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    Survey of Biochemistry: Amino Acids and Proteins

    Uploaded by

    Usama
    The document discusses amino acids and proteins, including the 20 standard amino acids. It covers topics like peptide bonds, torsion angles, alpha helices, beta sheets, and protein purification techniques including salting out, gel filtration chromatography, ion exchange chromatography, and affinity chromatography. It also discusses ways to assess protein purification, including Beer's Law calculations and Bradford protein assays.

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    © All Rights Reserved
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    Survey of Biochemistry: Amino Acids and Proteins

    Uploaded by

    Usama
    20 views29 pages
    The document discusses amino acids and proteins, including the 20 standard amino acids. It covers topics like peptide bonds, torsion angles, alpha helices, beta sheets, and protein purification techniques including salting out, gel filtration chromatography, ion exchange chromatography, and affinity chromatography. It also discusses ways to assess protein purification, including Beer's Law calculations and Bradford protein assays.

    Original Description:

    Original Title

    Bradford Assay Protein

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    The document discusses amino acids and proteins, including the 20 standard amino acids. It covers topics like peptide bonds, torsion angles, alpha helices, beta sheets, and protein purification techniques including salting out, gel filtration chromatography, ion exchange chromatography, and affinity chromatography. It also discusses ways to assess protein purification, including Beer's Law calculations and Bradford protein assays.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    20 views29 pages

    Survey of Biochemistry: Amino Acids and Proteins

    Uploaded by

    Usama
    The document discusses amino acids and proteins, including the 20 standard amino acids. It covers topics like peptide bonds, torsion angles, alpha helices, beta sheets, and protein purification techniques including salting out, gel filtration chromatography, ion exchange chromatography, and affinity chromatography. It also discusses ways to assess protein purification, including Beer's Law calculations and Bradford protein assays.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 29

    SURVEY OF

    BIOCHEMISTRY

    Amino Acids and Proteins

    1
    Amino Acids

    Can you give the 1-letter and 3-letter names


    for all 20 amino acids within 5 minutes?

    Can you draw a oligopeptide of any given


    sequence?
    With correct stereochemistry?

    2
    Amino Acids:
    Nonpolar Side Chains

    3
    Amino Acids:
    Polar, Uncharged Side Chains

    4
    Cysteine & Disulfide Bonds

    5
    Cysteine & Disulfide Bonds

    6
    Amino Acids:
    Polar, Uncharged Side Chains

    7
    Amino Acids:
    Polar, Uncharged Side Chains

    8
    Amino Acids:
    Polar, Charged

    Be able to draw,
    name and give
    1-letter/3-letter
    codes for
    all 20
    amino acids!

    9
    pKa and pI

    • pI = Isoelectric point
    the pH at which a molecule carries no
    net electric charge

    pKa2 pKa1

    1
    pI = [pKa1 + pKa2]
    2
    10
    Polypeptide Sequence
    Nomenclature

    Be able to draw
    a polypeptide! Tetrapeptide: AYDG

    11
    Peptide Bond

    Peptide bond links one amino acid to another.

    12
    Torsion Angles in Proteins

    Psi()
    Peptides
    Phi() typically
    assume
    the trans
    conformation

    13
    Torsion Angles Defined…

    • Psi: angle made from atoms


    N-Calpha-Ccarbonyl-Nn+1

    H O
    N
    N N
    H O H O
    14
    Torsion Angles Defined…

    • Phi: angle made from atoms


    Ccarbonyl,n-1-Nn-Calpha-Ccarbonyl,n

    H O
    N
    N N
    H O H O
    15
    Alpha Helix

    Sequence coils in a right-handed


    manner.

    Notice hydrogen bonding along the


    helical axis from carbonyl oxygen
    (of residue n) to the amino hydrogen
    of residue (n+4).

    Hydrogen bonding stabilizes the


    alpha helical structure!

    16
    Beta Sheets

    Notice the
    hydrogen
    bonding
    from the
    carbonyl
    oxygen to the
    amino
    hydrogen

    17
    Protein Purification Overview

    Courtesy of

    18
    Salting Out: (NH4)2SO4

    19
    Gel Filtration Chromatography

    Separation based on Size

    20
    Ion Exchange Chromatography

    Separation based on Charge

    21
    Affinity Chromatography

    Affinity What could be


    to cause proteins
    to elute off of an
    affinity column?

    22
    Ways to Assess Protein
    Purification

    • Total Protein Concentration Assays


    – Beer’s Law
    – Colorimetric Assays

    • Specific Protein Assays


    – Activity Assays
    – Immunoassays
    • ELISA
    • RIA

    23
    Beer’s Law

    A  bc
    What is the molar concentration of a solution of
    Bovine Serum Albumin (BSA) that exhibits an
    A280 of 0.75 with a path length of 1 cm?

    24
    Beer’s Law

    A  bc
    What is the molar concentration of a solution of
    Bovine Serum Albumin (BSA) that exhibits an
    A280 of 0.75 with a path length of 1 cm?

    0.75 Must know


    Conc. =
    (43,824 M-1cm-1) x (1 cm) the value of
    Conc. = 0.00001711 M = 17 µM 
    25
    Bradford Protein Assay

    Suppose you have a protein mixture and you need to


    determine the protein concentration.

    You cannot use Beer’s Law.


    Because you would not know the extinction coefficient
    for the protein mixture at 280 nm

    Alternative approach:
    Bradford Protein Assay

    26
    Bradford Protein Assays

    Bradford Protein Assay reagent


    contains Coomassie brilliant blue
    which reacts with basic (esp. Arg)
    and aromatic amino acids to yield a
    blue color with intensity
    proportional
    to the protein concentration.

    How is data generated?


    How is data analyzed?
    27
    Data Generation: Bradford Assay

    Reference contains only buffer

    Create a series of protein


    standards at known increasing
    concentrations and mix with the
    Bradford reagent
    2.5 5 10 15 20 25 µg/mL

    Treat the protein mixture of unknown concentration


    in the same manner as the standards and compare -
    visually and spectrophotometrically!
    28
    Bradford Assay Data Analysis

    Graph the absorbance


    at 595 nm as a function
    of [standard] in µg/mL.

    Fit the line and use it


    to deduce the [protein]
    in the mixture based on
    its absorbance.

    29

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