- Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg(2+), as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg(2+) to an active site because Mg(2+) is spectroscopically silent and Mg(2+) binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg(2+) with Mn(2+):Mn(2+) that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn(2+) is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn(2+) that is free in solution and Mn(2+) bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.