Purification of the Inducible Nitric Oxide Synthase

Regulation of the inducible nitiric oxide synthase (iNOS) isozyme is distinct from the constitutively expressed endothelial (e) and neuronal (n) NOS isozymes in that it occurs at the level of gene expression and not by Ca⁺² /calmodulin (CaM) (reviewed in ref. 1 ). These distinctions have direct bearing on the purification of iNOS. First, the source of iNOS is typically a cell line that can be induced to synthesize iNOS in response to one or more of a number of factors. Second, because iNOS binds CaM essentially irreversibly in the absence of Ca⁺² , the purification of iNOS does not include CaM-affinity chromatography. As is true for the constitutive NOS isozymes, the purification of iNOS depends on 2′,5′-adenosine diphosphate (ADP)-affinity chromatography. And because iNOS cannot bind CaM resins, purification of iNOS to homogeneity requires either an anionic-exchange step or gel-filtration step, or both (2 ,3 ).