Exercise 7 - Exercise 7/Chapter 7 CHAPTER 7: Protein...

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Exercise 7/Chapter 7 CHAPTER 7: Protein Extraction, Purification, and Quantification Purification of proteins from cells has several stages: o First, Bulk Extraction methods release proteins and other molecules from cells o Next, additional techniques identify distinct classes or species of proteins within bulk extract Electrophoresis separates proteins into different classes based on their size and/or their net electrical charge o Proteins can then be transferred to a membrane surface to make them accessible for antibody detection methods Concentration of proteins can be determined by using spectrophotometry to detect peptide bonds or the R-groups of aromatic amino acids Diverse Functions of Proteins in Cells Enzymes: catalyze complex metabolic reactions that go on in all parts of each cell Hormones (many are proteins): activate or inhibit the initiation of specific metabolic pathways in cells Receptors: proteins in membrane that act as specific binding sites for hormones or growth factors that activate or inhibit internal cell processes. Many have a binding site on extracellular side and an enzyme on their cytoplasmic side (dual functions) Cytoskeleton: A fibrillar network of microtubules, microfilaments, and intermediate filaments – each composed of proteins Pumps and Channels: Responsible for selective movement of molecules and ions across every cell membrane o Pumps hydrolyze ATP to move mol. and ions against concentration gradient o Channels facilitate selective movement of mol. and ions along conc. Gradient Structural Support Molecules (ex. Collagen): secreted from cells into extracellular matrix; form network of fibers that support tissues and link them together; act like I-beams to support structures Preparation of Crude Proteins from Whole Cells:
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Broken cells are subjected to centrifugation immediately after being disrupted so that particulate matter (unbroken cells, organelles, membrane fragments, large nucleic acids) can be removed During extraction, the peptide bonds that stabilize the primary function of polypeptides are very strong and are usually not broken Secondary level is easily altered (alpha helices and beta pleated sheets) o Due to relatively weak hydrogen bonds that are disrupted by changes in pH, temperature, and other factors Tertiary level also easily altered: held together by ionic bonds, hydrogen bonds, hydrophobic interactions, disulfide bonds o Can be disrupted by changes in pH, temperature, others -SH functional groups (“thiol” groups in protein structure) o When in cell: proteins mostly have free –SH groups on cysteine o Secreted cells: fortified with extra cross-linking bonds; have disulfide bonds (cross links between –SH groups) which causes the loss of hydrogens and a covalent bond directly linking 2 sulfur atoms by oxidation – ALTERS SHAPE OF PROTEIN, making it nonfunctional To avoid oxidation , a reducing agent is added o Donate electrons and hydrogens to oxidized molecules
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This note was uploaded on 03/20/2008 for the course BIO 205L taught by Professor Hanson during the Fall '07 term at University of Texas.

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Exercise 7 - Exercise 7/Chapter 7 CHAPTER 7: Protein...

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