Affinity Labeling with 4-Azidophthalimide (AzPI): Relation between Labeling Rate and Fluorescence Intensity

We recently developed 4-azidophthalimide (AzPI) as a compact fluorogenic photoreactive tag that can be attached to ligands to achieve selective fluorescence labeling of target proteins even in the presence of a large excess of non-target proteins. To further establish the utility of the AzPI tag, we focused here on streptavidin labeling with biotin-AzPI conjugates, and evaluated the relation between the amount of covalently labeled streptavidin (labeling rate) and fluorescence intensity. The labeling rate was proportional to the fluorescence intensity under standardized photo-irradiation conditions. Prolongation of the photo-irradiation time led to a marked increase in the labeling rate, but this was accompanied by a gradual decrease in the fluorescence intensity, which appeared to be due at least in part to photo-induced degradation of the target streptavidin. These findings should be helpful for achieving sensitive fluorescence detection of target proteins by using the AzPI tag.