Purification and physico-chemical characterization of the antigonadotropic fractions obtained by ultrafiltration from the pineal extract.
In order to isolate the antigonadotropic factor from a pineal extract prepared from bovine pineals the authors first applied ultrafiltration, which yielded three fractions: F1 greater than 10 kDa, 1 kDa less than less than F2 less than 10 kDa, and F3 less than 1 kDa. Examination of the biologic activity showed that only F1 and F2 have a statistically significant antigonadotropic activity. Ion exchange chromatography of F1 yielded two subfractions: one, F11, which was not retained, and another one, F12, which was retained by the exchanger. Both subfractions showed biologic activity. While F11 is closer to F2 less than 10 kDa, F12 has a molecular weight around 13.7 kDa, as resulted after applying the SDS-PAGE procedure. The F1 fraction has a proteic content of 903 micrograms/ml, F11 = 308 micrograms/ml and F12 = 247 micrograms/ml. Amino acids assay showed that the F12 subfraction has the same composition as F1 and the F11 subfraction also has free amino acids which indicates a process of adsorption of small molecules on large molecules. The F2 less than 10 kDa fraction, biologically active, has a proteic content of 433 micrograms/ml and an amino acids composition similar to F11 except for cysteine and methionine. The F3 less than 1 kDa fraction with a proteic content of 79 micrograms/ml having an amino acid composition in which prevails the glutamic acid shows poor antigonadotropic activity. The results of this study, as well as the fact that the antigonadotropic activity is concentrated in fractions both above and under 10 kDa, suggests at least two hypotheses: either that the pineal antigonadotropic factor exists under various molecular forms in which the activity is preserved, or that the antigonadotropic factor is a peptide with strong aggregation properties as demonstrated in our study.