biochem 5

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  1. what do we use to test for function of protein; since many proteins are enzymes, this is often related to ability of enzyme to catalyze particular reaction
  2. what are the steps in purification of a protein?
    • break cells open (ultrasound or mechanical)
    • centrifuge leaving solution with DNA, RNA and proteins
    • concentrate proteins (using ammonium sulfate or polyethylene glycol)
    • chromotography
    • check purity by gel electrophoresis
  3. what is dialysis?
    using pure buffers to equilibrate concentrations inside and outside tube of protein, which allows small molecules not big molecules through
  4. describe ion exchange chromatography
    • bead is attached to ionic molecule
    • most protein are neg at most pHs
    • anion exchange resident that binds anions and allows for separation
    • add high concentration of anions, neg charged proteins displace the anions attached to columns (more anions take place of proteins bound to column)
    • longer the column better the result
  5. describe gel filtration chromatography
    carbohydrate polymer beads, large molecules cannot enter, small molecules enter the aqueous spaces within beads
  6. describe affinity chromatography
    • glass bead attach linker (chemically attach group with affinity for protein)
    • protein binds glucose (construct a residue)
    • add high concentration of glucose, which replaces attached glucose, increase in salt-protein with less affinity comes off first, higher affinity comes off later
  7. what are the two common types of gels used in gel electrophoresis?
    • agarose and polyacrylamide
    • polyacrylamide is used for smaller molecules (not DNA)
  8. what is an elution column?
    resin matrix with ligands coupled to it.
  9. how do you make something sink to the bottom of the well in gel electrophoresis?
    mix it with something dense (sucrose glycerol)
  10. what is the effect of SDS on gel electrophoresis?
    • coats the protein in neg charge so the intrinsic charge of the protein becomes unimportant. migration of the protein is based on charge
    • SDS can also be used to access the purity of the protein
  11. how would you use SDS to determine mass of protein relative to mobility?
    log of mass and relative mobility give a linear line
  12. why are mild conditions used in SDS gel electrophoresis to probe quaternary structure?
    so two independent molecules won't link together 
  13. which is faster? sequence DNA or determine a protein sequence directly?
    sequence DNA
  14. describe the ninhydrin reaction
    all amino aicds react with ninhydrin at 100 degrees celsius forming blue product, except for proline (yellow)
  15. why are there no peaks for Asn or Gln? what are other aa that gives no results
    • b/c acid hydrolysis converts these residues to Asp and Glu
    • Trp is also destroyed in hydrolysis under acid conditions, and Cys does not give reliable results
  16. describe the parts of an amino acid analyzer
    • ion exchange column, ninhydrin tank, spectrometer (neg first, pos @end)
    • integrate areas underneath spectrometry
  17. what is the purpose of dabsyl chloride, dansyl chloride and fluorodinitrobenzene and what does it attack to?
    • fluorescent compounds make it easy to analyze amino acids, gives empirical formula; obtain molecular formula by SDS
    • attaches at the N terminal of alpha amino group and side chain of e-amino group of Lys
  18. what is one disadvantage of dabsyl chloride reaction?
    determine only single terminal a.a. lost the rest of the sequence
  19. what is the role of phenyl isothiocyanate reaction?
    breaking of the next peptide bond so the peptide chain is one residue shorter, put on bead so the one sequence shorter peptide chain stays in filter
  20. what is the role of edman degradation?
    cleaves any amino acid on c side of N terminus, excpet blocked N-terminal
  21. what is the role of carboxypeptidases?
    removes carboxy-terminal amino acid from a polypeptide chain, one residue at a time
  22. what is the average molecular weight of an amino acid?
  23. where does CNBr cut?
    c side of met
  24. where does trypsin cut?
    C side of lysine or arginine, Rn+1 is not proline
  25. what is the effect of maleic anhyride
    makes lysine no longer available to trypsin cleavage
  26. what does ethylenimine do?
    reacts with cysteine to amke the molecule trypsin cleavable
  27. what are some way to break disulfide bonds?
    • performic acid (harsh-oxidation: not reversible)
    • 2-meracaptoethanol creates SH groups, then in idoacetic acid (alkylating agent)
  28. what are the advantages of chemical synthesis?
    don't have to use naturally occurring amino acids, don't have to use L a.a
  29. describe the process of chemical synthesis
    t-boc is used to block N terminal, activate with DCC which gives good leaving group on carboxyl and mixing activated amino acids and resin (by passing solution through resin) and allowing N to act as nucleophile to attach C's kicks off byproduct and extend protein. 
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biochem 5
2013-02-11 02:20:47

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