[Isolation and purification of pregnancy-associated plasma protein A].
We tried to purify pregnancy-associated plasma protein A (PAPP-A) from normal term maternal serum in this study by means of a three step chromatographic procedure. At first 30 ml of maternal serum samples were applied to a column for sieve chromatography and eluted according to molecular weight. PAPP-A was detected in the high molecular weight fraction area by radioimmunoassay for PAPP-A, and 91% of the total amount of PAPP-A in the maternal samples was recovered. Secondary PAPP-A containing fractions were applied to a Heparin-Sepharose column and eluted by a stepwise increase in NaCl 0.15, 0.30 and 0.60 M in 0.05 M Tris-HCl buffer, pH 7.8. Ninety-three percent of PAPP-A in applied samples was recovered under the 0.6 M NaCl condition. Thirdly the pooled fractions which contained PAPP-A after Heparin-Sepharose affinity chromatography were applied to DEAE-Sephacel Chromatography and eluted by a stepwise increase in NaCl 0.15, 0.30 and 0.45 M in 0.01 M acetate buffer, pH 5.5. Ninety-one percent of PAPP-A in applied samples was recovered under the 0.3 M NaCl condition. Finally PAPP-A containing fractions were concentrated 10 times and 3.8 IU (1.7 mg) of PAPP-A was isolated. The purification schedule removed approximately 99% of total protein in the maternal serum while 74% of PAPP-A was recovered. The purification factor (fold), which was calculated as the increase in specific activity (mIU:PAPP-A/mg:protein) in comparison with the starting value in the maternal serum, was 526. And the purity (mg:PAPP-A/mg:protein) of the final product was 68.4%. Analysis of the final purified material by SDS-PAGE showed a single band of 200kDa, and western blot analysis showed that the main purified protein was PAPP-A. The immunological identity of the PAPP-A purified in this study to the PAPP-A donated by Teisner et al., was recognized by crossed immunoelectrophoresis and tandem crossed immunoelectrophoresis. We hope that the new PAPP-A purified in this study can be utilized for the development of a sensitive and convenient assay for PAPP-A and its standard material, and also for basic study to clarify the biological function of PAPP-A in the human body.
Suzuki
Y
Y
Department of Obstetrics and Gynecology, Tokyo Medical College.
Takada
J
J
Isaka
K
K
Takayama
M
M
Grudzinskas
J G
JG
D015153:Blotting, Western; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D007122:Immunoelectrophoresis; D007123:Immunoelectrophoresis, Two-Dimensional; D008970:Molecular Weight; D011247:Pregnancy; D011266:Pregnancy-Associated Plasma Protein-A / Q000302:isolation & purification*
D011266:Pregnancy-Associated Plasma Protein-A