Chapter 3 - Protein Isolation and Determination of Amino Acid Sequence

Abstract

Proteins are separated on the basis of differences in their size, shape, charge, solubility, and binding affinity for other molecules. Chemicals used to extract proteins from membranous particles include dissociating agents, such as urea and mercaptoethanol, chelating agents such as ethylene diaminetetraacetic acid (EDTA), and organic detergents, such as sodium deoxycholate, sodium lauryl sulfate, and Triton X-100. The initial purification steps usually separate proteins according to general classification, such as fibrous (insoluble) or globular (soluble). The fibrous and globular designations are related to shape and solubility. Globular proteins are spherical or ellipsoidal, and make up the majority of known proteins. Fibrous proteins contain one or more polypeptide chains and their molecules are elongated and asymmetrical with lengths that may be many times their diameters. Protein can also be separated on the basis of their size by dialysis, gel filtration, and membrane filtration. Chromatographic separations of proteins are based on the differential partitioning of solute molecules due to their differences in affinity between a moving solvent phase and a fixed or supportive phase. Determination of the amino acid sequence of a purified protein begins with determination of the amino acid composition, which is the number of moles of each amino acid residue present in per mole of protein. Determination of the amino acid sequence of a protein involves the identification of the N- and C-terminal amino acid residues, cleavage of any disulfide bonds present, limited cleavage of the peptide into overlapping smaller fragments, purification of the fragments, and their stepwise cleavage into individual amino acid residues.