Characterization of the metal-binding site of human prolactin by site-specific metal-catalyzed oxidation

Site-specific metal-catalyzed oxidation (MCO) was applied to characterize the metal-binding site (MBS) of recombinant human prolactin (hPRL), which belongs to the hematopoietic cytokine family. Copper and ascorbate of various concentrations were used to initiate the oxidation of hPRL, and the oxidation-sensitive motifs were characterized and quantitated by mass spectrometry. Based on the results obtained with 10 μM Cu2+ and 0.3–2.0 mM ascorbate, we propose that the MBS in hPRL is composed of His27, His30, and His173. This result shows the similarity of hPRL to human growth hormone (hGH), a member of the same family as hPRL, where the MBS is composed of His18, His21, and Glu174.