Welcome to 5610A: Advances in Analytical
Biochemistry
A survey course: We will cover many of the shiny new
analytical tools available to biochemists
Focus on Proteins
Core of the course: Analytical Techniques
UV/visible
Fluorescence
Circular Dichroism
Cellular Level Analysis
Mass Spectrometry
Nuclear Magnetic
Resonance
Separations
Assignments (12.5% ea): Jan 24th, Feb 7th, Feb 21st and March 13th
Big Project / Exam (40%): 15min presentation, 7 page review
What is Analytical Biochemistry?
Not a well defined field, but the main questions are…
Analytical Chemistry questions:
i) What’s there?
ii) How much?
Analytical Biochemistry Specific Questions:
iii) Where is it?
iv) Where did it come from?
v) How is it shaped?
vi) How does it move?
vii) What does it interact with?
In theory, Analytical Biochemistry stops there, but usually
these questions are asked to support hypotheses about
biological function
Diving Right in: Biochemistry is… Different
Life uses only a select few elements:
Mol % (Dry)
H = 46.7%
C = 42.0%
N = 6.4%
O = 4.75%
The distribution of elements in biological systems differs from
the distibution of elements in non-biological systems
The First Analytical Biochemistry Question…
First ever analytical biochemistry question: What kinds of
molecules do these elements form?
Sugars
Fatty Acids
Nucleic Acids
Emil Fischer
1852 - 1919
Bunsen and Kirchhoff
Spectroscope
Michel Chevreul
Johann Miescher
1786 - 1889
1844 - 1895
Elemental Analysis by: solvent
solubility, crystallization,
saponification, melting point,
distillation, and salt
fractionation
Invented alkaline extraction /
acid precipitation of DNA
(which he called Nuclein).
Elemental analysis was likely
by the Bunsen method.
The First Analytical Biochemistry Question…
Amino acids…
Henri Braconnot
1780 - 1855
Isolated Glycine
Heinrich Hlasiwetz and
Josef Habermann - 1867
Leucine and Tyronsine from Casein
http://web.lemoyne.edu/~GIUNTA/hlasiwetz.html
Other Amino Acids
Karl Ritthausen – 1866
Glutamic and Aspartic
Acid
Serine – 1865
Phenylalanine - 1881
Alanine – 1888
Arginine – 1895
Histidine – 1897
………
Cysteine – 1935!! (still debated!)
First Analytical Biochemistry Techniques…
These discoveries have one thing in common: Elemental
Analysis
Robert Bunsen
(1811-1899)
Gustov Kirchoff
(1824-1887)
Joseph Gay-Lussac (1778-1850)
and Louis Thenard (1777-1857)
Made chemistry-based elemental analysis
much more practical and accurate
He Spectrum
http://web.lemoyne.edu/~GIUNTA/thenard.html
http://www.scitechantiques.com/1959/
Macromolecules: Polysaccharides
So the principal biological molecules are: Sugars, Fatty Acids,
Nucelic Acids and Amino Acids
Sugars:
Disaccharides
GC-MS
Polysaccharides
(i) nature and molar ratios of the contained
monosaccharide building blocks?
(ii) positions of the glycosidic linkages?
(iii) monomer sequences and identification
of repeating units?
http://scholle.oc.uni-kiel.de/lind/iteach/kh_struct/kh_struct_eng_kap3.pdf
Macromolecules: Phospholipids
Fatty Acids
Fats
Sterols
Analytical Questions:
i) What types of phospholipids?
ii) Under what conditions do
Liposomes, Micelles and
Bilayers form?
iii) Membrane physics (how do
the monomers move around)?
iv) Interactions with proteins
Phospholipids
Analytical Tools:
i) HPLC/UV-visible (monomers)
ii) Fluorescence (membranes)
iii) NMR (micelles/proteins)
iv) MS (monomers-bilayers)
Macromolecules: DNA/RNA
Nucleotides
RNA
DNA
Macromolecules: DNA/RNA Con’t…
DNA is the hereditary molecule: Analytical biochemistry at it’s
best…
Hershey and Chase Experiment - 1952
Macromolucules: DNA/RNA Con’t…
Biological Roles:
Analysis:
X-ray crystallography
DNA: Heredity
p31 NMR
Genome encoding
Hybridization
Gene Expression (methylation)
MS/MS
RNA: Heredity (virus)
Intron excision (genome decoding)
mRNA (intermediate DNA – protein)
tRNA (genome decoding)
Ribosomes (genome decoding)
Metabolism (e.g. mRNA IRE)
Macromolecules: Proteins
Amino Acids
M
L
V
G
S
T
F
H
D
K
Peptides
Proteins
Proteins are definitely absolutely and totally the most important
and coolest biological molecule!!
Analytical Questions:
i) Which amino acids?
ii) In what order?
iii) Structure?
iv) Biological Function?
v) Dynamics?
vi) Quantitation / Localization?
What is Analytical Biochemistry?
Biological Roles…
Structural: hair/nails, cytoskeleton, muscle, cartilage
Keratin, Actin/Tubulin, Actin/Myosin, Collagen/Elastin
Signaling and Transport:
Insulin, Transferrin, Ion Chanels, Receptors/Kinases
Storage:
Ferritin (Fe2+), Calsequesterin (Ca2+), Seed Storage
Catalysis:
Gazillions (I’m not even going to try)
Proteins: Primary Structure Analysis
Gerardus Mulder
1802-1880
Elemental analysis
of whole proteins,
coined the term
‘protein’.
http://web.lemoyne.edu/~GIUNTA/mulder.html
Fibrin: C400H620N100O120S1P1
Ratios:
C1H1.55N.25O.33S.025P.025
Fibrin:
C2103H3108N550O642S20
Ratios: C1H1.47N.26O.31S.01
Primary Structure
S-Q-D-A-G-M-Q-Q-G-A-D-M-D-Q-V-S-A
Frederick Sanger (1918-1997)
Sequenced insulin using
limited Proteolysis and
paper chromatography!
Enzymatic
hydrolysis
Dipeptides
Proteins: Secondary Structure
Secondary structure is how the amino acid chain arranges itself
Alpha Helix
Beta Sheet
The primary structure contains all of the information needed
to predict how a protein will fold
The discovery of Secondary Structure…
Linus Pauling 1901-1994
http://www.nap.edu/readingroom/books/
biomems/lpauling.html
Trained in theoretical physics, at
the center of early X-ray
crystallograhy
Recognized the importance of the
H-bond in stabilizing protein
structure
“I think that enzymes are molecules that are complementary in structure to the activated
complexes of the reactions that they catalyse, that is, to the molecular configuration that is
intermediate between the reacting substances and the products of reaction for these
catalysed processes. The attraction of the enzyme molecule for the activated complex would
thus lead to a decrease in its energy, and hence to a decrease in the energy of activation of
the reaction, and to an increase in the rate of the reaction” - 1948
Published proposed -helix and -sheet in 1951
Proteins: Primary to Secondary Structure…
Secondary structure is caused by the tendency of amino acids
to ‘prefer’ specific rotations around the N-C () and C-C ()
bonds
Gopalasamudram
Narayana Iyer
Ramachandran
(1922-2001)
Ramachandran Plot
Primary Structure to Secondary Structure Con’t…
Certain amino acids prefer ‘helical’ / angles and some prefer
‘beta strand’ / angles.
Proteins: Tertiary Structure…
Tertiary structure is the organization of secondary structures
Helix Bundle
Beta Barrel
Greek Key
Primary and Tertiary Structure are Linked…
Hydrophobicity and Tertiary Structure
Chymotrypsinogen
Proteins: Quaternary Structure…
Quaternary structure describes how protein subunits that are
separate peptide chains interact with each other
Tetramer
Cooperativity
Heptamer
Size
Gigantic Complex
Multiprocess function
Quaternary Structure and Activity Control…
Blood Clotting
Prothrombin (II)
Thromboplastin (III)
Aggregation / Amyloidosis
Proaccelerin (V)
Fibrin
Diving Right In: What is Analytical Biochemistry
Volume = 7 x 109 cm3
Volume = 2 x 10-8 cm3
These are about as different as you can get…
But on the molecular level…