Heterogeneity of human luteinizing hormone. Detection and identification of alpha- and beta-subunits in international reference preparations.

The charge heterogeneity of three international reference preparations containing biologically active human luteinizing hormone (LH) or its free alpha- or beta-subunits, respectively, was investigated with isoelectric focussing in sucrose density gradients. The immunoreactive profiles throughout the pH-gradient were assessed with radioimmunoassay (RIA) systems specific for intact, i.e. undissociated, LH or free alpha- or beta-subunits. For the preparation of intact LH (MRC 68/40), two peaks were found at pH = 7.69 and 8.05; for the alpha-subunit preparation (NIBSC 78/554), the peak values were 4.76, 5.04, 5.94, 6.70, 6.96, 7.35, 8.02, 8.72 and 9.32; and for the beta-subunit preparation (NIBSC 78/556), the values were 7.60, 8.40, 8.55 and 9.61. These results are in excellent agreement with those obtained for a highly purified LH preparation (NM14) which was prepared by one of us and on which we have reported earlier. In addition, with the above-mentioned techniques, the spontaneous dissociation of MRC 68/40 into subunits upon incubation at 37 degrees C in phosphate buffer was clearly demonstrated by the increased immunoreactivity in the profiles assessed with both RIA-subunit systems. It is concluded that charge heterogeneity of LHi, alpha- and beta-subunits, as observed for different preparations, is confined to a limited population of forms. Differences between preparations are only quantitative. A single preparation, therefore, can be used as a general model for the study of human luteinizing hormone.