Preparation of Rat Growth Hormone

Abstract

WE have devised a simple method for the preparation of rat growth hormone from fresh frozen anterior pituitary glands. The hormone preparation was obtained in high yield and showed good biological activity while being essentially free of contamination by other pituitary hormones. Disk electrophoresis on polyacrylamide gels indicated the hormone to be relatively homogeneous. In a typical experiment 300 rat pituitary glands were homogenized for 1 min in 4 ml. of ice cold 0.3 M potassium chloride using a glass homogenizer with a ‘Teflon’ pestle. The homogenate was adjusted to pH 5.5 and stirred for 20 min at 5° C and afterwards centrifuged for 10 min at 14,000 r.p.m. The resultant supernatant was brought to an ethanol concentration of 30 per cent at −20° C and immediately centrifuged at 14,000 r.p.m. for 5 min. Additional growth hormone (10–12 mg) was recovered by extracting the residue in 0.3 M potassium chloride at pH 10. However, inconsistent biological activity has been obtained with the material so extracted. The ethanol supernatant was then dialysed, lyophilized and when assayed was found to contain the gonadotropic hormones. The ethanol precipitate was taken up in 1 ml. of tris-hydrochloric acid buffer, pH 8.6 (0.2 M tris, 0.05 M hydrochloric acid), and dialysed against four 500 ml. changes of this same buffer for a period of 4 h at 5° C. The dialysed solution was applied to a block of ‘Pevikon C-870’ (92 cm × 5 cm × 0.5 cm) equilibrated with tris-hydrochloric acid buffer, pH 8.6. A current of 400 V and 80 m.amp was applied for 18 h. After the completion of electrophoresis, the ‘Pevikon’ block was divided into 2-cm segments and each individual fraction was eluted with three 5-ml. portions of buffer using small sintered glass funnels and suction. ‘Pevikon’ particles, which passed through the filter, were removed by centrifugation. The equipment as well as the procedure used for zone electrophoresis was similar to that described by Müller-Eberhard¹. The effluent diagram of a typical zone electrophoresis is shown in Fig. 1. Protein concentration was estimated by measuring absorption at 280 mµ The effluent portions were concentrated by partial lyophilization and combined into Fractions A, B and C after analysing individual eluates by disk electrophoresis. Each of the combined fractions was lyophilized and then desalted by gel-filtration on ‘Sephadex G-25’. Fraction A was found to contain from 20–25 mg of rat growth hormone. Fraction B (10–12 mg protein) contained some growth hormone, but was also found to be contaminated with Fraction C. This latter fraction, yielding 8–10 mg of protein, demonstrated no growth hormone activity. Zone electrophoresis of Fraction B resulted in the recovery of an additional quantity (5–7 mg) of rat growth hormone. Zone electrophoresis studies have also been carried out at pH 10 (carbonate Γ/2 = 0.1) with no improvement in resolution. It was noticed that additional electrophoretic components were formed on exposure to pH 10 as compared with the electrophoretic pattern which resulted from isolation at pH 8.6. The yield and biological profile of rat growth hormone (Fraction A] is summarized in Table 1. Of particular interest is the absence of significant prolactin activity.