Separation and quantitation of metallothioneins by high-performance liquid chromatography coupled with atomic absorption spectrophotometry
A rapid, reproducible, and sensitive high-performance liquid chromatography (HPLC) method for the determination of the concentrations of metallothionein-I (MT-I) and metallothionein-II (MT-II) in rat liver has been developed. Metallothioneins (MTs) were separated and quantitated by anion-exchange high-performance liquid chromatography coupled with atomic absorption spectrophotometry (AAS). Purified rat liver MT-I and MT-II, used as standards for developing the method, were easily resolved, eluting at 7.5 and 10.4 min, respectively. To establish standard curves, protein concentrations of solutions of the purified MTs were determined by the Kjeldahl method for the determination of nitrogen, after which the standards were saturated with Cd (final concentration of 50 ppm Cd). Rat liver cytosols obtained from untreated and Cd- or Zn-treated rats were prepared for HPLC-AAS analysis by saturation with Cd (50 ppm Cd) followed by heat denaturation (placing in a boiling water bath for 1 min). Based on the method of standard additions, recovery of MTs exceeded 95% and repeated injection of a sample yielded a coefficient of variance of approximately 2%. A detection limit of 5 ..mu..g MT/g liver was established for the method. Only MT-II was detected in untreated rats, whereas following exposure to Cd or Zn, both forms of MTs were detected. Concentrations of total MTs in liver of untreated and Cd- or Zn-treated rats were also determined by the Cd/hemoglobin radioassay (which fails to distinguish MT-I from MT-II) and indicated that results obtained with the HPLC-AAS method compared favorably to the Cd/hemoglobin radioassay.
- Research Organization:
- Univ. of Kansas, Kansas City
- OSTI ID:
- 5569700
- Journal Information:
- Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 153:2; ISSN ANBCA
- Country of Publication:
- United States
- Language:
- English
Similar Records
Isolation and quantitation of metallothionein isoforms using reversed-phase high-performance liquid chromatography
The rapid isolation of highly purified cadmium metallothioneins using HPLC
Related Subjects
400104* -- Spectral Procedures-- (-1987)
550200 -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
ABSORPTION SPECTROSCOPY
ANIMALS
ANIONS
BODY
CADMIUM
CARBOXYLIC ACIDS
CHARGED PARTICLES
CHROMATOGRAPHY
COMPARATIVE EVALUATIONS
DIGESTIVE SYSTEM
ELEMENTS
GLANDS
GLOBIN
HEMOGLOBIN
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
ION EXCHANGE CHROMATOGRAPHY
IONS
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MAMMALS
METALLOPROTEINS
METALLOTHIONEIN
METALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PIGMENTS
PORPHYRINS
PROTEIN DENATURATION
PROTEINS
RADIOASSAY
RATS
RODENTS
SEPARATION PROCESSES
SPECTROSCOPY
STANDARDIZATION
VERTEBRATES