Preparation of Bio- and Immunoactive Human Prolactin in Milligram Amounts from Amniotic Fluid in 60% Yield1

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Abstract
Bio- and immunoactive human prolactin (hPRL) was isolated from amniotic fluid. The overall yield is a single passage through the procedure was 60%; 5 l 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 was stable for at least 1 yr at -70.degree. C, suitable for iodination, and gave rise to RIA [radioimmunoassay] 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: hollow-fiber dialysis to concentrate amniotic fluid; gel filtration on Sephadex G-50 to remove bulk contaminants; 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 2 separate occasions, required each time 3 mo. of a single investigator''s time, inclusive of assay work.