Conformational studies on lipoamide dehydrogenase from pig heart. 3. The binding of sodium dodecylsulphate.

Abstract

Lipoamide dehydrogenase binds sodium dodecylsulphate in its dimeric state at 25 °C in two phases. The first phase, 0–0.3 g/g protein, is bound purely to hydrophobic sites; the second binding phase, 0.3–1.4 g/g protein is less well characterized and is influenced by the ionic strength and the dissociation of a protonated group with pK 6.6. The effects can be reversed by cooling to 0 °C. The spectral and kinetic parameters are only affected during the second binding phase, maximal effects are obtained, with the exception of fluorescence parameters, at 1.4 g/g protein. The changes in the absorption spectrum indicate that different parts of the flavin absorption spectrum are influenced by unequal dodecylsulphate concentrations. Fluorescence polarisation shows that FAD becomes less tightly bound. Upon prolonged incubation it dissociates. Binding of dodecylsulphate has a large influence on the hydrodynamic parameters. Viscosity measurements indicate the existence of asymmetrical molecules; the sedimentation constant declines from 5.8 S to 2.6 S after an increase to 8.9 S. The results show that a dimeric dodecylsulphate · enzyme complex can be reactivated by removal of dodecylsulphate at 0 °C, the monomeric dodecylsulphate · protein complex remains inactive.