Ultracentrifugal studies of human luteinizing hormone and its subunits: dependence on protein concentration and ionic...

Purified human pituitary luteinizing hormone (hLH) and its alpha and beta subunits have been examined by physical methods. Freshly prepared hLH showed three closely adjacent bands on electrophoresis in polyacrylamide gels under alkaline conditions, but on standing even in the freeze-dried condition additional bands appeared. alpha and beta subunits gave bands which were quite different from the main hLH bands but comparable with the additional bands, as well as traces of hLH bands. hLH was investigated at three ionic strengths (0.1, 0.2, 0.5) at pH 5.9 +/- 0.1. Sedimentation velocity experiments demonstrated a complex system of association-dissociation which was further investigated by sedimentation equilibrium. Association occurred at the higher protein concentrations at each ionic strength, but to a significantly higher level at I = 0.5. Only at I = 0.1, pH 5.8, was there a clear indication over a range of protein concentration of the occurrence of a species of molecular weight 32 000 +/- 2000, in fair agreement only with the sum of the molecular weights of the alpha and beta subunits. At higher ionic strengths, there were indications of dissociation at low protein concentration (less than 0.10 g/100 mL) as well as association at higher values (greater than 0.20 g/100 mL). In view of the occurrence of molecular weights less than 28 000 at I = 0.2, HLH was treated in terms of a monomer of molecular weight 14 000, and some evidence was obtained for tetramer formation (4M = M4). At higher ionic strength this model does not apply and it is thought that indefinite association may be occurring to some extent also. The alpha subunit gave indications of association from its sedimentation coefficient vs. concentration plot, and sedimentation equilibrium (at pH 5.9 I = 0.1) demonstrated molecular weights increasing with increasing concentration. Evidence for tetramer formation also was obtained. The beta subunit, in spite of an apparently simple sedimentation coefficient-concentration curve, showed molecular weights varying from well below 14 000 to beyond 20 000. There is evidence to suggest that the isolated alpha and beta subunits, even on standing as dry solid, are not stable but give rise to lower molecular weight products. Aged freeze-dried hLH did not show such impurity.