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Title: Isolation and quantitation of metallothionein isoforms using reversed-phase high-performance liquid chromatography

Abstract

Reversed-phase HPLC (RP-HPLC) was used to isolate and quantify metallothionein (MT) isoforms from a variety of animal species and tissues. Separations were performed on C/sub 18/ radially compressed cartridge columns, eluted with a 2-step linear gradient of acetonitrile in 10 mM sodium phosphate, pH 7.0. Isoforms were detected by UV absorbance (214 nm) and by on-line interfacing with an atomic absorption spectrophotometer (HPLC-AA) to determine bound Zn, Cd and Cu. Rabbit liver and horse kidney MT's exhibited 7 distinct peaks on RP-HPLC, 2 of which were predominant (MT1 and 2). Pig liver and kidney MT2 yielded 2 subspecies on RP-HPLC, while MT1 yielded a single peak. Avian liver MT was unique from mammalian MT's in that MT2 was about tenfold more abundant than MT1. RP-HPLC and HPLC-AA were used to isolate and quantitate MT isoforms and their Zn content directly from cytosol. Quantitation was achieved by peak area integration and extrapolation from a standard curve of purified avian liver MT2. Both RP-HPLC and HPLC-AA had a lower detection limit of 1 /sup +/g of peptide and .1 ..mu..g of Zn. Recoveries (92-98%) were determined with labeled (/sup 35/S) MT and MT of known Zn content. Cytoplasmic MT-Zn in avian embryomore » hepatocytes cultured with added Zn was quantitated using HPLC-AA. In conclusion, both RP-HPLC and HPLC-AA are rapid and powerful separation techniques for the isolation, quantitation and characterization of the isoproteins comprising the MT gene family.« less

Authors:
; ;
Publication Date:
Research Org.:
Dept. of Agriculture, Beltsville, MD
OSTI Identifier:
6914109
Report Number(s):
CONF-8604222-
Journal ID: CODEN: FEPRA; TRN: 87-006129
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 45:4; Conference: 70. annual meeting of the Federation of American Society for Experimental Biology, St. Louis, MO, USA, 13 Apr 1986
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 59 BASIC BIOLOGICAL SCIENCES; CADMIUM; TISSUE DISTRIBUTION; COPPER; METALLOTHIONEIN; LIQUID COLUMN CHROMATOGRAPHY; ZINC; ABSORPTION SPECTROSCOPY; BIRDS; CYTOPLASM; EXTRAPOLATION; HORSES; KIDNEYS; LIVER; RABBITS; SULFUR 35; SWINE; TRACER TECHNIQUES; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BODY; CELL CONSTITUENTS; CHROMATOGRAPHY; DAYS LIVING RADIOISOTOPES; DIGESTIVE SYSTEM; DISTRIBUTION; DOMESTIC ANIMALS; ELEMENTS; EVEN-ODD NUCLEI; GLANDS; ISOTOPE APPLICATIONS; ISOTOPES; LIGHT NUCLEI; MAMMALS; METALLOPROTEINS; METALS; NUCLEI; NUMERICAL SOLUTION; ORGANIC COMPOUNDS; ORGANS; PROTEINS; RADIOISOTOPES; SEPARATION PROCESSES; SPECTROSCOPY; SULFUR ISOTOPES; TRANSITION ELEMENTS; VERTEBRATES; 560300* - Chemicals Metabolism & Toxicology; 550501 - Metabolism- Tracer Techniques

Citation Formats

Richards, M P, Darcey, S E, and Steele, N C. Isolation and quantitation of metallothionein isoforms using reversed-phase high-performance liquid chromatography. United States: N. p., 1986. Web.
Richards, M P, Darcey, S E, & Steele, N C. Isolation and quantitation of metallothionein isoforms using reversed-phase high-performance liquid chromatography. United States.
Richards, M P, Darcey, S E, and Steele, N C. Wed . "Isolation and quantitation of metallothionein isoforms using reversed-phase high-performance liquid chromatography". United States.
@article{osti_6914109,
title = {Isolation and quantitation of metallothionein isoforms using reversed-phase high-performance liquid chromatography},
author = {Richards, M P and Darcey, S E and Steele, N C},
abstractNote = {Reversed-phase HPLC (RP-HPLC) was used to isolate and quantify metallothionein (MT) isoforms from a variety of animal species and tissues. Separations were performed on C/sub 18/ radially compressed cartridge columns, eluted with a 2-step linear gradient of acetonitrile in 10 mM sodium phosphate, pH 7.0. Isoforms were detected by UV absorbance (214 nm) and by on-line interfacing with an atomic absorption spectrophotometer (HPLC-AA) to determine bound Zn, Cd and Cu. Rabbit liver and horse kidney MT's exhibited 7 distinct peaks on RP-HPLC, 2 of which were predominant (MT1 and 2). Pig liver and kidney MT2 yielded 2 subspecies on RP-HPLC, while MT1 yielded a single peak. Avian liver MT was unique from mammalian MT's in that MT2 was about tenfold more abundant than MT1. RP-HPLC and HPLC-AA were used to isolate and quantitate MT isoforms and their Zn content directly from cytosol. Quantitation was achieved by peak area integration and extrapolation from a standard curve of purified avian liver MT2. Both RP-HPLC and HPLC-AA had a lower detection limit of 1 /sup +/g of peptide and .1 ..mu..g of Zn. Recoveries (92-98%) were determined with labeled (/sup 35/S) MT and MT of known Zn content. Cytoplasmic MT-Zn in avian embryo hepatocytes cultured with added Zn was quantitated using HPLC-AA. In conclusion, both RP-HPLC and HPLC-AA are rapid and powerful separation techniques for the isolation, quantitation and characterization of the isoproteins comprising the MT gene family.},
doi = {},
url = {https://www.osti.gov/biblio/6914109}, journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:4,
place = {United States},
year = {1986},
month = {3}
}

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