Formation of Biologically Active 125I-Prolactin by Enzymatic Radioiodination

A method is described for the preparation of I-prolactin that is biologically active. Ovine prolactin was radioiodinated with lactoperoxidase to yield I-prolactin that had SA of approximately 130 nCi/M-g, and that retained 60% of the biological activity of the native hormone as determined by in vitro assay. Biological activity of the I-prolactin prepared by this method was further demonstrated by hormone-specific, saturable binding to particles prepared from mammary gland but not from several other mouse organs. (Endocrinology 91: 1545, 1972) INVESTIGATION of hormonal binding to -*• specific cellular receptor sites requires a radiolabeled hormone with high enough SA to be detected at the picomolar concentrations which, correspond to physiological levels. However, radioiodination reactions may alter those chemical properties of polypeptide hormones which determine the high affinity and high degree of specificity with which such hormones interact with binding sites in their target cells. Radioiodination of polypeptide hormones generally utilizes chloramine T to oxidize the iodide (1). Prolactin iodinated by this method retains a high degree of immunoreactive specificity (24); however, we have observed that the biological activity of the iodinated hormone preparation, as determined by criteria described below, is nearly completely lost. Therefore, we have adapted the enzymatic method developed by Marchalonis (5) for iodination of immunoglobulins in order to prepare biologically active Iprolactin. This method yields I-prolactin with high specific activity and high biological Received April 28, 1972. 1 Present address: Department of Physiology, Michigan State University, East Lansing, Michigan 48823. activity, as indicated by specific and saturable binding to mouse mammary epithelial cell particles and by a specific in vitro bioassay technique. Materials and Methods Iodination of prolactin. Ovine prolactin (P-S-9, Endocrinology Study Section, NIH) was iodinated by the chloramine T method as described by Kwa, et al. (2). The iodination reaction was carried out for periods varying between 20 and 60 sec, and was terminated by the addition of stoichiometric amounts of sodium metabisulphite. The iodinated prolactin was separated from other reactants by gel nitration on Sephadex G75, and was purified further by gradient chromatography as described below. For iodination of prolaction by the lactoperoxidase method, carrier-free Na I (New England Nuclear Corporation), ovine prolactin, lactoperoxidase (CalBiochem., Los Angeles, Lot No. 00612), and dilute hydrogen peroxide (Mallinkrodt, St. Louis) were allowed to react in 40mM sodium barbital buffer, pH 7.0 (see Table 1). These reagents were drawn into polyethylene 50 tubing in 25-nl aliquots, each separated by an air space, and then ejected into the I solution to initiate the iodination reaction. After 5 or 10 min a second aliquot of H2O2 was injected for a second activation period equal to the first. The reaction was terminated by washing onto a Sephadex G75 column with 1 ml of 2% bovine serum albumin and 1 ml of IM sucrose solution. The single peak of iodinated 1546 NOTES AND COMMENTS Endo Vol91 1972 No 6