Table 2.

Comparison of the methods used here to examine inhibition mechanism.

Technique	Enzyme Cone.	Information Content	Limitations
Equilibrium Dialysis	8 μM	Does inhibitor strip metal from the enzyme?	No direct evidence for ternary complex. Long dialyses may lead to false positive of metal stripping.
ITC	50 μM	What is the binding affinity (and associated thermodynamic parameters)?	Can yield binding parameters even for compounds that strip metal ions.
UV-visible	300 μM	Does MBL inhibitor binding affect the coordination number of the Co(II) ion(s)? Does MBL inhibitor binding change the electronic properties of the Co(II) ion(s). Does MBL inhibitor strip metal ions from MBL?	Requires non-native, Co(II)- substituted protein; requires large amounts of protein; relatively low-resolution information.
EPR	1 mM	Does MBL inhibitor binding affect the spin coupling between the Co(II) ions? Does MBL inhibitor strip metal ions from MBL?	Requires non-native, Co(II)- substituted protein; requires large amounts of protein; requires a non-standard piece of equipment in most research programs.
NMR	1 mM	Does MBL inhibitor binding change the positioning of metal binding ligands? Does MBL inhibitor strip metal ions from MBL?	Requires non-native, Co(II)- substituted protein; requires large amounts of protein;
Nano-ESI-MS	5 μM	Is a ternary (enzyme-metal-inhibitor) complex present in the sample?	Under most cases, technique is not quantitative; can only yield information on the presence of a ternary complex but not its concentration.
Computational	N/A	Does the MBL inhibitor bind? Technique can provide information about binding modes, potential active site interactions, and be used to interpret other data.	Technique requires a model of the enzyme, usually a crystal structure