Über den einfluss von harnstoff auf die molekulare struktur von lactat-dehydrogenase aus dem schweineherz

Abstract

The physico-chemical properties of lactate dehydrogenase from pig heart were investigated in a range of o−8 M urea in order to elucidate the problem of dissociation of DPN-dehydrogenases into subunits due to the action of urea. Native lactate dehydrogenase has a molecular weight of 115000 ± 6500. Increasing concentration of urea leads to a decrease of the sedimentation coefficient ( s) and diffusion constant ( D) accompanied with an increase in viscosity as well as levorotation and to a rapid decrease of the constant of optical rotatory dispersion ( λ c ). In the range of small urea concentration, aggregation occurs as a result of intermolecular hydrogen-bonds. This process is reversed above an urea concentration of 3 M. In about 6 M urea, s and D reach a constant value approximately corresponding to the molecular weight of the native enzyme; λ c approaches the value characteristics for the random coil. The anisotropy of the molecule increases and leads via a maximum (“opening” of the molecule, less than 3 M urea) to a stationary value (random coil). The structural changes are partially reversible after removal of the urea; the enzymic activity is irreversibly lost. The behaviour may be interpreted as a consequence of electrostatic interactions between the groups with equal charges of the lactate dehydrogenase molecule which is highly charged at about pH7. This explanation appears justified since the iufluence of charge (pH) in absence of urea leads to a corresponding effect. The assumption of a dissociation process into subunits proposed by a number of authors for the interpretation of analogous investigations will not result in an explanation completely free from contradictions.