Purification of selenoprotein P from human plasma.
Selenoprotein P was partially purified (> 1000-fold) from human plasma in four chromatographic steps using 75Se-labeled selenoprotein P secreted by HepG2 cells in culture as a marker. The purified preparation was injected into mice and monoclonal antibodies, which precipitated the labeled protein, were generated. Neither of two different monoclonal antibodies had cross-reactivity with plasma from five animal species. Antibodies were coupled to agarose, and selenoprotein P was purified from human plasma by immunoaffinity chromatography followed by chromatography on heparin agarose. With two different matrix-bound monoclonal antibodies, the purification procedure gave two bands on SDS-PAGE with mobilities corresponding to 61 and 55 kDa. Both bands stained for carbohydrate and showed increased electrophoretic mobility after enzymatic deglycosylation. Immunoaffinity chromatography removed approx. one-third of the selenium from plasma or 0.4 mumol Se/l at a total selenium concentration of 1.1 mumol/l, indicating that selenoprotein P constituted this proportion of total plasma selenium in healthy US blood donors.
Akesson
B
B
Department of Medicine, Vanderbilt University, Nashville, TN 37232-2279.
Bellew
T
T
Burk
R F
RF
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002846:Chromatography, Affinity; D018197:Hepatoblastoma / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D012643:Selenium / Q000097:blood / Q000302:isolation & purification; D051149:Selenoprotein P; D051140:Selenoproteins
D000911:Antibodies, Monoclonal; D011506:Proteins; D051149:Selenoprotein P; D051140:Selenoproteins; D012643:Selenium