Purification of selenoprotein P from human plasma using immunoaffinity chromatography

Conference · Mon Mar 11 00:00:00 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-; CODEN: FAJOE
• Journal Information: FASEB Journal (Federation of American Societies for Experimental Biology); (United States), Vol. 5:4; Conference: 75. annual meeting of the Federation of American Societies for Experimental Biology (FASEB), Atlanta, GA (United States), 21-25 Apr 1991; ISSN 0892-6638
• Country of Publication: United States
• Language: English

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FRACTIONATION
SELENIUM
METABOLISM
BLOOD PLASMA
ELECTROPHORESIS
ION EXCHANGE CHROMATOGRAPHY
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ELEMENTS
EVEN-ODD NUCLEI
INTERMEDIATE MASS NUCLEI
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
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NUCLEI
ORGANIC COMPOUNDS
PRIMATES
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560300* - Chemicals Metabolism & Toxicology