Early Pregnancy Factor: Its Characterization and Purification from Pregnant Swine Serum.

Abstract

The objective of the present investigation was to purify the early pregnancy factor (EPF) from swine pregnancy serum using diafiltration, ion-exchange chromatography, HPLC-gel permeation, and affinity chromatography. EPF activity was determined by using RIT after the pregnancy serum obtained from a mature swine 25 days after mating was divided into 2 parts (below M.W. 100KD and above M.W. 100KD) by the diafiltration. Using RIT, EPF activity was found in the fraction below M.W. 100KD. When this fraction was applied onto the DEAE-Shepharose column, the EPF activity was only found in the unadsorbed fraction. Next, this fraction was applied onto the CM-Sepharose column and was divided into 3 parts. A fraction which was eluted by 50 mM ammonium acetate buffer containing 50 mM NaCl had the EPF activity and the other fractions did not have this activity. The fraction which was eluted with 50 mM NaCl was analysed by SDS-PAGE and contained 6 bands between M.W. 30KD and 20KD. When the pattern of this SDS-PAGE was compared with the bands obtained from non-pregnant swine serum (day 0 of cycle, estrus), the 3 bands were not confirmed in the non-pregnancy serum. The molecular weight of these bands were 21KD, 23.7KD, and 26KD, respectively. On the HPLC analysis, EPF activity was found between M.W. 14 and 21KD. Therefore, it was thought that the swine EPF active material of M.W. 21KD might be the major active form of the swine EPF in this gestation period. In addition, the affinity gel which was coupled with Rabbit anti nonpregnancy swine whole serum lgG was effective for the purification of EPF.