THE GLYCOSYLATION AS SOURCE OF THE VARIABILITY IN PROLACTIN PATTERNS OF INDIVIDUAL HUMAN AMNIOTIC FLUIDS

Whilst it is known that prolactin has size heterogeneity in human amni otic fluid, no studies exist concerning the heterogeneity between indi vidual amniotic fluids and the molecular properties of the prolactin f orms which are connected with such a variability. To elecit this, prol actin forms from amniotic fluid taken from 6 full-term deliveries and processed separately, were fractionated by ConA-Sepharose, and charact erized by SDS-PAGE, immunoblotting, and glycan detection of blotted pr olactins. Recording the absorbance at 280 nm, dimers in the wash of th e ConA-Sepharose column and in the elution with methyl-alpha-D-glucosi de were evident in 3 fluids. The proteins of the chromatographically m ore rapid and retarded migrating fractions, generating the first dimer , of unbound material consisted on average of 74-83% and 85-90% of pro lactin forms. The eluted fractions (bound material) contained 10-27% o f the prolactin amount of the unbound fractions. The electropherograms using 12.5% gels indicated identical patterns of bands in repeated ex periments per fluid but variability between the charges. Thus, the app arent molecular masses (kDa) and the proportions to the total prolacti n (%) were 14.5-16 (8-12%), 20-23 (3-5%), 24 (4-8%), 25 (6-10 %), 27 ( 19-25%), 28-29 (6-9%), 39-43 (0.5-1.0%), 60-64 (12-19%), 87-90 (8-11%) and 117-140 (2-7%) in nonreducing electrophoretical conditions. Using the glycan moiety of transferrin as reference in the calculation of t he glycan rate, the glycosylation degrees in the bands mentioned above were (ng glycan/mug protein) 80-140, 0, 55-70, 20-25, 20-35, 400-600, 250-350, 80-110, 90-140, suggesting that the glycan moiety varied in the prolactin isoforms and that dissimilarities in the individual prol actin patterns may be attributed in part to differences in the glycosy lation degree.