Ion-exchange chromatography used to isolate a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma.
Mammalian seminal plasma contains among others, proteins called spermadhesins, which are the major proteins of boar and stallion seminal plasma. These proteins appear to be involved in capacitation and sperm-egg interaction. Previously, we reported the presence of a protein related to spermadhesins in goat seminal plasma. In the present study, we have further characterized this protein, and we propose ion-exchange chromatography to isolate this seminal protein. Semen was obtained from four adult Saanen bucks. Seminal plasma was pooled, dialyzed against distilled water and freeze-dried. Lyophilized proteins were loaded onto an ion-exchange chromatography column. Dialyzed-lyophilized proteins from the main peak of DEAE-Sephacel were applied to a C2/C18 column coupled to an RP-HPLC system, and the eluted proteins were lyophilized for electrophoresis. The N-terminal was sequenced and amino acid sequence similarity was determined using CLUSTAL W. Additionally, proteins from DEAE-Sephacel chromatography step were dialyzed and submitted to a heparin-Sepharose high-performance liquid chromatography. Goat seminal plasma after ion-exchange chromatography yielded 6.47 +/- 0.63 mg (mean +/- SEM) of the major retained fraction. The protein was designated BSFP (buck seminal fluid protein). BSFP exhibited N-terminal sequence homology to boar, stallion and bull spermadhesins. BSFP showed no heparin-binding capabilities. These results together with our previous data indicate that goat seminal plasma contains a protein that is structurally related to proteins of the spermadhesin family. Finally, this protein can be efficiently isolated by ion-exchange and reverse-phase chromatography.
Teixeira
Dárcio Italo Alves
DI
Laboratório de Fisiologia e Controle da Reprodução, Universidade Estadual do Ceará, Fortaleza, CE, Brazil.
Melo
Luciana Magalhães
LM
Gadelha
Carlos Alberto de Almeida
CA
Cunha
Rodrigo Maranguape Silva da
RM
Bloch
Carlos
C
Rádis-Baptista
Gandhi
G
Cavada
Benildo Sousa
BS
Freitas
Vicente José de Figueirêdo
VJ
D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006041:Goats; D008297:Male; D012661:Semen / Q000737:chemistry*; D029589:Seminal Plasma Proteins / Q000235:genetics / Q000302:isolation & purification*
D029589:Seminal Plasma Proteins; C425042:spermadhesin