Purification and Properties of Rat Prolactin

Abstract

Prolactin was isolated from fresh, whole rat pituitary glands by the following procedure: 1) sequential extraction to remove other hormones, 2) extraction of prolactin with 60% ethanol at pH 9.5 and precipitation with 80% ethanol at pH 6, 3) filtration on Sephadex G-100 at pH 9, and 4) chromatography on DEAE-cellulose with gradient elution at pH 8. The yield was about 1 mg/g of glands at a potency of 28 IU/mg by the local crop assay and 11 IU by the systemic assay. The preparation was 95% homogeneous by electrofocusing in 2M urea and showed a pI =5.46. Analysis by sedimentationequilibrium gave a mol wt of 21,600. Best- analysis of the amino acid composition gave mol wt of 22,500; only 2 residues of methionine were present. Leucine constituted the amino terminus. Immunodiffusion showed that antiserum to rat prolactin cross-reacted strongly with hamster and weakly with mouse pituitary extracts, but not with extracts from pigeon, pig, or guinea pig. Complement fixation was absent when antiserum to rat prolactin was tested against bovine, ovine and rabbit prolactin, well as human and rat growth hormone. (Endocrinology85: 886, 1969)