The intracellular pathways of zinc trafficking leading to the formation of native Zn-proteins are poorly understood. Maret et al (J Biol Inorg Chem (2006) 11, 1049-62) employing the colorimetric reagent for Zn2+, Zincon (ZI), reported the existence of adventitious protein binding sites for Zn2+. This result suggested that proteomic binding sites may compete for intracellular Zn2+ or act as an intermediate along the pathway to constitute Zn-proteins. Our study focused on the possibility that ZI forms ternary adducts with Zn2+ and proteomic binding sites. The reaction of proteome (LLC-PK1 cells) with Zn2+ and ZI (Kd ~ 10 -5 M) yielded a biphasic titration curve. Initially, the sensor did not compete for added Zn2+ with the proteome’s higher affinity Zn2+ binding sites, which amounted to four times the concentration of native Zn-proteins in the sample. This was followed by an increase in absorbance which was red-shifted relative to Zn-ZI. It was hypothesized that proteome-Zn-ZI adducts had formed. In agreement, the Zn-ZI species chromatographed with the proteome, whereas ZI displayed little affinity for protein. Preincubation of the proteome with N-ethyl-maleimide abolished the first phase of the titration without affecting the second step, showing that the high affinity proteomic binding sites involved sulfhydryl ligands. Bovine serum albumin (BSA) served as a model to test the above hypothesis. Like proteome, BSA bound Zn-ZI but not ZI. The product absorbance spectrum was also red-shifted. Its maximum stoichiometry was BSA(Zn-ZI)8, consistent with the detection of eight binding sites for Zn2+ on BSA. Finally, the reaction between Zn-ZI and TSQ was studied; a fluorescence emission spectrum was observed at 470 nm, indicative of the formation of TSQ-Zn-ZI. Together, these findings indicated that Zincon forms ternary adducts when reacted with proteome and Zn2+.

Probing proteomic zinc binding with Zincon

256th National Meeting (2018)

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