Separation of the Gonadotropic Activity of Crude Choriogonadotropin from the Inhibitory Effect on Lymphocyte Transformation

Abstract

The effect of Choriogonadotropin of different purities on the transformation of peripheral human lymphocytes was studied. Various crude hormone batches inhibited lymphocyte transformation in a dose-dependent manner, both in the mixed lymphocyte reaction and in the phytohemagglutinin-induced stimulation. The inhibitory activity, however, was found not to be correlated with the gonadotropic activity of the crude hormone batches (2660-4300 lU/mg). Choriogonadotropin (13000 lU/mg), which was purified in 3 steps, showed no inhibitory effect except at high doses (> 5000 IU/m/ final dilution). More detailed investigations provided evidence that in the first step of the Choriogonadotropin purification procedure (batch adsorption of crude Choriogonadotropin on SP-Sephadex C-50), the inhibitory activity can be enriched in a fraction (Fract. I) which displays a very low gonadotropic activity (< 500 lU/mg). A further separation of Fract. I was achieved by isoelectric focusing as well as by chromatography on DEAESephadex A-25. By these means, the inhibitory potency could be enriched more than 100-fold. The substances which display inhibition of DNA synthesis in lymphocytes were proven to act in a nontoxic way. A preliminary characterization of the strongly inhibiting substances which show a dose-dependent suppression of lymphocyte transformation by about 99%, showed that this effect is probably exerted via non-dialysable sialoglycoproteins.By a fourth purification step entailing a chromatography of purified Choriogonadotropin (13000 lU/mg) on SP-Sephadex C-50, a highly purified Choriogonadotropin (14000 lU/mg) could be obtained which showed no inhibitory effect on lymphocyte transformation (in both mixed lymphocyte reaction and in phytohemagglutinin-induced stimulation) up to a dose of 43000 IU/m/. The components which were removed from Choriogonadotropin in this step seem to be immunologically identical with the strongly inhibiting substances isolated by isoelectric focusing.These investigations demonstrate that biologically active, highly purified Choriogonadotropin is unable to inhibit lymphocyte transformation. The inhibitory activity of crude hormone can be enriched in choriogonadotropin-free fractions. Therefore, it is concluded that the inhibitory activity of crude hormone is not a property of Choriogonadotropin itself.

Effects of Crude and Purified Human Chorionic Gonadotropin on Lymphocyte Response

“…083. As demonstrated elsewhere, we were able to enrich the inhibiting principle of crude hCG in a fraction (fraction I) with merely insignificant gonadotropic activity (Merz et al, 1979). Fraction I is com posed of several acidic substances.…”

“…In a previous study, we were able to show that various commercially available crude hCG preparations have displayed different inhibitory effects on lymphocyte transformation, irrespective of their gonadotropic activity (Merz et al, 1979) The data in table I show that crude hCG has a dosedependent inhibitory effect on lymphocyte reaction. However, highly puri- fted preparations of hCG have lost their inhibitory activity.…”

“…Highly purified hCG preparations (four purification steps) yielded no inhibition of lymphocyte transformation at concentrations 10,000 IU/mg, whereas a hormone preparation purified in three steps showed an inhibitory activity (Merz et al, 1979). In a previous investiga tion, we were able to demonstrate that the inhibiting principle of crude hormone can be enriched in fractions free of hCG (Merz et al, 1979). The major components of fraction I are additionally shown to be immunologically nonidentical with hCG as could be demonstrated by means of the double immunodiffusion test (see fig.…”

“…Gunden et al (1975) and thereafter Caldwell et al (1975) adduced inhibition of lymphocyte reaction to be due to a contaminanl in crude hCG preparations. We have been able to separate the inhibitory activity from commercial crude hCG batches (Merz et al, 1976(Merz et al, , 1979. In the present study, the inhibitory effect of crude chorionic gonadotropin on lymphocyte blastogen-esis after mitogenic (PHA, phytohemagglutinin) and allogenic stimulation (MLR, mixed lymphocyte reaction) is investigated.…”

“…Further purification is achieved in a fourth step (chromatography on SP Sephadex C 50). This procedure is described elsewhere (Merz et al, 1979). Biological activity of hCG was determined by means of the rat prostate assay (Merz et al, 1974).…”