Characterization of rat lutropin charge microheterogeneity using chromatofocusing

Chromatofocusing was utilized to separate rat lutropin isohormones. The pH gradients generated were highly reproducible, allowing accurate comparisons of isohormones in different elution profiles. Extracts of anterior pituitaries from intact male rats yielded at least seven species of immunoreactive lutropin after chromatofocusing. Five species exhibited apparent pI's in the range 8.97 to 9.25. Two additional peaks of rat lutropin were also observed: one in the void volume (pI greater than 9.8) and one which bound to the column but could be eluted with 1.0 M NaCl (pI less than 7.0). All seven lutropin isohormones were active in an in vitro bioassay. The biological-to-immunological (B:I) assay ratios were directly related to the apparent pI. The presence of both the basic and the acidic species of biologically active rat lutropin has not been previously observed with isoelectric focusing. Chromatofocusing should prove to be a valuable analytical tool in the isolation and characterization of gonadotropin isohormones.