Thyroid peroxidase of the pig, dog, rat, and mouse

Thyroid

Peroxidase

of the Pig,

Dog,

Rat, and Mouse

Solubilization and Identification of lsozymes by Isoelectric Focusing’,’ ZOILO

GONZALEZ-LAMA”

AND ROBERT

N. FEINSTEIN”

Thyroid peroxidase plays an important role in the biosynthesis of the thyroid hormone [for early review, see ( l)]. The enzyme has been studied chiefly in the thyroid of pig (2-6). sheep (7-8), beef (9), and human ( lo- 13). In these species, the enzyme occurs largely in an insoluble form. and a recurring problem has been its solubilization for study. Among the techniques employed for solubilization of the enzyme have been treatment with butanol (5, 14), alkali (5, IS), digitonin (3), trypsin (16-18), and combinations of these. Some problems exist in connection with all of these, and we believe we have now found more satisfactory techniques. We have found that dog thyroid peroxidase is also insoluble, and we have achieved some solubilization of dog and pig thyroid peroxidase by urea, Antaron R- 155 (an anionic detergent), and especially by chlorhexidine { 1,l ‘-hexamethylene bis[5-(4-chlorophenyl) biguanidel}, which was shown by Jensen et al. (19) to release several other membrane-bound enzymes. We have also investigated the thyroid peroxidase of the rat and of the normal and acatalasemic (20) mouse. These rodent thyroid peroxidases prove to be largely soluble, and the demonstration of specific thyroid peroxidases is therefore complicated by the presence of contaminant hemoglobin, which acts peroxidatically (21). and of catalase. ’ This work was supported by the tion, and by the Ministerio Education * The United States Government’s and

U. S. Energy Research and y Ciencia, Spain. right to retain a nonexclusive

to copyright covering this paper 3 Present address: Departamento

Granada, Spain. A TO whom all

correspondence

should

is acknowledged. Microbiologia,

Facultad

Development royalty-free Ciencias,

Administralicense

in

Universidad

be addressed.

292 Copyright @ 1977 by Academic Press. Inc. All rights of reproductmn in any form rexrved.

ISSN 0006.2944

THYROID

PEROXIDASE

MATERIALS

SOLUBILIZATION

293

AND METHODS

Frozen pig thyroid was purchased from Pel-Freez (Rogers, Ark.). Upon receipt, it was thawed, cut into small cubes. rinsed in cold distilled water, blotted, refrozen in dry ice-acetone, and stored at -70”. Dog thyroids were obtained surgically from healthy adult beagles of both sexes from the Argonne National Laboratory colony and frozen and maintained in plastic vials at -70” until use. Dog blood was obtained in heparinized syringes from the jugular vein of healthy animals of the same colony, without anesthesia. Rats used were Charles River adult males: the rats were sacrificed by ether inhalation, and heart blood samples and thyroid gland were removed. In one experiment a rat received, I hr before sacrifice, an intraperitoneal injection of 2000 mgikg of 3-amino-1,2.4triazole (AT), a known inhibitor of the catalase of solid tissues (22). and also known to inhibit the activity of thyroid peroxidase (23). Mice were 8-16 weeks of age, either C3HiCsYAnl (standard inbred C3H mouse) or C3H/Csb/Anl[C3H mouse in which the acatalasemic gene locus (24) has been introduced]. The Csb locus causes almost total loss of blood catalase activity (20) and the production of a labile tissue catalase (25). Effects of acatalasemia on tissue peroxidases are less well known. Mouse blood was obtained from the orbital sinus (26), the mouse was sacrificed with ether, and the thyroid was removed. Peroxidase assays were done using either 2,6-dimethoxyphenol (27) or benzidine (28) as substrate. Other substrates tested, all of which gave comparable results, were guaicol, o-dianisidine. 3.3’-diaminobenzidine, and homovanillic acid. These chemicals were all obtained from commercial sources and were not further purified. Antaron R- 155 (anionic), Antarox D- 100 (nonionic), and Antaron L- 13s (anionic detergent) were gifts of Antara Products Division of General Dyestuff Corp.: Tween-20 (nonionic detergent) was a gift of the Atlas Powder Company; cetylpyridinium chloride (cationic detergent) was a gift of the Wm. S. Merrell Company; and chlorhexidine, in the form of a 20% aqueous solution of the gluconate, was a gift of ICI United States, Wilmington, Del. Isoelectric focusing in polyacrylamide gel slabs was done on the Multiphor apparatus (LKB Produkter, Bromma, Sweden), modified as described elsewhere (29). Equilibrium, with a maximum voltage of about II00 V, was attained in about 3 hr, the gel bed being cooled with circulating ice-water. The pH was determined by use of a flat surface electrode at OS-cm intervals. Peroxidase isozymes were visualized on the gel strips by covering them with a solution containing 0.2% benzidine dihydrochloride and 0.06% H,O,. Other substrates, in a variety of buffers at several pHs, gave comparable results: benzidine, however. appeared to be the most sensitive indicator.

294

(K)N%AI..EI-LAMA

AND

FEINSI

PIN

RESULTS Table 1 demonstrates the solubilizing effect of several reagents on the peroxidase activity of pig thyroid. using benzidine as assay substrate. It will be noted that while several detergents, particularly Antaron R-155, significantly solubilized the thyroid peroxidase. all the detergents tested caused a decrease in total activity. Whether this was a dissociable inhibition or a permanent inactivation was not determined. Urea, however, brought about an appreciable solubilization with only a minor loss of total activity. Table 2 compares the solubilizing ability of urea and chlorhexidine on the thyroid peroxidase of dog and pig. These data are separated from those of Table I because benzidine rather than dimethoxyphenol was the substrate; chlorhexidine precipitates under the conditions of the dimethoxyphenol peroxidase assay. An acetate buffer (pH 4.4) was used: at pH values more alkaline than this, the benzidine begins to precipitate. Figure 1 shows the isoelectrically focused, soluble peroxidase activity of the thyroid of the rat and mouse (both normal and acatalasemic) and of blood of normal mice and rats. In these species, the true peroxidatic activity of the thyroid is clearly separated from the peroxidatic activity of and certain peroxidase activities appear contaminating hemoglobin, which cannot be due to hemoglobin. TABLE

SOLUBILIZING

EFFECTOF

SEVERAL

1

REAGENTSON

PIG THYROID

PEROXIDASE"

A 480 Soluble Reagent

Precipitate

Supernatant

Total

(5%)

0.290 0.112 0.019

0.076

0.366 0.120

0.034

0.116

0.055 0.150

21 7 6.F

0.059

0.080

0.139

0.039 0.074 0.028

0.025 0.036 0.307

0.064 0.1 IO

Water 1% Antarox 1% Antaron

D-100 L-135

1% Antaron R-155 I% Tween-20 1% Cetyl pyridinium chloride I% Digitonin 4 M Urea n Pig thyroid Willems polytron, the precipitate resuspended supernatant, peroxidase to be linear

0.008 0.036

(27). Assay with time.

as

was immediately the resuspended

mixture

was

incubated

for

(30 water) 5 min

33 15 41 38

39 33 92

0.335

recentrifuged (in cold

VT%)

77 58

was thawed. scissor-minced, disintegrated in 9 vol and centrifuged (30 min at 48,OOOg). The supernatant was resuspended to the original volume in the (cold)

precipitate as well

Recovery

I7 30 92

of cold water in the was discarded, and reagent listed. The

min at 48,OOOg), precipitate, was

at 37”.

Reaction

was

and the assayed demonstrated

new for

5

Hz0 4 M Urea 2% Chlorhexidine

Dog (n = 2)

AND

ON THE

2.04 2 1.51 0.75 t 0.46 0.40 2 0.51

2.50 2 0.71 0.28 -r- 0.30 0.05 -c 0.05

Precipitate

OF UREA

2 SOLUBILITY

0.27 -c 0.34 1.37 ? 1.26 1.77 t 1.43

0.12 ” 0.08 1.38 2 0.45 1.96 k 0.52

Supematant

A 340

AND

TABLE ACTIVITY

2.31 k I.86 2.12 + I.71 2.17 k 1.94

2.62 k 0.78 1.66 2 0.33 2.01 k 0.49

Total

OF PEROXIDASE

IN DOG

PIG

II.7 + 7.8 65 + II 82-t I2

4.6 lr 1.7 83 2 20 98k 4

Soluble (%I

AND

THYROID’

922 942

I I3

63 5 I6 77% 6

Recovered (96)

n Pig thyroid was disintegrated by a Willems polytron at a 10% concentration: dog thyroid at 5%. Each of the two dog thyroid experiments was performed on a pool of three dog thyroid half-glands (partial thyroidectomies). Other details are as in the legend to Table I, except that (a) benzidine was the substrate. and (b) assay incubation was carried out for 3 min at room temperature. Reaction was demonstrated to be linear with time. Figures given are mean ? SD.

Hz0 4 M Urea 2% Chlorhexidine

Reagent

OF CHLORHEXIDINE

Pig(n = 4)

Species

EFFECT

I

2

3

FIG. I. Thyroids and bloods of mice and gel. Drawings are counterparts of photographs of all samples applied was 25 ~1. Gels were Slide

I: Supernatant

of

10%

mice. Slide 2: Supernatant of catalase) mice. Slide 3: C3H/Cs” color Slide

homogenate

4: Supernatant

5

rats isoelectrically focused on polyacrylamide above. for easier visualization. The volume stained with benzidine-H,O, (details in text).

of pooled

10% homogenate whole blood.

of the thyroid extracts. Slide 5: Lysate of whole rat blood.

4

PH

thyroids

from

C3HICs”

(acatalasemic)

of pooled thyroids from C3H/Cs” (normal lysed in a volume of water to approximate the of 10%

homogenate

of single

rat thyroid.

Figure I shows only minor differences between the thyroids of normal catalase (Cs”) and acatalasemic (Csh) mice. Most noticeable, as expected, is the greater catalatic activity of Cs” in the cathodal area (indicated by the area of oxygen bubbles in the photograph). An apparent slight difference in the activity of the more anodal peroxidase isozyme, the Csb showing a greater activity, may not be real. Figure 2 provides further evidence for the existence of a true. nonhemoglobin peroxidase activity in the soluble portion of rodent thyroid. The figure compares peroxidase or peroxidase-like activity of

THYROID

I

2

PEROXIDASE

PH

297

SOLUBILIZATION

3

4

Fro. 2. Effect of aminotriazole (AT) on peroxidatic activity of rat thyroid and blood. Slide 1: Supematant of 10% homogenate of normal rat thyroid. Slide 2: Supematant of 10% homogenate of thyroid of rat injected I hr earlier with 2000 mg of AT/kg. Slide 3: Normal rat blood lysate. Slide 4: Blood lysate of AT-injected rat. Other details are as in Fig. I.

whole blood and of an aqueous extract of thyroid taken from (a) a rat sacrificed 1 hr after the intraperitoneal injection of 2000 mg of AT/kg, and (b) a rat sacrificed after an intraperitoneal injection of 10 ml of O.% NaCVkg. It can be seen that the hemoglobin lines (PI 6.9-8.0) are unaffected by injected AT, either in blood or as a contaminant in the thyroid extract. On the other hand, the true peroxidase lines (pl 5.4 and 6.5) are greatly diminished or eliminated in the thyroid of the rat receiving AT. The AT, of course, is not dissociated from the catalase molecule by the isoelectric focusing, because the linkage is covalent (30) and the inhibition

79x

(.iONLAl.k./.-I

,AMA

,4ND

I-EINS’I‘Elh

is considered irreversible (22. 30). Although attempts were made, we were not able to demonstrate AT inhibition directly on the focused gel slab. DISCUSSION

Jensen ef (I/. (19) found that chlorhexidine solubilizes rat liver acid phosphatase. urate oxidase, and /3-N-acetylglucosaminidase. In the case of the first two enzymes, increased chlorhexidine concentration caused not only solubilization but also an increased degree of activation; in the case of the acetylglucosaminidase. a lesser activation was observed with increased chlorhexidine concentration. Jensen it ~1. offered no meaningful explanation of these findings. We suggest the possibility that a pH effect, due to buffering by the chlorhexidine, may be involved; we, too. observed apparent activation to various degrees before we became aware of pH shifts caused by the chlorhexidine. When the pH was carefully controlled, no “activation” was to be observed. In the experience of Davidson rt trl. (14). those reagents that appear to solubilize pig thyroid peroxidase (as estimated by solubilization of yellow material) also irreversibly inactivated it. Urea is presumably included in this list. In the present work. after treatment of pig thyroid with 4 M urea we were able to recover an average of over 60% of the peroxidase activity, of which an average of 83% was soluble. Two experiments with dog thyroid indicated an average 925% recovery, with 6.5% solubility. Chlorhexidine treatment yielded appreciably better results in both respects, in both species. We have made no attempt at further purification of these solubilized enzyme preparations. SUMMARY Dog and pig thyroid peroxidase, which exist naturally in a largely insoluble form, can be solubilized by the use of 4 M urea, or of cblorhexidine, with small losses of total activity. In the mouse and the rat, the thyroid peroxidase occurs in a soluble form. The demonstration of these rodent thyroid peroxidases is therefore complicated by unavoidable contamination with peroxidatically acting hemoglobin and catalase: the demonstration of the presence of true peroxidase was achieved by isoelectric focusing on polyacrylamide gel slabs, which separates the various factors, and by the use of the catalase and peroxidase inhibitor 3-amino1,2,4-triazole. ACKNOWLEDGMENTS We wish to express supplying dog thyroids dissections.

our appreciation and bloods,

and

to Drs. to Mr.

Thomas Everett

E. Fritz and Calvin F. Staffeldt for the

M. Poole for mouse thyroid

THYROID

PEROXIDASE

SOLUBILIZATION

299

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