Affinity of ions to ADP, ATP
Contact: Dr. Christos Chinopoulos MD, PhD

The core of the methodology relies on the differential affinity of Mg2+ for ADP and ATP. It is thus important to dwell a bit on this.

Nucleotides and ions exhibit affinities to each other. This is depicted in the figure below:
The affinity constants between the nucleotides and ions are variable, and highly dependent on extraneous factors (pH, temperature, ionic strength, etc). Furthermore, the presence of multiple ions or nucleotides complicates the complexation (no pun intended) and chelation of the nucleotides with the ions.

Here, I will not describe the relation of complexes and chelations formation to extraneous factors. For this, the reader is referred to

I only wish to emphasize that the user of this method must keep the system free from fluctuations of extraneous factors, and make sure that the buffers are not contaminated with other nucleotides and/or ions. For example, please do not use creatine or creatine phosphate containing buffers and make sure that your chemicals are not contaminated with Ca2+, Ba2+, Mn2+, Sr2+ or other metals. Exact composition of the buffers is given under Buffers, chemicals.