Characterization of the major proteins of bovine seminal fluid by two‐dimensional polyacrylamide gel electrophoresis

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Abstract
Recently, we demonstrated that the major proteins from bovine seminal plasma BSP‐A1, ‐A2, ‐A3 and ‐30‐kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5–7.8 and at molecular weights (Mr) between 6 and 100 kDa. Many isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D‐PAGE. BSP‐A1 was found at a Mr of 16.5 kDa and in the range of pl of 4.7–5.0; BSP‐A2 at 16 kDa and at a pl of 4.9–5.2; BSP‐A3 at 15 kDa and at a pl of 4.8–5.2, and BSP‐30‐kDa at 28 kDa and at a pl of 3.9–4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP‐30‐kDa to 4.8–5.0 and decreased its Mr to 25 kDa, but no change was observed for BSP‐A1, ‐A2 and ‐A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their Mr nor their pl. Furthermore, when CBSP proteins were separated in 2D‐PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP‐30‐kDa is mainly due to its sialic acid content.