SDS-PAGE of immunoaffinity-purified rat selenoprotein P demonstrates a major band at 57 kDa and a less intense band at 45 kDa, suggesting the existence of more than one form of the protein. Separate experiments were carried out in which plasma from rats administered Se-75 and immunoaffinity-purified Se-75-labeled selenoprotein P were applied to a heparin-Sepharose column at pH 7. In each experiment three peaks of Se-75-labeled protein were eluted during a continuous gradient from pH 7 to pH 8.5. The remaining Se-75-labeled material was eluted as a single peak by 1 M NaCl. Phosphorimaging of the SDS-PAGE gel of the peaks revealed that the first peak (1a) contained two bands (45 and 57 kDa) while the three remaining peaks each contained one band (peak 1b, 45 kDa and peaks 2 and 3, 57 kDa). N-terminal sequencing of the first eight amino acids of each band revealed that all five have the same N-terminal amino acid sequence and therefore are forms of selenoprotein P. Staining using a digoxigenin-based staining procedure revealed that all five forms contain carbohydrate. This demonstrates that plasma-derived selenoprotein P can be separated into five forms based on SDS-PAGE migration and heparin-Sepharose affinity and that at least two isoforms of selenoprotein P exist based on the presence of 45- and 57 kDa forms. The five forms have been given designations based on their order of elution from the heparin-Sepharose column and their M(r) on SDS-PAGE: selenoprotein P-57A (peak 1a, 57 kDa), selenoprotein P-45A (peak 1a, 45 kDa), selenoprotein P-45B (peak 1b, 45 kDa), selenoprotein P-57B (peak 2, 57 kDa), and selenoprotein P-57C (peak 3, 57 kDa). (C) 1996 Academic Press, Inc.
