All solutions were prepared in Nanopure or glass distilled water and extracted with 1% Dithiozone in Chloroform. Metal free glassware, pipettes, and cuvettes were used through out the experiment. Buffers used in this study were 0.2M sodium biphosphate (Malliockrodt), pH 7.0, 0.2M sodium biphosphate with 0.1M pyridine-2,6-dicarboxylic acid, pH 7.0, and 0.05M Tris, pH 7.5. The concentration of the stock solution of carbonic anhydrase (Sigma) was 10.0mg/ml in the Tris, pH 7.5. The concentration of the stock substrate solution (p-nitrophenyl acetate) was 5X10-2M (Aldrich) in 100% acetonitrile (Optima).

 Preparation of Apo Enzyme

Half a milliliter of the stock solution of CA was transferred to 10-12,000 KDA dialysis tubing and dialyzed against 250 milliliters of 0.1 M sodium phosphate, 0.1 M dipicolinic acid, pH 7.5. Enzyme dialyzed against buffer alone served as a control for determining the extent of apo-enzyme formation. The enzyme was dialyzed for three hours and the buffer solutions were changed after each hour. Each dialyzed CA solution was passed through a G-10 size exclusion columns that were first washed with EDTA, and equilibrated with metal free Tris buffer. The enzyme was found to elute from the column in drops 70-120.

Enzyme Assay

Kinetic analysis of CA activity was assayed using a UV 160U Shimadzu spectrophotometer at 400nm. The reaction cocktail consisted 980µl of Tris Buffer, 20µl p-NPA, and 50µl enzyme (either apo- or holo-). Blank cuvettes contained 1.3ml of Tris buffer and 20µl p-NPA. The change in A400 was monitored at 30 second intervals over a period of 30 min. The protein concentration of each fraction was determined using a standard Coomassie Blue assay.