Ligand Binding in Allosteric Flavoproteins: Part 1. Quantitative Analysis of the Interaction with NAD + of the Apoptosis Inducing Factor (AIF) Harboring FAD in the Reduced State.

To perform their action, flavoproteins usually interact with a variety of low molecular weight partners, including electron transporters, yielding transient complexes whose tightness is often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe the quantitative analysis of the redox-dependent interaction of the mammalian apoptosis inducing factor (AIF) with its NAD ligand. In particular, we report a protocol for the spectrophotometric titration of AIF in its reduced state under anaerobic conditions with NAD, in order to determine the dissociation constant of the resulting complex.