Evgeniya E. Burkova1, Pavel S. Dmitrenok2, Sergey E. Sedykh1, Valentina N. Buneva1,3, Svetlana E. Soboleva1, and Georgy A. Nevinsky1,3
- SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
- Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, Vladivostok, Russia
- Novosibirsk State University, Novosibirsk, Russia
Human placenta is an organ which protects, feeds, and regulates the grooving of the embryo. Therefore, identification and characterization of placental components including proteins and their multi-protein complexes is an important step to understanding the placenta function. We have obtained and analyzed for the first time an extremely stable multi-protein complex (SPC, ∼1000 kDa) from the soluble fraction of three human placentas. By gel filtration on Sepharose-4B, the SPC was well separated from other proteins of the placenta extract. Light scattering measurements and gel filtration showed that the SPC is stable in the presence of NaCl, MgCl2, acetonitrile, guanidinium chloride, and Triton in high concentrations, but dissociates efficiently in the presence of 8 M urea, 50 mM EDTA, and 0.5 M NaCl. Such a stable complex is unlikely to be a casual associate of different proteins. According to SDS-PAGE and MALDI mass spectrometry data, this complex contains many major glycosylated proteins with low and moderate molecular masses (MMs) 4–14 kDa and several moderately abundant (79.3, 68.5, 52.8, and 27.2 kDa) as well as minor proteins with higher MMs. The SPC treatment with dithiothreitol led to a disappearance of some protein bands and revealed proteins with lower MMs. The SPCs from three placentas efficiently hydrolyzed plasmid supercoiled DNA with comparable rates and possess at least two DNA-binding sites with different affinities for a 12-mer oligonucleotide. Progress in study of placental protein complexes can promote understanding of their biological functions.
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Placenta samples were obtained from healthy mothers and then cut into small pieces before being washed with buffer (20 mM Tris-HCl pH 7.5, 125 mM KCl, 0.5 mM EDTA-NaOH pH 7.5, 0.5% sodium citrate) in order to remove blood. The washed pieces were then homogenized in cold buffer (4 °C; 425 ml, 250 mM sucrose, 20 mM Tris-HCl (pH 7.5), 125 mM KCl, 20 mM MgCl2, 0.5 mM Na-EDTA (pH 7.5), 0.5% sodium citrate) before being centrifuged for 30 minutes at 13000 rpm. The pellet of insoluble complexes was removed and the placenta soluble components extract was diluted twice against distilled water before being diluted again against a buffer (50 mM Tris-HCl (pH 7.5), 0.1 mM NaCl). The extract was concentrated to 0.5 ml through air flow in the dialysis bag at 4 °C before undergoing gel filtration and elution with TBS buffer. Fractions of the SPC (MM of 10-15+ kDa) were collected. 1 μl of the reaction mixture was mixed with 2 μl of 0.2% trifluoroacetic acid and 2 μl of matrix (saturated solution of sinapic acid in 0.1% acetonitrile and trifluoroacetic acid (1:2)). 1 μl of the final mixture was spotted on a MALDI plate and air dried before being analyzed using a MALDI-TOF mass spectrometer that had been modified with a CovalX HM1 detection system.