Isolation of Isohormones of Human Prolactin from Amniotic Fluid

Abstract

Human prolactin (hPRL) with high immunological and biological activity was isolated from amniotic fluid as a mixture of isohormones. The isolation procedure consisted of: 1) gel filtration of a 50% sucrose concentrate of amniotic fluid on Sephadex G-50, with a resolving power of approximately 2,000 theoretical plates; 2) isoelectric focusing on a density gradient (IF) in the apparent isoelectric point (pI)-range 5–3, with a combined yield from steps 1) and 2) of 50%; 3) removal of the bulk of Ampholine and exchange to volatile buffer by gel filtration on Sephadex G-25, followed by lyophilization. All fractionation steps were monitored by radio immunoassay (REA) and analytical polyacrylamide gel electrophoresis (PAGE). Gel filtration on Sephadex G-50 yielded a peak of hPRL which comprised an ascending Limb characterized on IF by a pI' *5.2, a center with a pI' *5.7 and a descending limb with a pI' *6.2. HPRL with pI'* 6.2 fractionated upon PAGE (pH = 8.16, 0°) into 3 isohormones, designated as isohormones A, B and C. The hormone with a pI' *5.7 yielded, in addition to C, isohormone D with increased free mobility. isohormone C appeared to be a smaller molecular form (MW 27,000), while isohormones A, B and D were similar in molecular size at pH 8.16 (MW 36,000). The isolated hormone preparations were comprised of either hPRL A, R and C or hPRL C and D. Each preparation exhibited a dose response curve in RIA parallel to the curve obtained by human pituitary prolactin. Its bioactivity as determined by the local pigeon crop-sac bioassay was estimated at 45 IU/mg, of immunologically active pituitary hPRL (Lewis) relative to an ovine prolactin standard. The relative availability of amniotic fluid: (compared to human pituitaries) makes the preparation of hPRL from this source practical for any Laboratory.