Quantitative and Qualitative Composition of Rat Placental Cryoextracts prior to and after Lyophilization

Abstract

The purpose of the study was to investigate a peptide composition of cryoextracts from rat placental tissue using a gel-penetrating chromatography prior to and after low temperature storage and lyophilization. Materials and methods. Cryoextracts were harvested from tissue homogenates of the rat placenta with a method of freezing (-20oC, 1 day), subsequent thawing and centrifugation. The following types of cryoextracts were analyzed: cryoextracts-1 – immediately after preparation; cryoextracts-2 – frozen down to -196oC and stored for 1 month at this temperature; cryoextracts-3 – subjected to cryosublimation after storage at -196oC and thawing. Separate fractions of cryoextracts were obtained with a method of gel chromatography using columns of 27x2 cm with a Sephadex G-100. Protein load was measured with a method of spectrophotometry. The quantitative and qualitative evaluation of a peptide content in the substances with molecular masses from 100 to 12,000 Da was done with the help of gel-penetrating chromatography with a column of 400x16 mm, filled with a polyvinyl gel. Results and discussion. The analysis of gel chromatograms showed the volume ratio of proteins with molecular masses from 20 to 150 kDa in placental cryoextracts to be 80.28%, while total concentration of protein equaled 23.81 ± 1.03 mg/ml. A portion of low molecular fractions with molecular masses from 12 to 4 kDa comprised 19.60%. The data, obtained by gel-penetrating chromatography, demonstrated the extracts to contain from 7 to 10–12 protein-peptide fractions with molecular masses from 447 to ≥12,000 kDa. The basic points were the peaks Pr with molecular masses ≥12000 Da, and also in the following groups: A (molecular masses from 7,500 to 2,000 Da), B (molecular masses from 1,413 Da), C (molecular masses from 888 to 949 Da), D (molecular masses from 706 to 694 Da), E (molecular masses of 846 Da), F (molecular masses of 556 Da) and H (molecular masses of 447 Da). The cryoextracts-1 were represented mainly by 5 fractions of the group A, while comprised 7.59% from the total volume of a cryoextract. The volume ratio of low molecular peptides with molecular masses from 1,249 to 706 Da was 20.82%. In cryoextracts, stored at -196oC (cryoextracts-2), the volume ratio of the group A fractions comprised 7.76%, while concentration of low molecular fraction was as high as 25.15%. In cryoextracts, subjected to sublimation (cryoextracts-3), 5 high molecular peptide fractions of the group A were missing. The total volume ratio of low molecular fraction was significant and comprised 24.97%. It contained 6 low molecular peptide fractions – peaks B, C, E, F, H with molecular masses from 1,873 to 447 Da. Conclusion. Cryoextracts, subjected to freeze-drying, demonstrated an increase in the volume ratio of both high molecular fractions and peptides with molecular masses up to 1,000 Da, which may be due to coagulation of proteins as well as their destruction in the process of evaporative drying. Freezing down to -196oC and lyophilization of cryoextracts resulted in a strong tendency to increase the content of low molecular compounds of a peptide nature