Abstract
With the exception of calcium very little is known about metal binding characteristics of either human salivary or porcine pancreatic amylase. In order to learn more about these protein-metal binding interactions, calcium-free human salivary and porcine pancreatic amylase [P(protein)] were obtained by carboxymethylcellulose chromatography of the partially purified proteins. Because these proteins acquired small amounts of calcium after further preparatory studies, they were dialyzed against 1 mM EDTA, pH 7.4, at 22 degrees C, which removed essentially all acquired calcium. The calcium-free amylases were then subjected to equilibrium dialysis against copper or zinc solutions with or without added glycine. The experimental data were fitted to appropriate mathematical equations, and binding constants of the metal complexes were calculated. Both human salivary and porcine pancreatic amylase were found to have two metal ion binding sites, only one of which was selective for calcium. Copper or zinc appeared to bind to the second site forming the species CuCaLP (or ZnCaP), where L, a ligand, is the glycine anion. Neither copper nor zinc displaced calcium from human salivary amylase, although copper bound to both binding sites in human salivary apoamylase to form the species Cu2L2P in which the amylase molecule appeared to form a bridge between the two copper atoms. In the case of the zinc-human salivary apoamylase system, the experimental data could not be analyzed quantitatively since the protein formed an insoluble complex species. Copper displaced calcium from porcine pancreatic amylase and formed a mixed ligand species similar to that formed with human salivary apoamylase. Zinc bound to both metal binding sites of porcine pancreatic apoamylase, forming species ZnP and Zn2P, although it did not displace calcium from the protein. While calcium in amylase is known to be critical for its amylolytic activity, little is known about the function of either zinc or copper in amylase albeit both of these metals are important in biological systems.
Highlights
With the exception of calcium very little is known and bound and could not be removed by about metal binding characteristics of either human dialysis against chelating agents [2]
Metal salt solutions (0.8 ml) of different concentrations were introduced by a syringe into the half-cells (Al-5) on one side of the dialysis apparatus, and a protein solution of known concentration was introduced into the opposite half-cells (B1-5).The openings used for introducing solutions were sealed with parafilm, and solutions were equilibrated by shaking on an automatic shakerfor 24 h at room temperature (22 "C).At the endof the experiment, concentrations of metal ions in solutions of each half-cell were determined byflame aspiration atomic absorption spectrophotometry, and concentrations of amylase were determined by measurement of absorbance at 280 nm
When protein solutions aredialyzed against solutions containing metal ions and chelating agents, distribution of the metal ionsbetween the two compartments of the dialysis cell depends upon the effectiveness with which the protein competes with the chelating agent in binding the mioentsa.lThis is governed by factors such as: (i) total concentrationsof the metal ([MITI, protein ([PIT), and the chelating agent ([LIT); (ii)pK, values of the chelating agent; (iii) stability constants of the metal complexes formed with the chelating agent and the protein; and (iv) pH of thesolution.Frommaterial balance, total concentration of the metal bound to the protein, [MIp,bcean expressed as
Summary
From the Center for Molecular Nutrition and Sensory Disorders,Georgetown University Medical Center, Washington, D. In order to developed an electrodialysis method for complete removal of learn more about these protein-metal binding interac-calciumfrom a-amylaseobtained fromvarioussources intions, calcium-free human salivary and porcine pan- cluding human saliva. This method is cumbersome and recreatic amylase [P(protein)] were obtainebdy carbox- quires specialized apparatus. Quantitation of loss of amylolytic though copper bound to both binding sites in human activity is difficult since spontaneous reactivation of the ensalivaryapoamylasetoformthe species Cu2L2Pin zyme by calcium can occur especially since calcium is which the amylase molecule appeared to forma bridge presentin variable amountsas a contaminantin several between the two coppeartoms.
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