Bovine thyrotropin has a specific bioactivity 5- to 10-fold that of previous estimates for highly purified hormone

Bovine TSH (bTSH) was subjected to reverse phase HPLC using a Synchropak RP-P C18 column. Elution was with a linear gradient of 0-50% 1-propranol in 0.1% trifluoroacetic acid. Individual fractions were assayed for TSH bioactivity by the cultured thyroid cell cAMP response assay. Optimum separation of biologically active TSH was obtained at a mobile phase pH of 5. Under these conditions, bTSH purified by conventional chromatographic techniques to 25 U/mg protein was purified further to approximately 200 U/mg protein. These data indicate that bioactivity is only present in a relatively small fraction of the molecules in a highly-purified TSH preparation. The implications of these findings are discussed.