Purification of selenoprotein P from human plasma using immunoaffinity chromatography

Conference · Mon Mar 11 04:00:00 EST 1991 · FASEB Journal (Federation of American Societies for Experimental Biology); (United States)

OSTI ID:5372698

Aakesson, B; Bellew, T; Burk, R F ^[1]

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1. Vanderbilt Univ., Nashville, TN (United States)

Selenoprotein P was purified from rat plasma using immunoaffinity chromatography. The same approach was used with human plasma. HepG2 cells were labeled with {sup 75}Se. The labeled medium, containing proteins secreted by the cells, was added to human plasma and the {sup 75}Se was used as a marker for {gt}1,000-fold purification of the major {sup 75}Se-containing protein. This material was used to produce 2 monoclonal antibodies. In a competitive assay, human plasma, but not plasma from 5 other species, inhibited binding of {sup 75}Se by these 2 antibodies. The antibodies were coupled to agarose and columns were made. Human plasma was processed in 2 steps. Step 1 was an antibody column and step 2 was a heparin-agarose column. SDS-PAGE demonstrated bands at 61 and 55 kDa. Both bands stained with PAS. Amino acid analysis of carboxymethylated material indicated that selenocysteine was {gt}1% of the total amino acids. N-terminal sequencing revealed a strong similarity to rat selenoprotein P. Immunodepleted human plasma and control plasma were chromatographed on Sephacryl S200 and selenium was measured in the eluted fractions. Immunodepletion removed one-third of the selenium. The elution pattern of control plasma revealed a broad peak of selenium just ahead of and including the albumin peak. Most of this peak was absent from the immunodepleted serum and a graph of the difference between the 2 chromatograms was a single peak of selenium well separated from the albumin peak.

• OSTI ID: 5372698
• Report Number(s): CONF-9104107--
• Conference Information: Journal Name: FASEB Journal (Federation of American Societies for Experimental Biology); (United States) Journal Volume: 5:4
• Country of Publication: United States
• Language: English

Related Subjects

560300* -- Chemicals Metabolism & Toxicology
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD PLASMA
BODY FLUIDS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ELEMENTS
EVEN-ODD NUCLEI
FRACTIONATION
INTERMEDIATE MASS NUCLEI
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
MAN
MATERIALS
METABOLISM
METALLOPROTEINS
MONOCLONAL ANTIBODIES
NUCLEI
ORGANIC COMPOUNDS
PRIMATES
PROTEINS
RADIOISOTOPES
RATS
RODENTS
SELENIUM
SELENIUM 75
SELENIUM ISOTOPES
SEMIMETALS
SEPARATION PROCESSES
TRACER TECHNIQUES
VERTEBRATES