxidized form of each sample was recorded and then sodium dithionite was added and the reduced spectrum obtained. Oxidized forms are designated by OX and reduced forms by RED.

arations when assayed by iodination, guaiaco1oxidation, or coupling. The specific activities in Table II based on The specific activity of TPO VII compared to that of previous preparations (V and VI) heme content were calculated from measuredetermined by several different assays is ments of A,,, of the pyridine hemochromoshown in Table II. Specific activities were gens, assuming an extinction coefficient calculated both on the basis of units per equal to that reported for the (Y band of milligram of protein [method of Lowry et al. horseradish peroxidase [E,~ = 32 (28)]. (13)] and on the basis of heme content. Based However, as pointed out previously (8) and on units per milligram of protein TPO VII as confirmed in Fig. 6 the heme in TPO was clearly more active than previous prep- appears to be different from that in HRP. Enzyme Activity

TABLE II SPECIFIC ACTIVITY OF TPO PREPARATIONS BASED ON VARIOUS ASSAYS

Units of enzyme activity Iodide oxidation assay Enzyme preparation V VI VII

Guaiacol oxidation assay

Iodination assay

Coupling assay

Year of Per mg Per nmol Per mg Per nmol Per mg Per nmol Per mg Per nmol prepaheme protein heme protein heme hemeb protein ration A410/AZ80 protein” 1970 1971 1974

0.40 0.38 0.54

1920 1344 1584

369 293 213

1520 1523 2100

292 332 282

29.0 38.2 59.7

5.6 8.3 8.0

u Protein determined by method of Lowry et al. * Heme concentration based on A 562of pyridine hemochromogen, as described in text c Not determined.

ND’ 980 1700

ND 214 228

252

RAWITCH ET AL

The specific activity values in Table II based on heme content, therefore, should be regarded as approximations. Nevertheless the relative values for the different preparations are of interest. Comparison of TPO VI and TPO VII shows that per nanomole of heme TPO VI was more active than TPO VII based on the iodide and guaiacol oxidation assays. However, the two preparations were about equally active in the iodination and coupling assays, which most closely reflect the physiological action of the enzyme. In the iodide oxidation assay, TPO V was the most active, per nanomole of heme as well as per milligram of protein. It would be expected that the specific activities in any given assay based on heme content would be approximately equal for the different preparations, if the only heme present were that derived from TPO. Possible reasons for the differences in assay values for the different preparations are presented at the end of the Discussion.

was 1 x lo-” M I-, compared to a previously reported value of 2 x lo-’ M for TPO VI (12). Values for V,,,,l, for TPO VI and TPO VII, respectively, were 89 and 133 PEq of I/ml bound/min/mg of enzyme. Based on heme content these values correspond to 1.9 x lo4 (TPO VI) and 1.8 x 10” (TPO VII) pmol of Ilmlimin bound/Fmol of enzyme heme. Assuming 1 mol of heme per mole of enzyme, the turnover number for TPO is approximately 1.8 x 10” Eq of I bound/ minim01 of enzyme. This is higher than the corresponding value (7.4 x 10:‘)observed in a previous study with TPO VI. This discrepancy may be related to the use of a new preparation of goiter Tg in the present study. In other experiments also we have noticed that initial rates of iodination were faster with the new goiter thyroglobulin than with previous preparations. Specific Activity qf’ TPO VII Compared to Thyroid Peroxidase Preparations jrom Other Laboratories

K,,l for Iodide in Zodinatiow, oj’ Th yroglobulin

Comparison of specific activities of TPO preparations from different laboratories is Figure ‘7 shows Lineweaver-Burk plots complicated by differences in assay procefor TPO VI- and TPO VII-catalyzed iodina- dures. Nevertheless, at least based on the tion of goiter thyroglobulin over a range of guaiacol assay, TPO VII is considerably iodide concentrations from 10 to 500 PM. more active than any previously reported The rate of iodination was faster with TPO preparation, including the most active fracVII than with TPO VI at all levels of iodide. tion described by Alexander (10). Based on The K,,, value for both enzyme preparations a millimolar extinction coefficient of 5.57 for guaiacol oxidation at 470 nm (29) it may be calculated from the specific activity in Table II (2100 Uimg) that TPO VII oxidized guaiaco1at a rate of 794 ~moliminimg. Using the same extinction coefficient Alexander reported a comparable figure of 460 ~moliminl mg for his most active fraction. Since the molecular weight of TPO VII is very close to that reported by Alexander for his most active preparation, the relative guaiacol activities calculated per micromole of enzyme are very similar to those calculated per milligram of enzyme. DISCUSSION

FIG. 7. Lineweaver-Burk plots for TPO-catalyzed iodination of goiter thyroglobulin with varying iodide concentration. Comparison of TPO VII with TPO VI.

Thyroid peroxidase, solublized from hog thyroid membranes by deoxycholate-trypsin treatment, was purified to near homogeneity by a combination of gel filtration, DEAE-

PROPERTIES

OF THYROID

cellulose chromatography, and hydroxylapatite chromatography. The resulting TPO preparation, designated TPO VII, shows a higher specific activity for guaiacol oxidation than any previously reported thyroid peroxidase and it is very stable upon storage at -20°C in dilute phosphate buffer. Disc gel electrophoresis of TPO VII indicated a purity of at least 80% and possibly as high as 94%, if the second most intense band (peak III in Fig. 1) represents active peroxidase. A purity of about 95% is suggested by the results obtained by electrophoresis in SDSgels under nonreducing conditions. As shown in Fig. 2, a single major band representing 95% of the staining material was observed under these conditions, corresponding to a molecular weight of about 80,000. The results of isoelectric focusing were consistent with a purity of 86%. Taken together, these results indicate that TPO VII is at least 80% pure and possibly in excess of 90% pure, depending on the criterion used for homogeneity. Isoelectric focusing experiments suggest that TPO VII is microheterogeneous, consisting of a family of proteins with very similar physical and chemical properties. Such microheterogeneity is not unexpected in view of the proteolytic solubilization procedure required in this purification. The molecular weight of TPO VII, determined by sedimentation equilibrium, was 93,000. SDS-gel electrophoresis under reducing conditions gave rise to approximately equimolar amounts of two polypeptides, with molecular weights near 60,000 and 24,000, and to a small peak, probably representing undissociated TPO, corresponding to a molecular weight of 87,000. These results indicate that TPO VII is a molecule of approximate molecular weight 90,000, composed of two peptide chains of unequal size, with the larger about 2.5-fold the size of the smaller. Since dissociation of the chains in SDS occurs only after reduction of the protein we conclude that the two chains of TPO VII are covalently crosslinked by disulfide bonds. Earlier studies (‘7, 8) have indicated a molecular weight of 64,000 for TPO, based on gel filtration through a calibrated column of Bio-Gel P-100. When this procedure was applied to TPO VII (using Bio-Gel P-150)

PEROXIDASE

253

an apparent molecular weight of 67,000 was observed. TPO preparations V and VI also showed apparent molecular weights in this range when measured by this procedure. The reason for the discrepancy between these results and those obtained by sedimentation equilibrium is not clear but may indicate some atypical interaction between TPO and the polyacrylamide matrix of the Bio-Gels. It should be noted that sedimentation studies on an earlier TPO preparation (1) of 5.7 S, a value significantly yielded an sqO,~~ larger than that expected for a globular protein with a molecular weight of 64-67,000. It is of interest, in this regard, that the molecular weight of partially purified trypsinsolubilized hog thyroid peroxidase, based on gel filtration through calibrated columns of Sephadex G-200 (a crosslinked dextran gel), was reported both by Hosoya and Morrison (6) and by Pommier et al. (9) to be approximately 100,000. This value is in good agreement with the results obtained in the present study by sedimentation equilibrium and SDS-gel electrophoresis (approximately 90,000), especially since molecular weights of glycoproteins determined by Sephadex gel filtration tend to be anomalously high. It should be noted that the estimated purities of TPO V and VI (40 and 36%, respectively) reported in a previous publication (12) were based on an assumed molecular weight of 64,000. However, based on the results obtained in the present study it is likely that the molecular weights of TPO V and TPO VI, like that of TPO VII, were close to 90,000. Use of this value raises the purity estimates of TPO V and TPO VI to 56 and 51%, respectively. The same calculation (based on heme absorbance) applied to TPO VII yields a purity of 81%. The revised purity estimates for TPO V and TPO VI are in better accord with their A,,,IA,,, values (0.38-0.40) relative to that of TPO VII (0.54). However, as indicated previously (12) the purity calculations based on heme absorbance must be considered only as approximations, since they are based on the assumption that the heme in TPO is ferriprotoporphyrin IX. Our results (see below) do not support this conclusion. The amino acid composition of TPO is characterized by a high content of proline,

254

RAWITCH

a predominance of arginine over lysine residues, and an [Asp] + [Glu]/[Lys] + [Arg] ratio of over 2. The latter is consistent with the acidic pl (near 5.75) found in gel isoelectric focusing experiments. TPO VII contains 13 mol of tryptophan in addition to 12 mol of tyrosine. When TPO VII was excited at 282 nm the emission spectrum was primarily that of tryptophan as is usually the case in the tryptophan-containing proteins. However, the tyrosines were not completely silent, giving rise to an easily recognizable shoulder at 305 nm. Circular dichroism measurements in the far uv are consistent with approximately 18% (Yhelix and 20% p structure in TPO. The relatively low (Y helix content of the enzyme is consistent with the unusually high proline content found upon amino acid analysis. Carbohydrate is an integral component of several hemoprotein peroxidases (30) including horseradish peroxidase, chloroperoxidase, and lactoperoxidase. It has been previously suggested that TPO is also a glycoprotein (1, 31), but unequivocal evidence for the presence of carbohydrate in TPO had to await the availability of a highly purified preparation such as TPO VII. In the present study we have shown that hog TPO contains approximately 10% carbohydrate, an amount comparable to that observed in lactoperoxidase (32) but much less than that observed in plant peroxidases (30). Mannose (27 residues) and N-acetyl glucosamine (12 residues) were the predominant hexose forms in TPO VII. In contrast, Rombauts et al. (32) reported much smaller amounts of neutral sugar in lactoperoxidase as well as significant quantities of galactosamine. No galactosamine was found in TPO VII. The presence of two residues of galactose and nearly one residue of fucose in TPO VII suggests that at least one of the oligosaccharide moieties in this enzyme may be of the complex type seen in many extracellular glycoproteins. The presence of eight residues of glucose per mole of TPO VII is an unusual finding in mammalian glycoproteins outside of the collagen-like family. However, since glucose contamination from a large variety of sources is possible, this observation must be viewed with caution. It is interesting to speculate,

ET AL.

however, since TPO is a membrane-bound enzyme and since recent reports from Spiro (33,34) and from others (35) have implicated glucose-containing oligosaccharides in glycoprotein processing through membranes, that TPO may indeed contain covalently bound glucose which has simply not been removed due to its continued association with the membrane. The total weight recovery from amino acid and carbohydrate analyses was in excess of 85% based on protein determinations by the Lowry procedure. While the Lowry procedure for protein determination is a good approximation, it may not provide a valid measure of actual protein weight for TPO, since BSA was used for the protein standards. The recovery of 85%, therefore, should not be interpreted to mean that 15% of the TPO VII structure was unaccounted for. It would have been of interest to perform lipid analysis on the purified enzyme, but unfortunately there was insufficient material for this purpose. The nature of the heme in thyroid peroxidase has been a subject of controversy. Hosoya and Morrison (6) concluded that the heme in TPO is ferriprotoporphyrin IX (protoferriheme), the same heme found in horseradish peroxidase. This view was also favored by Krinsky and Alexander (36). However, studies reported by Taurog et al. (8) suggested that the hemes in TPO and in HRP are not identical. The results reported in the present study with a more highly purified TPO preparation confirm the earlier report of Taurog et al. The spectral curves in Fig. 6 show significant differences between the pyridine hemochromogens of TPO and HRP, suggesting that the heme in TPO is not ferriprotoporphyrin IX. This observation is consistent with results obtained with other animal peroxidases. Neither lactoperoxidase (30, 32) nor myeloperoxidase (30, 37) is thought to contain ferriprotoporphyrin IX as the prosthetic group. However, ferriprotoporphyrin IX is present in all plant peroxidases that have so far been isolated (30). Unfortunately, limitations in the amount of TPO VII precluded more extensive studies on the nature of the heme in thyroid peroxidase. The results in Table II show that the dif-

PROPERTIES

OF THYROID

PEROXIDASE

255

8. TAUROG, A., LOTHROP, M. L., AND ESTABROOK, ferent catalytic activities associated with R. W. (1970) Arch. Biochem. Biophys. 139, TPO did not bear a constant relationship 221-229. to each other when different TPO preparaJ., DE PRAILAUNE, S., AND NUNEZ, J. tions were compared. The variation was 9. POMMIER, (1972) Biochimie (Paris) 54, 483-492. greater when comparisons were made per 10. ALEXANDER, N. M. (19’77) Endocrinology 100, nanomole of heme than per milligram of pro1610- 1620. L, J,, THOMPSON, J. E,, AND DUNN, tein. Possible explanations for these obser- 11, DEGRooT, vations may involve the following factors: A. D. (1965) Endocrinology 76, 632-645. (i) Different catalytic activities might be 12. TATJROG, A., DORRIS. M. L., AND LAMAS. L. (1974) Endocrinolo& 94, 1286-1294. mediated by different active sites on the enzyme (38), and (ii) variable degrees of in- 13. LOWRY, 0. H., ROSEBROUGH, N. H., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chew. 193, activation and/or denaturation of the enzyme 265-275. protein may have occurred during the rather lengthy isolation procedure, for example, 14. ALEXANDER, N. M. (1962) Amal. Biochem. 4, 341-345. in the steps involving treatment with rela- 15. HOSOYA, T., KONDO, Y., AND UI, N. (1962)J. Biotively high concentrations of detergent, and d/em. (Tokyo) 52, 180-189. (iii) since trypsin digestion of the thyroid 16. TAUROG, A., RIESCO, G., AND LARSEN, P. R. particulate fraction might be expected to (1976) Endocrinology 99, 281-290. produce a heterogenous group of active 17. WEBER, K., AND OSBORNE, M. (1969) J. Biol. Chem. 244, 4406-4412. peroxidase components (5, lo), the three 18. DAVIS, B. (1964) AWL N. Y. Acad. Sci. 121, 404preparations may have differed significantly 427. in the nature of the peptide fragments that 19. AWDEH, Z. L. (1969) Sci. TOO/S16, 42-43. appeared in the finally purified material, ACKNOWLEDGMENTS The authors are indebted to Dr. Russell Prough, Biochemistry Department, University ofTexas Health Science Center for help with the heme spectral studies and to Mr. Carl A. Luer, University of Kansas Medical Center, for assistance in obtaining circular dichroic spectra. Valuable assistance with the enzyme purification procedure was provided by Marguerite Gunder and by medical student summer fellows, Howard Winer and Rolland W. Jenkins. We also acknowledge with gratitude the assistance of Dr. Toshiro Nakashima with some of the peroxidase assays. REFERENCES 1. TAUROG, A. (1970) Recent Progr. Horm. Res. 26, 189-247. 2. LAMAS, L., DORRIS, M. L., AND TALJROG,A. (1972) Endocrinology 90, 1417-1426. 3. DAVIDSON, B., NEARY, J. T., SCHWARTZ, S., MALOOF, F., AND SOODAK, M. (1973) Prep. Biothem. 3, 473-493. 4. NEARY, J. T., DAVIDSON, B., ARMSTRONG, A., MALOOF, F., AND SOODAK, M. (1973) Prep. Biothem. 3, 495-508. 5. NEARY, J. T., DAVIDSON, B., ARMSTRONG, A., STROUT, H. V., MALOOF, F., AND SOODAK, M. (1976) J. Biol. Chem. 251, 2525-2529. 6. HOSOYA, T., AND MORRISON, M. (1967) J. Biol. Chem. 242, 2828-2835. 7. COVAL, M., AND TAUROG, A. (1967) J. Biol. Chem. 242, 5510-5523.

20. YPHANTIS, D. A. (1964) Biochemistry 3,297-317. 21. SCHACHMAN, H. K. (1957)iTT Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds), Vol. 4, p. 32, Academic Press, New York. 22. BREDDERMAN, P. J. (1974) Anal. Biochem. 61, 298-301. 23. KIM, J. H., SHOME, B., LIAO, T. H., AND PIERCE, J. G. (1967) Anal. Biochem. 20, 258-274. 24. CASSIM, J. Y., AND YANG, J. T. (1969) Biochemistry 8, 1947-1951. 25. CHEN, Y. H., YANG, J. T., AND MARTINEZ, H. H. (1972) Biochemistry 11, 4120-4131. 26. CHEN, Y. H., YANG, J. T., AND CHAU, K. H. (1974) Biochemistry 13, 3350-3359. 27. GREENFIELD, H., AND FASMAN, G. D. (1969) Biochemistry 8, 4108-4116. 28. ANTONINI, E., AND BRUNORI, M. (1971) Hemoglobin and Myoglobin in their Reactions with Ligands, p. 11, North-Holland, Amsterdam/ London. 29. HOSOYA, T. (1960) J. Biochem. (Tokyo) 47, 794803. 30. DUNFORD, H. B., AND STILLMAN, J. S. (1976) Coord. Chem. Rev. 19, 187-251. 31. NEARY, J. T., KOEPSELL, D., DAVIDSON, B., ARMSTRONG, A., STROUT, H. V., SOODAK, M., AND MALOOF, F. (1977) J. Biol. Chem. 252, 1264-1271. 32. ROMBAUTS, W. A., SCHROEDER, W. A., AND MORRISON, M. (1967) Biochemistry 6, 29652977. 33. SPIRO, M. J., SPIRO, R. G., AND BHOYROO, V. D. (1976) J. Biol. Chem. 251, 6400-6408.

RAWITCH 34. SPIRO, R. G., SPIRO, M. J., AND BHOYROO, V. D. (1976) J. Biol. Chem. 251, 6409-6419. 35. LIU, T., TURCO, S., HUBBARD, C., STETSON, B., ANDREWS, W., WIRTH, D., RIVES, P., AND ROBBINS, P. W. (19’77) Fed. Proc. 36 (No. 3), Abstract No. 2484.

ET AL. 36. KRINSKY, M. M., AND ALEXANDER, N. M. (1971) J. Biol. Chem. 246, 4755-4758. 37. WV, N. C., AND SCHULTZ, J. (1975) FEBS Lett. 60, 141-144. 38. TAUROG, A., AND DORRIS, M. L. (1971)Fed. PTOC. 30 (No. 3, P. II), Abstract No. 1570.

PROPERTIES

002M

OF THYROID

257

PEROXIDASE

Poe+OO‘lM

07M

PO,----

,o 9 O8 P 06 04 02 0

FRACTION

25 drops (-1.7ml) NVMBER

2 I

Hog thyroid peroxidase: physical, chemical, and catalytic properties of the highly purified enzyme.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 1, April 15, pp. 244-257, 1979 Hog Thyroid ALLEN Peroxidase: Physical, Chemical, and Catalyti...
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