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Chapter 4

BIOL2020 Ch40 notes.docx

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BIOL 2020
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BIOL2020 – CHAPTER 40 Techniques in Protein Biochemistry  Proteins must be separated from all other constituents of cell so their biochemical properties can be identified and characterized ; aka protein must be purified 40.1 THE P ROTEOME IS THEFUNCTIONAL R EPRESENTATION OF THE GENOME  Completely sequenced human genome contains 3 billion bases and about 25000 genes  Proteome – level of functional information, which encompasses types, functions and interactions of proteins that yield a functional unit  Genome provides list of gene products that could be present but only a subset of these gene products will actually be expressed in a given biological context  Proteome tells us what is functionally present ; not a fixed characteristic of cell  Proteome – varies with cell type, developmental stage and environmental conditions 40.2 THE P URIFICATION OFP ROTEINS IS THEF IRSTSTEP INU NDERSTANDING T HEIR FUNCTION  To understand a protein, we need to isolate it from the thousands of other proteins in the cell  Assay – used to identify protein which we are interested in and we use this test after every stage of purification to see if purification is working ; tells us how much enzyme activity is present, not how much enzyme protein is present  We also need total amount of protein present in mixture being assayed ; this measurement of total amount of protein includes enzyme of interest as well as all other proteins present but is not a measure of enzyme activity  Specific activity – ratio of enzyme activity to amount of protein in enzyme assay at each step of purification ; will rise as protein mixture used for assay consists of a greater and greater extent of protein of interest  Point of purification is to remove all proteins except protein in which we are interested and quantitatively it means we want to maximize specific activity PROTEINS M UST BER EMOVED F ROM THE CELL TOBE PURIFIED  Now we must break open cell and release cellular contents ; Disruption of cell membranes yields a homogenate, a mixture of all of the components of the cell but no intact cells  Mixture is centrifuged at low centrifugal force; pellet of heavy material at bottom and lighter solution above called supernatant  Pellet and supernatant called fractions  Supernatant centrifuged at greater force to get another pellet and supernatant ; procedure called differential centrifugation & yields several fractions of decreasing density ; each fraction still contains many proteins which are assayed for activity being purified  One fraction will have more enzyme activity than others and that will be material to which discriminating purification techniques are applied ; this fraction called crude extract PROTEINS CAN BE PURIFIEDACCORDING TO SOLUBILITY, IZE, HARGE AND B INDINGA FFINITY  Salting out o most proteins need some salt to dissolve – process called salting in o most proteins precipitate out of solution at high salt concentration – effect called salting out o salting out due to competition between salt ions and protein for water to keep protein in solution o salt concentration at which a protein precipitates differs from one protein to another o many proteins lose activity in presence of high salt concentrations o salt can be removed by process of dialysis ; protein-salt solution placed in small bag made of semipermeable membrane with pores; proteins too large to fit through pores of membrane but salts can and they emerge in medium outside the bag  Separation by size o Gel filtration chromatography (aka molecular exclusion chromatography) – separates proteins based on size o Sample applied to top of column consisting of porous beads made of insoluble polymer; small molecules can enter beads so larger molecules follow shorter path to bottom of column and emerge first o Molecules that are of a size to occasionally enter bead will flow from column at an intermediate position and small molecules take longer more circuitous path and will exit last  Ion-exchange chromatography – separated based on net ionic charge o If protein has net positive charge at pH 7, will usually bind to column of beads containing negatively charged carboxylate groups; negatively charged protein won’t bind at the column o Positively charged protein bound to column can be released by increasing concentration of salt in buffer poured over column; positively charged salt ions compete with positively charged groups on protein for binding to column o Protein with net negative charge will be bound to ion-exchange beads carrying positive charged; can be eulted from column with use of buffer containing salt  Affinity chromatography o Takes advantage of fact that some proteins have high affinity for special chemical groups/specific molecules o EX) Pass crude extract through column of beads containing cov
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