PARENT SESSION
Cryopreservation
530
NOVEL PURIFICATION METHOD FOR MAMMALIAN SEMINAL PLASMA PHOSPHOLIPID-BINDING PROTEINS.
Manjunath, Puttaswamy1, Ménard, Martin1, Nauc, Veronica1, Lazure, Claude2, 1 2
ABSTRACT- A family of bull seminal plasma (BSP) phospholipids-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The analogous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma phospholipids-binding proteins. The method is based on the interaction of this family of proteins with egg yolk low-density fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF (isolated by ultracentrifugation on KBr solution, density 1.21 g/ml) with alcohol precipitates of bull, boar and stallion seminal plasma solubilized in phosphate-buffered saline. LDF were re-isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized and delipidated. BSP analogous proteins were finally purified by p-aminophenyl phosphorylcholine-Agarose and gelatin-Agarose chromatographies, and analyzed by SDS-PAGE. With this new protocol, phospholipid-binding proteins of bull, boar and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio-immunoassay data showed that 10 mg of LDF binds all BSP proteins present in 120 mg of alcohol precipitated bull seminal plasma proteins. These results confirm the efficiency of the method and that the LDF step could be used for the purification of all BSP proteins analogs from different mammalian species.
KEY WORDS: capacitation, egg yolk, seminal plasma phospholipid-binding proteins, purification