1. Biochemistry: an Evolving Science

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1. Noncovalent bond (transient interaction)
electrostatic , hydrogen, van der Waals,
hydrophobic interaction 2. General protein
features 3. Amino acids nonpolar, neutral
polar, charged polar 4. Protein structure
primary, secondary, tertiary, quaternary 5.
Periodic structures a-helix, ß-pleated sheet
6. Formation of three-dimensional structure of
RNase A The amino acid sequence specifies the
complex three-dimensional structure. In many
cases, the native form is the thermodynamically
most stable structure. 7. Protein folding a
highly cooperative process protein misfolding
and aggregation mad cow, Altzheimer, Parkinson
diseases
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EXPLORING PROTEINS AND PROTEOMES
1. Genome vs. proteome 1) genome - genome gene
chromosome - Caenorhabditis elegans 19,000
genes/ 97,000,000 bp - Drosophila melanogaster
14,000 genes/180,000,000 bp - Human 25,000
genes/3,000,000,000 bp - functional genomics,
structural genomics - bioinformatics - biochip
DNA chip, protein chip 2) proteome - proteins
expressed by the genome - functional expression
of information - characterization and cataloging
of proteins - MALDI (matrix-assisted laser
desorption-ionization) spectrometry

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• 2. Protein purification
• 1) low resolution ? high resolution
• 2) assay
• - protein properties UV absorption
  (chromophores tryptophan, tyrosine,
  phenylalanine)
• - enzyme properties unit of enzyme (1
  µmole/min), activity (unit),
•   specific activity (unit/mg)
• - lactate dehydrogenase 340 nm  
• 3) best source
• 4) solubilization
• 5) stabilization
• 6) isolation

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• 3. Purification methods
• 1) protein source
• - cell disruption
• - homogenate
• - differential centrifugation
• 2) differential precipitation
• - ammonium sulfate (salting out) high specific
  activity, high yield
• - pH, temperature, organic solvent, polycations,
  polyethylene glycol
• 3) composition
• - charge ion exchange chromatography (DEAE
  diethylaminoethyl, CM carboxymethyl),
  electrophoresis
• - polarity adsortion chromatography, paper
  chromatography, reverse-phase chromatography,
  hydrophobic interaction chromatography
• - pI isoelectric focusing (polyampholytes small
  multicharged polymer with many pI values ? pH
  gradient)

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• 4) size
• - gel electrophoresis
• v Ez/f
• E electric field strength
• z net charge of protein
• f friction coefficient
• - exclusion chromatography (gel filtration)
• - dialysis semipermeable
• - ultracentrifugation
• Fc m'w2r m(1-v?)w2r
• Fc centrifugal force
• (1-v?) buoyancy factor

v Fc/f m(1-v?)w2r/f v sedimentation
velocity f friction coefficient
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• S v/w2r m(1-v?)/f
• S sedimentation coefficient
• Svedberg unit (10-13 sec)
• sedimentation velocity ? mass, density of
  particle, shape, density of solution
• zonal ultracentrifugation difference of
  sedimentation coefficient
• equilibrium density gradient ultracentrifugation
  difference of densities
• sedimentation equilibrium determination of
  molecular weight of proteins
• 5) binding specificity
• - affinity chromatography
• - immunoprecipitation

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Solubilities of several proteins in ammonium
sulfate solutions
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