34] Isolation of the iron—sulfur-containing polypeptides of NADH: Oxidoreductase ubiquinone

Abstract

This chapter describes the methodology employed for the stepwise fragmentation of Complex I and isolation of subunits containing FeS clusters. Complex I is structurally composed of three distinct fragments, a water-soluble FeS-flavoprotein (FP), a water-soluble FeS-protein (IP), and a water-insoluble fraction containing phospholipids and hydrophobic polypeptides (HP). The resolution of Complex I into FP, IP, and HP is achieved in the presence of chaotropes that are also used for further fragmentation of FP and IP. Chaotropes destabilize membranes and increase the water solubility of hydrophobic proteins by lowering the transfer entropy of hydrophobic structures from a lipophilic surrounding into the destructured water of the aqueous phase. Improvements in the resolution of IP are required, as none of the resolved proteins is completely pure. The procedures are optimized for pH, the detergents used, and the concentrations of detergent, Na trichloroacetate (NaTCA), urea, and ammonium sulfate.