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

Conference · Wed Mar 05 04:00:00 EST 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6914109

Richards, M P; Darcey, S E; Steele, N C

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.

• Research Organization: Dept. of Agriculture, Beltsville, MD
• OSTI ID: 6914109
• Report Number(s): CONF-8604222-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:4
• Country of Publication: United States
• Language: English

Related Subjects

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560300* -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ABSORPTION SPECTROSCOPY
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIRDS
BODY
CADMIUM
CELL CONSTITUENTS
CHROMATOGRAPHY
COPPER
CYTOPLASM
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DISTRIBUTION
DOMESTIC ANIMALS
ELEMENTS
EVEN-ODD NUCLEI
EXTRAPOLATION
GLANDS
HORSES
ISOTOPE APPLICATIONS
ISOTOPES
KIDNEYS
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MAMMALS
METALLOPROTEINS
METALLOTHIONEIN
METALS
NUCLEI
NUMERICAL SOLUTION
ORGANIC COMPOUNDS
ORGANS
PROTEINS
RABBITS
RADIOISOTOPES
SEPARATION PROCESSES
SPECTROSCOPY
SULFUR 35
SULFUR ISOTOPES
SWINE
TISSUE DISTRIBUTION
TRACER TECHNIQUES
TRANSITION ELEMENTS
VERTEBRATES
ZINC