Purification and characterization of a large, tryptic fragment of human thyroid peroxidase with high catalytic activity

Alvin Taurog, Martha L. Dorris, Naokata Yokoyama, Clive A. Slaughter

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Thyroid peroxidase (TPO) was purified from human thyroid tissue, obtained at surgery from patients with Graves' disease, by a procedure similar to one that we had previously used for the purification of porcine TPO. The membrane-bound enzyme was solubilized by treatment of the thyroid particulate fraction with trypsin plus detergent. After precipitation with ammonium sulfate, the enzyme 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. Although a high degree of purification was achieved, the finally isolated product was considerably more heterogeneous than the TPO obtained from porcine thyroids. Several pools of active enzyme differing in values for A412 A280 and in specific activity were collected. Gel electrophoresis was performed under native, denaturing [sodium dodecyl sulfate (SDS)] and denaturing plus reducing conditions. Native gel electrophoresis indicated that the active enzyme (93 kDa) was heavily contaminated with an inactive 60-kDa fragment, which we were unable to remove by HPLC. The inactive fragment was highly antigenic when tested on immunoblots with an antibody to TPO. The presence of the inactive fragment greatly reduced values for A412 A280 in the finally purified human TPO. Two of the pools, with A412 A280 values of 0.159 and 0.273, were used for further testing. Catalytic activity was very similar in these two pools when measured on the basis of heme content by several different assays. Moreover, the specific activities of both, based on heme content, were very similar to those observed with a porcine TPO preparation with A412 A280 = 0.48. These findings indicate that the inactive 60-kDa fragment most likely did not contain heme. On SDS-polyacrylamide gel electrophoresis under reducing conditions, the 60-kDa fragment completely disappeared and was replaced by a 36- and a 24-kDa component. Amino terminal sequence information obtained on these components indicated that the 24-kDa component represents the amino terminal portion of the active 93-kDa fragment, whereas the 36-kDa fragment represents the carboxyl terminal portion. A model is proposed suggesting that the 60-kDa fragment was generated by trypsin cleavage of native TPO at two internal sites within a disulfide loop (res ~300 and res 564) and at one further internal site (res 280). In addition, trypsin cleavage is proposed at sites near the amino and carboxyl ends common to both the active 93-kDa and the inactive 60-kDa fragments. According to this model, the inactive 60-kDa fragment is missing that portion of the polypeptide chain extending from res ~300 to res 564. Since the 60-kDa fragment lacks heme, this region of the molecule appears to be important for heme binding and may contain the heme binding site.

Original languageEnglish (US)
Pages (from-to)333-341
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume278
Issue number2
DOIs
StatePublished - May 1 1990
Externally publishedYes

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Iodide Peroxidase
Purification
Heme
Catalyst activity
Electrophoresis
Gels
Trypsin
Thyroid Gland
Swine
Enzymes
Chromatography
Sodium Dodecyl Sulfate
DEAE-Cellulose
Graves Disease
Ion Exchange Chromatography
Ammonium Sulfate
Durapatite
Disulfides
Detergents
Surgery

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Purification and characterization of a large, tryptic fragment of human thyroid peroxidase with high catalytic activity. / Taurog, Alvin; Dorris, Martha L.; Yokoyama, Naokata; Slaughter, Clive A.

In: Archives of Biochemistry and Biophysics, Vol. 278, No. 2, 01.05.1990, p. 333-341.

Research output: Contribution to journalArticle

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