Purification of the Constitutive Nitric Oxide Synthase

Constitutive nitric oxide synthase (NOS) exists as two isoforms: endothelial (e) and neuronal (n) (reviewed in
ref.1
). The first reports on the purification of eNOS and nNOS were by Schmidt et al. (
2
) and Bredt and Snyder (
3
), respectively; all subsequent reports have been modifications of these procedures. The purification of both of the constitutive isoforms and the inducible (i) NOS depends on the use of 2′,5′-adenosine diphosphate (ADP) affinity chromatography. The 2′,5′-ADP ligand is a structural mimic of the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) that is required for NOS activity. Therefore, the biospecific elution is accomplished by the presence of NADPH. The purification of both constitutive isoforms, unlike iNOS, also depends on a second affinity-chromatography step, calmodulin (CaM)-affinity chromatography. Exogenously-added CaM is essential for both purified eNOS and nNOS activity. In the presence of calcium, CaM binds both constitutive isoforms in a tight (K
d
∼ 10 n
M
) but reversible complex, thereby activating NOS. In contrast, CaM binds to iNOS in the absence of added calcium and dissociates only in the presence of a protein denaturant. Thus, during the 2′,5′-ADP-affinity step, CaM is removed from eNOS and nNOS, but not iNOS. The binding of eNOS and nNOS to the CaM resin is performed in the presence of calcium, and elution of NOS is achieved by the absence of calcium and presence of ethylenediaminetetraacetic acid (
EDTA
) and high salt. Depending on the source of enzyme, near homogenous NOS is often obtained by utilizing these two affinity steps.