Purification and Characterization of Equine Relaxin*

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Abstract
It has been previously determined that the equine placenta is the sole significant source of relaxin during pregnancy and that relaxin immunoactivity is also present in term placentas. Therefore, placentas obtained at the time of foaling were selected for starting material for purification of equine relaxin. Frozen whole placentas were ground and then extracted with 0.5 N HCl-85% acetone. Relaxin was precipitated by raising the acetone concentration to 97%. Equine relaxin was further purified by stepwise elution ion exchange, gel filtration, and gradient elution ion exchange chromatographies and trichloroacetic acid precipitation. Equine relaxin purified in this manner was shown to be heterogeneous with one major form (R-1) and several minor forms (two of which are identified as R-2 and R-3). About 1.5 mg R-1 were obtained per kg placenta. Purity was assessed by slab and disc gel electrophoresis and dansyl end group analysis. R-1 had a potency of 28 U/mg, as measured in the mouse interpubic ligament bioassay and displayed a dose-response curve parallel with porcine relaxin. Amino acid analysis indicated the presence of tyrosine, histadine, and proline, amino acids absent in porcine relaxin. Dansyl end group analysis indicated the blockage of N-terminal groups on R-1 and the presence of Lys on R-3. A lower molecular weight was indicated by the electrophoretic migration of relaxin reduced with 2-mercaptoethanol suggesting that equine relaxin consists of two chains.