BCMLch3
Biochemistry-Lehinger CH3
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Intersection
Biochemistry
- Methionine – thioether
- Proline – imino
- Tryptophan – 280nm stronger absorbance
- Tyrosine – 280nm stronger absorbance
- Phenylalanine – 280m lesser extent
- Cysteine – sulfhydryl, readily oxidized to form a covalently linked dimeric amino acid
- Arginine – guanidine
- Histidine – imidazole, only common AA ionizable side chain near neutral
- Peptide bonds – 1/2t = 7yrs
- Oligomeric: at least two identical subunits
- Protomers: identical subunits
- Conjugated: contain permanently associated chemical components
- Prosthetic group: nonAA conjugated protein (lipid, sugar, metal)
- Separation Techniques
- Fractionation – extract is separated into different fractions based on
- Salting Out – NH2SO4 to make proteins insoluble due to high salt conc
- Dialysis – bag/tube with semipermeable membrane, exch of salt NOT PROTIEN
- SIZE - Retains larger proteins within
- Column Chromatography –
- SIZE, CHARGE, BINDING AFFINITY
- Cationexchange Chromatography –
- CHARGE – (-) stays slower
- Affinity Chromatography
- BINDING – binders are slower
- Chromatography is used later
- Electrophoresis –
- CHARGE – migration; SHAPE – electrical potential (E) and electrophoretic mobility (mu)
- SDS – purity and molecular weight
- ANALYSIS – binds to protein proportional to molecular weight of protein
- SDS 1: 2 AA (-) charge, alters native conformation
- Elect & SDS – determines molecular weight (Coomassie blue dye)
- Separates subunits – each band = different protein/subunit
- SMALLER MORE RAPIDLY – at bottom
- Isoelectric focusing – determines pI
- Activity: total units of enzyme in solution
- Specific activity: number of enzyme units per mL of total protein [enzyme concentration]
- PURITY – increases and constant when pure
- Polymorphic: AA seq is varies in human pop
- Sanger reagent (FDNB) – labels the amino terminal residue [dansyl chloride/dabsyl chloride]
- Edman degradation – labels and removes only the amino terminal residue from a peptide leaving rest intact
- Sequenator – machine that does the E.deg.
- DNA sequencing is faster and more accurate than sequencing the protein
- Analytes – molecules analyzed in mass spec
- How 2Obtain a peptide: Tissue; Genetic engineering; Direct chemical synthesis
