Purificação parcial das ram seminal vesicle proteins (RSVP14 e RSVP22) no plasma seminal ovino

Abstract

The present study aimed to develop a methodology to purify the RSVPs (Ram Seminal Vesicle Protein) of 14 kDa and 22 kDa. The work was developed with 16 animals of the Morada Nova breed, kept in an intensive production system, which the samples of semen were collected weekly by means of electro-ejaculator, to separate seminal plasma from the sperm cells through centrifugation. Seminal plasma proteins were precipitated with cold ethanol and 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a Gelatin-Sepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four peaks (P1, P2, P3 and P4), which P1 and P2 contained proteins with low or none affinity for gelatin and P3 and P4 encompassed proteins with fibronectin II domains with high affinity to gelatin. An aliquot of 20 μg of proteins from each peak was subjected to the 12.5% SDS-PAGE technique and another aliquot containing 20 μg of proteins was subjected to Western Blot technique using primary ovine anti-BSPs. The molecular weights of proteins separated by electrophoresis were determined with the aid of the Quantity One Software, which 5 protein bands were detected in P4, with molecular weights between 12 and 30 kDa. The Western blot technique demonstrated specific labeling for RSVP at all four peaks, whereas P1 and P2 labeling was less intense, due to some aggregate or phospholipid-associated form of RSVP that resulted in a slight immunodetection labeling, and at P3 and P4 labeling was specific. The last peak, P4, was subjected to a second chromatographic step on the HiTrap ™ Heparin HP column. Therefore, P4 originated two well-separated chromatographic peaks (P1 and P2), where P1 encompassed the proteins with low or none affinity to heparin and P2, the proteins with affinity to heparin. Proteins from the two new peaks were submitted to 12.5% SDS-PAGE and Western Blot, which it was possible to detect the presence of 3 protein bands in P1 and 3 protein bands in P2. It is believed that RSVP14 and RSVP22 are present in the fraction with no affinity to heparin and fraction with affinity to gelatin, respectively, requiring further testing to prove our initial theories.