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

Abstract

Purified human pituitary luteinizing hormone (hLH) and its .alpha. and .beta. subunits were examined by physical methods. Freshly prepared hLH showed 3 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, and traces of hLH bands. hLH was investigated at 3 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 [I], 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 MW 32,000 .+-. 2000, in fair agreement only with the sum of the molecular weights of the .alpha. and .beta. subunits. At higher [I], there were indications of dissociation at low protein concentration (< 0.10 g/100 ml) and associated at higher values (0.20 g/100 ml). In view of the occurrence of MW less than 28,000 at I = 0.2 hLH was treated in terms of a monomer of MW 14,000, and some evidence was obtained for tetramer formation (4M [monomer] .dblarw. M4). At higher I this model does not apply, and 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, despite an apparently simple sedimentation coefficient-concentration curve, showed MW varying from well below 14,000 to beyond 20,000. The isolated .alpha. and .beta. subunits, even on standing as dry solid, are probably not stable but give rise to lower molecular weight products. Aged freeze-dried hLH did not show such impurity.