A new radiochemical method to determine the stability constants of metal chelates attached to a protein

A new method was developed to determine the stability constants of bifunctional chelates of indium (In) coupled to a protein. This method utilizes the displacement reaction between an indium complex and ferric ion. By measuring the position equilibrium constant 'K' of this reaction and knowing the stability constant of the corresponding ferric chelate, the overall formation constant of the indium chelate can be determined. Human serum albumin (HSA) was conjugated with ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) using their cyclic dianhydrides. A new method was developed to couple triethylenetetraminehexaacetic acid (TTHA) with HSA using Woodward's Reagent K. The chelating agents coupled to HSA were complexed with indium-114m-(114mIn) labeled indium and purified by dialysis or microcentrifugation. The stability constants of these indium complexes were determined at physiologic pH using ferric chelate of nitrilotriacetic acid (Fe-NTA) as the source of ferric ion. No significant differences were found between the stability constants of the indium chelates conjugated to protein and those of unconjugated species.