Preparation of bio- and immunoactive human prolactin in milligram amounts from amniotic fluid in 60% yield.
Bio- and immunoactive human prolactin (hPRL) was isolated from amniotic fluid. The overall yield is a single passage through the procedure was 60%; 5 liters of amniotic fluid (10 mg hPRL) yielded 3 mg (30%) of a mixture of hPRL isohormones A, B (both minor), C and D (both major), and less than 10% contamination with extraneous protein. A further 3 mg hPRL was less highly purified. Two mg hPRL was recycled. The product has been stable for at least 1 year at--70 C, suitable for iodination, and gave rise to RIA dose-response curves indistinguishable from those obtained with pituitary hPRL. The isolated hormone, after stoichiometric iodination, is active in binding to both mammary gland and liver cell membrane receptors and exhibits a biological activity, in the local pigeon crop-sac assay, of 47.3 +/- 6.1 IU/mg. The isolation procedure consisted of 3 steps: 1) hollow-fiber dialysis to concentrate amniotic fluid; 2) gel filtration on Sephadex G-50 to remove bulk contaminants; 3)isoelectric focusing in a density gradient, taking advantage of the elevated isoelectric points, relative to contaminants, of hPRL isohormones (6.0-6.5, corrected). Production of this preparation, carried out on two separate occasions, required each time 3 months of a single investigator's time, inclusive of assay work.
D000653:Amniotic Fluid / Q000032:analysis*; D000818:Animals; D001681:Biological Assay; D002850:Chromatography, Gel; D010856:Columbidae; D003426:Crop, Avian / Q000187:drug effects; D004355:Drug Stability; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D008722:Methods; D011247:Pregnancy; D011388:Prolactin / Q000302:isolation & purification* / Q000494:pharmacology; D011863:Radioimmunoassay; D011869:Radioligand Assay
D011388:Prolactin