Characterization and purification of iodinated bovine thyrotropin by high performance liquid chromatography

Journal Article · Fri Aug 01 04:00:00 EDT 1986 · Endocrinology; (United States)

DOI:https://doi.org/10.1210/endo-119-2-887· OSTI ID:5094974

Stanton, P G; Hearn, M T

Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of (/sup 125/I)bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact (/sup 125/I)bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.

• Research Organization: Monash Univ.y, Clayton, Australia
• OSTI ID: 5094974
• Journal Information: Endocrinology; (United States), Journal Name: Endocrinology; (United States) Vol. 2; ISSN ENDOA
• Country of Publication: United States
• Language: English

Related Subjects

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY
400702 -- Radiochemistry & Nuclear Chemistry-- Properties of Radioactive Materials
550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
ANIMALS
BETA DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CATTLE
CHEMICAL REACTIONS
CHEMISTRY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DIIODOTYROSINE
DOMESTIC ANIMALS
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
HALOGENATION
HORMONES
HYDROLASES
HYDROXY ACIDS
INTERMEDIATE MASS NUCLEI
IODINATION
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LABELLING
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MOLECULAR WEIGHT
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC IODINE COMPOUNDS
PEPTIDE HORMONES
PEPTIDE HYDROLASES
PITUITARY HORMONES
PURIFICATION
RADIOCHEMISTRY
RADIOISOTOPES
RADIOPHARMACEUTICALS
RADIORECEPTOR ASSAY
RUMINANTS
SEPARATION PROCESSES
SERINE PROTEINASES
TRACER TECHNIQUES
TRYPSIN
TSH
VERTEBRATES