Interaction of azotobactin with blocking and mobilizing agents in NTBI assay

Accurate estimation of non-transferrin bound iron (NTBI) is an important tool in monitoring effects of chemotherapy and iron chelation therapy in various conditions of iron overload and transfusion related thalassemias. A key factor in its estimation, is its extraction from putative ligands in the serum, barring transferrin and ferritin and blocking of unsaturated binding sites of the same. The molecular interactions between azotobactin and blocking agents Co(3+) and Ga(3+) were studied by UV/visible spectrophotometry. The role of different mobilizing agents in modulating Fe(3+) binding to Azotobactin was monitored with fluorescence emission studies. The fluorescence spectrum of Azotobactin is Exc(lambda) 380 nm/Em(lambda) 490 nm. In the presence of Ga(3+), the emission peak underwent a blue shift to 465 nm with a significant decrease in the intensity, whereas, Co(3+) did not show any shift or decrease in the fluorescence emission spectrum. With the addition of EDTA to the azotobactin-Fe (Az-Fe) complex, there is an immediate regain in the fluorescence of azotobactin whereas addition of nitrilotriacetate (NTA) did not show any regain in the fluorescence. Results illustrate that the citrate complex of cobalt and NTA are suitable blocking and mobilizing agents in the azotobactin assay of NTBI in biological fluids like human serum, since they do not affect either the spectroscopic properties of azotobactin or the binding kinetics of azotobactin and Fe(3+).