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Step 1: Complete Hydrolysis
HCl 6.0M, 110 C, 16-72 Hrs
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Edman Degredation
- Determine 1st residue (Nth terminal)
- pH 9.0
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Dansylation
- Determine 1st residue (Nth terminal)
- Less harsh, faster than Edman
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2-ME or DTT
Cleave disulfide bond
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Iodoacetate
Prevent formation of disulfide bond
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CNBr
Cleaves on C-side of methionine
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Trypsine
Cleaves lys, arg on C-side
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Chemotrypsin
Cleaves phe, tyr, try on C-side
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Endopeptidase S. Aureus V8
Cleaves asp, glu on C-Side
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Elastase
Cleaves gly, ala, val, syr on C-side
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Carboxypeptide A
- C-side of all except lys, arg, pro
- Will not work if R(n-1) = pro
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Carboxypeptide B
- C-side of all
- Will not work if R(n-1) = pro
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Carboxypeptide C
- Cleaves all on C-side
- Requires acidic conditions (pH 3.5)
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Carboxypeptide Y
- Cleaves all on C-side
- Slow with gly
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Pepsin
- Cleaves leu, phe, trp, tyr on N-side
- Will not work if R(n-1) = pro
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UV-Absorbance @ 280nm
Phe, tyr, trp
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