Pattern of immunoreactivity after reverse-phase high performance liquid chromatography of ovine lutropin at different...

Unlabelled and radioiodinated ovine lutropin (oLH) was fractionated using reverse-phase high pressure liquid chromatography, employing a 5 micron Spherisorb C18 column. Following chromatography of unlabelled oLH at pH 3.3, 4.3 and 5.3, all eluted fractions were tested by radioimmunoassay of oLH, oLH alpha and oLH beta. Regardless of pH, the material corresponding to the first peak contained only alpha-subunit immunoreactivity. With increasing pH, the oLH alpha, oLH and oLH beta immunoreactivity separated into two groups of fractions. The retention times of the radioiodinated alpha- and beta-subunits of oLH corresponded to those of the first (descending part) and last peaks from unlabelled oLH, respectively. Following chromatography of radioiodinated oLH at pH 3.3, 5.3 and 6.5, all eluted fractions were analyzed using antiserum against either alpha- or beta-subunits of oLH. At pH 3.3 two radioactive peaks were detected: the first corresponded to the fraction with maximum binding to alpha-subunit antiserum, and the second corresponded to the fraction with maximum binding to beta-subunit antiserum. At pH 5.3 and 6.5, maximum radioactivity occurred in conjunction with maximum binding to both antisera. A free alpha-subunit in the radioiodinated oLH was even detected after chromatography at pH 6.5.