Preparation of Biologically Active 125 I LH-RH Suitable for Membrane-Binding Studies

Since the availability of the synthetic decapeptide, LH-RH, several groups of investigators have produced iodine-labeled hormone for immunological studies. Both the chloramin-T (1-3) and the lactoperoxidase iodination methods have been used (3). However, few data are available on the biological activity of the iodinated product, or on the contamination of ¹²⁵I LH-RH with unlabeled hormone. In 1973, a group (3) reported that the ¹²⁵I LH-RH possessed 13% of the bioactivity of the native hormone, but did not comment on possible contamination with unlabeled hormone. Again in 1973, another group (4) produced bioactive ¹²⁵I LH-RH using a lactoperoxidase technique, and were able to separate labeled from unlabeled hormone by means of polyacrylamide gel electrophoresis.

The aim of the present study was to prepare biologically active ¹²⁵I LH-RH free of carrier hormone which would be suitable for both immunological and pituitary cell membrane-binding studies.

Materials and Methods. Preparation of ¹²⁵I LH-RH. The iodination system was based on the lactoperoxidase method using a hydrogen peroxide generating system described by Miyachi et al. (4). Reagents were added to the iodination vial in the following order: 15 μl 0.01 M PBS (0.01 M phosphate buffer, 0.15 M saline, pH 7.5); 5 μg synthetic LH-RH (Abbott) in 5 μl of 0.01 M PBS; 1.5 mCi of ¹²⁵I (Industrial Nuclear); Lactoperoxidase (Sigma) 50 ng in 10 μl of 0.1 M sodium acetate (pH 5.6); 5 μl glucose oxidase (Miles); 25 μl of 0.1% glucose. All reagents were at room temperature (21°C) when added except for the glucose oxidase (4°C). The reaction was allowed to continue for 1 1/2 min at room temperature, mixing being achieved by finger-flicking. Then, 0.5 ml of 0.01 M phosphate (pH 7.5) was added, and the iodination mixture transferred to 200 mg of Dowex I × 10 (200-400 mesh) in 0.5 ml of 0.01 M phosphate to remove excess unreacted iodine. This was then mixed for 10 sec and centrifuged.