-
Trypsin
- Rn-1= positively charged residues: Arg, LYS;
- Rn NOT Pro
- highly specific
-
Chymotrypsin
- Rn-1= bulky hydrophobic residues: Phe,Trp, tyr
- Cleaves more slowly for Rn-1=Asn, His, Met, Leu
- Rn NOT pro
-
Elastase
- Rn-1= small neutral residues: Ala, Gly,
- Ser, Val
- Rn NOT Pro
-
Thermolysin
- Rn=Ile, Met, Phe, Trp, Tyr, Val
- Rn-1 NOT Pro
- Occasionally cleaves at Rn=Ala, Asp, His, Thr
- heat stable
-
Pepsin
- Rn =Leu, Phe, Trp, Tyr
- Rn-1 NOT Pro
- Also others; quite nonspecific;
- pH optimum =2
-
Endopeptidase V8
Rn-1=Glu
-
-
dansyl chloride
- bonds with primary amine
- help determine N-terminus
-
FDNB
- bonds with primary amine
- help determine N-terminus
-
Carboxypedptidase
C-terminus determination
-
8 steps for sequencing protein
- 1) Seperate subunits
- 2) break disulfide bonds
- 3) Amino Acid Composition
- 4)End group analysis
- 5) "Molecular Scissors"
- 6)repeat 5 w/ differenet scissors
- 7) sequence each purfied peptide fragment with Edman Degradation
- 8) reconstruct sequence
-
How do you seperate subunits of protein?
use: extreme pH; 8M urea; 6M guanidine HCl; high salt concentration
-
Break disulfide bonds
- 1) reduce by DTT or 2-mercaptoethanol, block with iodoacetate OR
- 2)oxidize by performic acid
-
Amino Acid composition
acid and enzymatic hydrolysis of polypeptides then amino acid compostion analyzed by HPLC
-
Amino Acid grp determination
- N-terminus: Dansly Chloride & FDNB
- C-terminus: carboxypeptidase or other exopeptidase
-
cleave chains into smaller fragments
Trypsin, Chymotrypsin, elastase CNBr
-
sequence peptides produced from cutting in 5&6
use Edman Degredation: -PHENYLISOTHICYANATE
-
Reconstruction
comapre and align overlapping peptide pieces
-
what cleaves large argomatics and where?
Pepsin cleave at N terminus; Chymotrypsin cleaves at C terminus (Rn-1)
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