Ch 3 Text 1

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  1. What is a major goal of biochemistry?
    ·         to determine how amino acid sequences specify the conformations, and hence functions, of proteins. Other goals are to learn how individual proteins bind specific substrates and other molecules, mediate catalysis, and transduce energy and information
  2. o Study of protein occurs apart from other components. Steps? 
    • §  Purify the protein of interestàamino acid sequence determined (which computers or documentation have or through mass spectrometry) àdetermine the function within a physiologically relevant context
    • · Antibodies are good for locating proteins and measuring quantities
    • Finally, X-ray crystallography and NMR elucidate the 3D structure
  3. ·         The __is the functional representation of the genome
    o   The complete DNA base sequences, or genomes, are now available. However, these genomes are __ that could be __. Only a subset of the proteins encoded by these genes will actually __

    inventories of the genes

    expressed within a cell under specific conditions

    be present in a given biological context
  4. Explain the proteome
    §  The proteome—derived from proteins expressed by the genome—of an organism signifies a more complex level of information content, encompassing the types, functions, and interactions of proteins within its biological environment
  5. §  The proteome is not a __ characteristic of the cell. Because it represents a __, it varies with __, __, and __, such as the presence of hormones

    functional expression of information

    cell type, developmental stage, and environmental conditions
  6. §  The proteome is much larger than the genome because almost __. Furthermore, these proteins do not __; they often interact with one another to form __ with __. 

    Whereas the genome is “__,” the proteome is highly __
    all gene products are proteins that can be chemically modified in a variety of ways

    exist in isolation

    complexes with specific functional properties

    hard wired

  7. §  An understanding of the proteome is acquired by investigating, characterizing, and cataloging proteins, all of which begins by __
    separating a particular protein from all other biomolecules in the cell
  8. The __ of Proteins is an Essential First Step in Understanding their Function
  9. ·         Pure proteins: amino acid sequence can be knownà then what? 
    ·         Purification should yield a sample with only one type of molecule
    function --> map evolutionary relationships --> crystallography that shows functionà structure
  10. ·         A protein can be purified by subjecting the impure mixture of the starting material to __ based on __. To monitor, an __is done for some __. The more specific the assay, the more __.
    o   A positive result= __
    a series of separations based on physical properties such as size and charge


    unique identifying property of the protein

    effective the purification

    protein present
  11. o   For enzymes, the assay usually measures __, usually measured indirectly
    §  Ex: measuring the enzyme lactate dehydrogenase can be done by measuring the __ in one minute since NADH absorbs light at 340 nm
    ·         For purification, the __ must be known
    enzyme activity

    increase in the absorbance of light at 340 nm

    amount of protein present in the mixture being assayed
  12. ·         With these two experimentally determined numbers—__ and __—we then calculate the __, the ratio of enzyme activity to the amount of protein in the mixture
    o   Ideally, the __ will rise as the __ and the protein mixture being assayed consists of a greater and greater extent of lactate dehydrogenase
    ·         In essence, the overall goal of the purification is to __, which for a pure enzyme, will have a __
    enzyme activity and protein concentration

    specific activity

    specific activity

    purification proceeds

    maximize the specific activity

    constant value
  13. ·         __then reduces the cell into components. We determine which component is enriched in the protein of interest     
    Steps: __

    form homogenate through disrupting the membrane--> fractionate through centrifugation--> dense pellet at the bottom and supernatant above--> centrifuge supernatant--> more fractions--> assay each fraction for the desired activity
  14. ·         Proteins can be purified according to __(3)__; and, protein mixtures are subjected to a series of __, each based on a different property. At each step, the preparation is __ and its __ is determined
    • solubility, size, charge, and binding affinity
    • separations
    • assayed
    • specific activity
  15. Salting out 

    o   Salting Out: being less soluble at high salt concentrations, which changes among proteins. It is useful for concentrating dilute solutions of proteins, including active fractions obtained from other purification steps. Dialysis can be used to remove the salt too

    o   Dialysis: occurs through a semipermeable membrane. Protein mixture is placed in dialysis bag, submerged in buffer that lacks the small molecules that will be separated. Smaller molecules and ions diffuse through the bag down their concentration gradients
  16. o Gel-filtration chromatography (aka: molecular exclusion chromatography)
    o   On the basis of size, it separates. The sample is applied to the top of a column containing porous beads made of an insoluble but highly hydrated polymer like agarose. Small molecules enter the beads; larger cannot. Small molecules are distributed in the aqueous solution both inside the beads and between them, whereas larger solutions are only located between. 
  17. o Gel-filtration chromatography (aka: molecular exclusion chromatography) in terms of density
    §  Larger molecules flow more rapidly through this column and emerge first because a smaller volume is accessible to them. The smaller the molecule, the larger the time. 
  18. Ion-exchange chromatography
    used to obtain proteins of high purity. One chromatography step isn’t great for high purity because other proteins in the crude mixture will co-elute with the desired materialà sequential separations based on distinct molecular properties (such as net charge) are used
  19. §  Ex: negatively charged proteins won’t bind to beads with __, but positive charged ones will. The bound protein can then be __(released) by increasing the concentration of __ or another salt in the __
    §  Proteins with a __ will emerge first, followed by those with __
    • carboxylate groups
    • eluted 
    • sodium chloride
    • eluting buffer
    • low density of net positive charge
    • higher charge density
  20. §  This is often referred to as __; and, positively charged proteins (__) can be separated by chromatography on __ columns. Negatively charged proteins (__) can be separated by __ on __ columns
    • cation exchange
    • Cationic
    • negatively charged carboxymethylcellulose

    • anionic
    • anion exchange
    • positively charged diethylaminoethylcellulose
  21. o   __: highly selective for the protein; takes advantage of the __ (example on p. 70). Useful for isolating __—proteins that regulate gene expression by binding to specific DNA sequences. A protein mixture is passed through a column containing __ attached to a __; proteins with a high affinity for the sequence will __ and __. In this instance, the __is released by washing with a solution of high salt concentration
    Affinity chromatography

    high affinity of proteins for specific chemical groups

    transcription factors

    specific DNA sequences


    bind and be retained

    transcription factor
  22. §  Useful for isolating a protein that recognizes group X by:
    · __
    · __
    · __
    Covalently attaching X or a derivative of it to a column

    Adding a mixture of proteins to this column, which was then washed with buffer to remove unbound proteins

    Eluting the desired protein by adding a high concentration of a soluble form of X or altering the conditions to decrease binding affinity
  23. §  Most effective when interaction of protein and bait molecule are __
    §  The process of __ can isolate proteins expressed from __, where extra amino acids are encoded in and can serve as an __ when expressed

    standard affinity chromatography

    cloned genes

    affinity tag
  24. o   __: an enhanced version of the column techniques, but the column materials are much more __ and, thus, possess more __ and __. Because of this finer material, __is applied to obtain __. The net result is both __ and __. Measures are taken with a __that monitors the __
    High Pressure liquid chromatography

    finely divided

    interaction sites and greater resolving power


    adequate flow rates

    high resolution and rapid separation


    absorbance at a particular wavelength
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Ch 3 Text 1
2014-09-07 03:52:36
Test One
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