The effects of calcium ion-chelating ligand (EDTA), heat, and enzyme concentration on the stability of a metalloenzyme (R-amylase, from Bacillus sources) solution have been examined by experimental as well as theoretical studies. A simple two-stage inactivation model has been presented that explicitly includes the calcium ion concentration and can explain all experimental results. The first stage involves a reversible inactivation process caused by the dissociation of a metal ion (Ca 2+ ) from the active enzyme molecule, followed by the second stage of inactivation in which the apoenzyme (protein without the metal) undergoes an irreversible thermal inactivation (denaturing). These studies also suggest that the diluted enzyme solution is more prone to inactivation than the concentrated enzyme solution.

According to a previous report, only the smaller anions, like chlorides,that readily fit into the anion binding site of alpha-amylase can cause an increased stability (relative to enzymes in aqueous solution), and the anions that are too large to fit into the binding site should have no effect on the enzyme. Even though the results on large benzoate ions are consistent with the above postulate, much larger citrate ions from sodium and potassium citrate show stabilization at moderate salt concentrations and follow an expected trend of low stability only at large salt concentrations. The citrate ions from ammonium citrate exhibit very little to almost no stabilization. In addition, low to moderate concentrations of NaCl that provide a large stability to the enzyme show almost no stability in the presence of EDTA. We put forward an inactivation model that involves a reversible dissociation of the anion bound to the protein, followed by a reversible inactivation step of calcium ion dissociation and an irreversible denaturation step of apoenzyme.

The iodine binding capacity (IBC) of glycogen is around 0.30% (w/w) at 3°C. The amount of iodine complexed comprises about 12.5% of the mass of glycogen that takes part in the glycogen–iodine (GI) complex formation. This suggests involvement of four iodine atoms for every 25 anhydroglucose units (AGU, C6H10O5). Since the chromophore is due to the I4 unit within the helix of 11 AGUs, only 44% of the AGUs (11 out of 25) are involved in the complex formation. The heat of formation of the GI complex is around −40 kJ/mol of I2 bonded. These results suggest remarkable similarities with those of the amylopectin–iodine (API) complex. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 1409–1412, 1997

Glycogen–iodine (GI) complex formation has been studied at different concentrations of iodine and glycogen. For each glycogen concentration (0.25, 0.125, 0.0625, 0.0313 g/L), the iodine concentration was varied from 0.0317 to 1.59 g/L and the absorbance readings were taken at 453 and 560 nm (GI wavelengths of maximum absorbance). The 453 nm absorbance curves for the GI solution (GI complex and unreacted iodine), and that of the pure iodine solution (without glycogen) level off at a high iodine concentration, and give a peak in the subtracted curve. The 560 nm curves consistently increase in absorbance, and no peak is noticed in the subtracted curve. The spectra of concentrated iodine solutions in water and alcohol suggest the formation of neutral iodine clusters. We suggest that these iodine clusters do not react with glycogen, and that the GI complex formation takes place by the addition of I2 molecules. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 927–931, 1997