Chapter3 - a.a. and primary structure of proteins

3.1 a.a. amino derivatives of carboxylic acids

20 standard amino acids
glycine has no chiral carbon
all aa in proteins are of the L configuration
aliphatic
aromatic
sulfur containing
alcohol
base
acid
amide
additional amino acid: hormones, neurotransmitters..

3.2 ionization of a.a.

pKa1 pKa2 pI

3.3 peptide bonds link a.a. in proteins

aa, dipeptide, tripeptide, oligopeptide(4~20), pp
residues: -ine, -ate 变为 -yl (eg: Glycyl, glutamyl)

3.4 Protein Purification & Analytical Techniques

3.4.1 protein quantification

determination of protein concentration:
[Total] Spectrophotometric (Beer-Lambert Law): A280, Bradford assay, BCA
[Target-specific] Immune-technology: ELISA

3.4.2 protein purification

0~4C 避免denaturation

1. prepare a solution of proteins： differential centrifugation
2. a relatively crude separation/fractionation (makes use of the different solubilities of proteins in salts solutions)
   crud separation: ammonium sulfate硫酸铵
3. column chromatography to fractionate the mixture of proteins that remains after ammonium sulfate precipitation沉淀 and dialysis透析.
   charge: ion exchange chromatography (IEX), CF
   size: GF
   hydrophobicity: HIC, RPC
   biorecognition(ligand specificity): AC
   other types of chromatography: paper chromatography, MCAC, HIC, HPLC

3.4.3 analytical techniques

electrophoresis separates proteins based on their migration in an electric field: qE = vf
polyacrylamide gel electrophoresis (PAGE) 凝胶电泳
Isoelectric focussing gel electrophoresis (IEF)
2-dimensional gel electrophoresis (2-DE)
Mass spectrometry (MS)
Electrospray ionization mass spectrometry (ESI)
Matrix-assisted laser desorption ionization (MALDI)
Tandem mass spectrometer (MS)

3.5 Protein Sequencing Strategies

summary of determination:

1. reduce the disulfide bonds and purify(separate the chains)
2. fragment with exo-, endopeptidase(CT, Trypsin) or CNBr, then purify(separate the fragments)
3. characterize each fragment by aa analysis or sequencing.
4. overlap fragments —> deduce entire sequence
5. deduce positions of disulfide bonds

3.5.1 cleaving and blocking disulfide bonds
cystine residue
3.5.2 peptide cleavage
chemical reagent: BrCN
proteases: Trypsin, Chymotrypsin
3.5.3 sequencing of peptide fragments
Edman degradation: the N-terminal residues are successively cleaved and identified
Sanger Reagent (Dansyl Chloride for End Group Analysis)
3.5.4 overlap fragments
other methods: mass spectrometry