| PURIFICATION AND CHARACTERIZATION OF PROTEINS
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| Protein purification procedures take advantage of separations based on charge, size, binding properties, and solubility. The complete characterization of the protein requires an understanding of its amino acid composition, and its complete primary, secondary, and tertiary structure and, for multimeric proteins, its quaternary structure.
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| POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS |
| Most proteins undergo some form of enzymatic modification after the synthesis of the peptide chain. The 'post-translational' modifications are performed by processing enzymes in the endoplasmic reticulum, Golgi apparatus, secretory granules, and extracellular space. The modifications include proteolytic cleavage, glycosylation, lipation and phosphorylation. Mass spectrometry is a powerful tool for detecting such modifications, based on differences in molecular mass. |
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| Figure 2.10 Three-dimensional structure of a dimeric protein. Quaternary structure of Cu,Zn-superoxide dismutase from spinach. Cu,Zn-superoxide dismutase has a dimeric structure, with a monomer molecular mass of 16 000 Da. Each subunit consists of eight antiparallel β-sheets called a β-barrel structure, in analogy with geometric motifs found on native American and Greek weaving and pottery. Courtesy of Dr Y Kitagawa. |
| In order to characterize a protein, it is first necessary to purify the protein by separating it from other components in complex biological mixtures. The source of the proteins is commonly blood or tissues, or microbial cells such as bacteria and yeast. First, the cells or tissues are disrupted by grinding or homogenization in buffered isotonic solutions, commonly at physiologic pH and at 4°C to minimize protein denaturation during purification. The 'crude extract' containing organelles such as nuclei, mitochondria, lysosomes,
microsomes, and cytosolic fractions can then be fractionated by high-speed centrifugation or ultracentrifugation. Proteins that are tightly bound to the other biomolecules or membranes may be solubilized using organic solvent or detergent.
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