Biochemistry - Ch 10 - Proteins & Primary Structure

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Yasham
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Biochemistry - Ch 10 - Proteins & Primary Structure
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2011-10-31 18:50:19
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Biochemistry Primary structure proteins
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Biochemistry - Ch 10 - Proteins & Primary Structure
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  1. Proteins can perform what 5 functions?
    • Enzymes - catalysis
    • Structural - Rigidity of the cells
    • Motor proteins - Convert chemical energy to mechanical work
    • Cell signaling and signal transduction - receptors, insulin, receptors, etc
    • Transport - transport fatty molecules, etc albumin and hemoglobin
    • Regulatory proteins - transcription factors for gene expression
  2. What is salting out of proteins?
    • Salting out is based on the decreased solubility of proteins at high salt concentrations.
    • Different proteins are precipitated at different salt concentrations.
  3. What does trypsin cleave after in polypeptides?
    Arg (R) and Lys (K), unless followed by Pro (P)
  4. What does chymotrypsin cleave after in polypeptides?
    Phe (F), Tyr (Y), Trp (W) unless followed by Pro
  5. What does elastase cleave after in polypeptides?
    After Ala (A), Gly (G), Ser (S), or Val (V) unless followed by Pro (P)
  6. What does endopeptidase v8 cleave after in polypeptides?
    Cleaves after Glu (E), even if there is a Pro (P) present!
  7. What is the pKa of Lysine (Lys, K)?
    10.79

    • Asp - D - 3.9
    • COOH - 4.0
    • Glu - E - 4.07
    • His - H - 6.04
    • NH3 - 8
    • Lys - K - 10.79
    • Arg - R - 12.48
  8. What is the pKa of Arginine (Arg, R)?
    12.48


    • Asp - D - 3.9
    • COOH - 4.0
    • Glu - E - 4.07
    • His - H - 6.04
    • NH3 - 8
    • Lys - K - 10.79
    • Arg - R - 12.48
  9. What is the pKa of Histidine (His, H)?
    6.04


    • Asp - D - 3.9
    • COOH - 4.0
    • Glu - E - 4.07
    • His - H - 6.04
    • NH3 - 8
    • Lys - K - 10.79
    • Arg - R - 12.48
  10. What is the pKa of Aspartic Acid (Asp,D)?
    3.9


    • Asp - D - 3.9
    • COOH - 4.0
    • Glu - E - 4.07
    • His - H - 6.04
    • NH3 - 8
    • Lys - K - 10.79
    • Arg - R - 12.48
  11. What is the pKa of Gluatamic Acid (Glu, E)?
    4.07


    • Asp - D - 3.9
    • COOH - 4.0
    • Glu - E - 4.07
    • His - H - 6.04
    • NH3 - 8
    • Lys - K - 10.79
    • Arg - R - 12.48
  12. What are proteins sensitive towards (including denaturing)?
    • pH
    • High temperatures
    • Presence of proteases
    • Oxygen (keep at high concentration)
    • Freezing (glycerol - water freezes and structure may tear apart)
  13. What does the gel filtration method exploit for sequencing?
    Based on size.

    • Exploits difference in protein size
    • Mixture of proteins run through a column packed with porous polymers
    • -small proteins enter the polymer via pores and are retarded inside
    • -large molecules travel through column without retardation. These are the first to be elucated.
  14. what is chromatography in general?
    • Separation of proteins by taking advantage of:
    • -size (gel filtration)
    • -charge (ion exchange)
    • -binding affinity to ligands ( affinity chromotography, ie Immobilized metal affinity chromotography)
  15. What does the Ion Exchange Chromatography exploit (anion)?
    It exploits the charge differences in proteins.

    • Anion exchanger consists of anions binding to cationic group in the column.
    • The postive proteins were first to leave the column.

    • Q-resin: R-NR'3+
    • DEAE-resin: R-NHR'2+
  16. What does the Ion Exchange Chromatography exploit (cation)?
    It exploits the charge differences in proteins.

    • Anion exchanger consists of cations binding to anionic group in the column.
    • The negative proteins were first to leave the column.

    • S-resin: RCH2-SO3-
    • CM-resin R-O-CH2-COO-
  17. What does the hydrophobic interaction chromatrography exploit?
    It exploits hydrophobic interactions to purify nonpolar proteins

    • Substrate used is lightly substituted with octyl or phenyl groups
    • High to low salt concentrations used

    Hydrophillic proteins are the first to leave
  18. What does affinity chromotagraphy exploit?
    Specific binding is exploited.

    • Exploits protein's ability to bind specifically but noncovalently
    • -Specific ligands with high affinity

    Proteins with no affinity to substrate pass first.
  19. What does immobilized metal affinity chromotagraphy (IMAC) exploit?
    Exploids affinity of proteins for metal ions that are tagged with a polysistine (tag of 6 - 8 histines)

    The His-tag has an affinity towards metal ions.

    Untagged proteins are first to leave.
  20. What is electrophoresis?
    Separation of molecules based on charge and size
  21. What does Polyacrylamide gel electrophoresis (PAGE) take advantage of?
    Mass and charge.

    • Mobility of small molecules is greater than large molecules (with same charge density).
    • -High pH is used so nearly all proteins are net negative

    More negative move towards positive electrode.
  22. What does SDS-PAGE exploit?
    Separates by mass only.

    • Disrupts tertiary structure by breaking noncovalent bonds
    • -proteins unforld into rod-like shape.

    Separation by mass based on distance traveled.
  23. What does capillary electrophoresis (CE) exploit?
    High voltage to separate based on charge

    • Fast and efficient separation with the use of high voltage
    • Both positive and negative ions travel through capillary by electroosmotic flow.
  24. What does Isoelectric focusing (IEF) exploit?
    pH gradient and electric field

    • Protein's charge will change based on pH
    • Electric field used to attract towards area but will stop when pH = pI

    Two dimensional (2D) gel electrophorsis used IEF and SDS page to separate by mass and by charge (as a function of pH)
  25. What are immunoassays?
    • Use of antibodies for specific binding and identification method.
    • ie - coloring in western blot or ELISA.
  26. What are the steps for Enzyme linked immunosorbent assay (ELISA)?
    • 1) Immobilize the first antibody on a solid support
    • 2) Wash excess antibodies not adhered
    • 3) Incubate with protein
    • 4) Was excess protein
    • 5) Expose to second antibody with assayable enzyme such as a detectable product.
  27. What is the principle of Western Blot (8)?
    • 1) Separate proteins by polyacrylamide gel electrophoresis (PAGE)
    • 2) Transfer proteins on nitrocellulose filter by electroblotting - the protein attach to the filter
    • 3) Incubate the membrane with a protein-containing buffer (skim milk or casein) - fills open spots
    • 4) Incubate the membrane with primary antibodies specific to the protein of interest. - specifically binds to the protein
    • 5) Wash out excess of primary antibodies.
    • 6) Incubate the secondary antibodies with conjugated with a label or enzyme. - affinity towards primary antibody (not protein like ELISA)
    • 7) Wash out excess of secondary antibodies.
    • 8) Detect the signal.

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