BCHM 310 Lecture 10: BCHEM 315 3:3
22 Dec 2018
School
Department
Course
Professor
BCHEM 315 3/3
Edman degradation
- Boiling protein in 6M HCl gets rid of all
-
ANS: B, mostly -vely charged aa (12%) only 4% +vely charged so must be below 7
How to deal w limitations of seq
1. Long seq: break into shorter polypep, use proteases that cut at only certain aa→ purify
resulting polypep→ seq polypep
a. Trypsin cuts after K/R (not if Pro is after)
b. Chymotrypsin cuts after F, Y, W (not if Pro is after)
c. V8 protease cuts after D, E
d. Asp-N cuts before D, E
e. CNBr cuts after M
also soln to blocked N-term (opens up new N-term)
To find seq of aa look for overlaps in seq of polypep to paste them together
2. Blocked N-term: cleave w protease to gen new free N-term, cannot directly seq original
N-term frag
3. Disulfide bonds: in ox state a Cys-S-S-Cys would result in a blank result for the 1st Cys so
therefor must reduce 1st to Cys-S S-Cys + carboxymethylated or I-CH2-COO- to prevent
re-ox→ Cys-S-CH2-COO- and -OOC-CH2-S-Cys→ seq
a. How to identify disulfide bond connections: keep protein ox
Alt connection
4. For multimers→ sep polypep
