Term
| 6 Steps for amino acid determination |
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Definition
1. If a heteromultimer, chains are separated and purified.
2. Disulfide bridges between cysteine are cleaved.
3. N-terminal and C-terminal residues are identified.
4. Polypeptide chain are cleaved into smaller fragments, and amino acid composition of each are determined.
5. Step 4 is repeated using different cleavage procedures to generate different and overlapping segments of peptides.
6. Overall sequence is constructed from sequences in overlapping fragments. |
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Term
| Separation of heteromultimers |
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Definition
Dissociation via pH extreems:
8M Urea
6M Guanidinium hydrochloride
high salt conc.
Separation via size and charge differences. |
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Term
| Cleavage of Disulfide bridges |
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Definition
*New S-S bonds can not be formed.
- performic acid forms two eq's of cysteic acid.
-2-mercaptoethanol or DTT reduces to SH, but must be protected by subsequent reaction with alkylating agent (e.g. iodoacetate, 3-bromopropylamine) |
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Term
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Definition
-Edmand degredation: phenylisothiocyanate [phenyl-N=C=S) reacts with free amino terminal to yield a PTH derivative which can be analysed.
10-20 cycles in larger
50 cycles in 100-200 protiens |
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Term
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Definition
Enzymatic Approach
-Carboxypeptidase A: (bovine pancrease) all residues except Proline, Arginine, Lysine.
-Carboxypeptidase B: (hog pancreas) Effective only on Arg or Lys at C-terminal.
-Carboxypeptidase C & Y: (citrus leaves, yeast) all amino acids. |
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