Amino Acids, Proteins and Porphyrins
Best method of separation of ammonium sulphate from protein solution ? (AIIMS May 2009)
|A||Ion exchange chromatography|
|B||Thin layer chromatography|
|D||Salting in and salting out|
a. Most proteins are soluble in water due to hydrophilic amino acids on their surfaces. The pH and ionic strength of a solution dictates the solubility of the protein. If the ionic strength of the solution the protein resides in is too strong or too weak, then it will precipitate.
b. Ammonium Sulphate is the most common salt used for precipitating proteins due to its stability in cold solutions. Ammonium Sulphate fractionation is widely used in the first stage of protein purification to remove non protein molecules.
c. Size Exclusion Chromatography
d. Size exclusion—or gel filtration—chromatography separates proteins based on their Stokes radius, the diameter of the sphere they occupy as they tumble in solution.
e. The Stokes radius is a function of molecular mass and shape. A tumbling elongated protein occupies a larger volume than a spherical protein of the same mass.
f. Size exclusion chromatography employs porous beads
g. . The pores are analogous to indentations in a river bank. As objects move downstream, those that enter an indentation are retarded until they drift back into the main current. Similarly, proteins with Stokes radii too large to enter the pores (excluded proteins) remain in the flowing mobile phase and emerge before proteins that can enter the pores (included proteins
a. Column chromatography of proteins employs as the stationary phase a column containing small spherical beads of modified cellulose, acrylamide, or silica whose surface typically has been coated with chemical functional groups. These stationary phase matrices interact with proteins based on their charge, hydrophobicity, and ligand-binding properties. A protein mixture is applied to the column and the liquid mobile phase is percolated through it. Small portions of the mobile phase or eluant are collected as they emerge
b. Ion Exchange Chromatography
c. In ion exchange chromatography, proteins interact with the stationary phase by charge-charge interactions. Proteins
d. with a net positive charge at a given pH adhere to beads with negatively charged functional groups and vice versa.
e. Proteins, which are polyanions, compete against monovalent ions for binding to the support—thus the term “ion exchange.”
f. Affinity chromatography exploits the high selectivity of most proteins for their ligands. Enzymes may be purified by affinity chromatography using immobilized substrates, products, coenzymes, or inhibitors. In theory, only proteins that interact with the immobilized ligand adhere. Bound proteins are then eluted either by competition with soluble ligand or, less selectively, by disrupting protein-ligand interactions using urea, guanidine hydrochloride, mildly acidic pH, or high salt concentrations.