AMINO ACID SEQUENCE DETERMINATION HELP SHEET
1. Acid hydrolysis of a protein is used to determine its amino acid composition:
only 17 AAs survive acid hydrolysis
Trp is destroyedAsn & Gln are converted to Asp & Glu
AA composition helps you find the number of cleavage sites for an enzyme or CNBr
For example, if a peptide contains 2 Lys & 1 Arg, then Trypsin digestion may yield 4 peptides
2. N-terminal residues can be determined with DansylCl method, which reacts with free amino groups. After acid hydroylsis, the Dansyl-AA can be determined.
3. C-terminal residues can be determined by enzymatic digestion with the enzyme carboxypeptidase, which releases amino acids sequentially.
4. Enzymatic and Chemical methods for peptide cleavage into smaller peptides are:
A. Enzymatic -- Trypsin -- cleaves on carboxyl side of Lys & Arg (unless Pro is next AA)
B. Enzymatic -- Chymotrypsin -- cleaves on carboxyl side of Aromatic AAs (Phe, Tyr, Trp)
(unless Pro is next AA)
C. Chemical -- CNBr cleaves on carboxyl side of Met & converts it to HomoSerLactone (HSL)
5. Edman degradation removes AA from the N-terminus, 1 at a time and can be repeated on the residual peptide remaining after the first AA is removed:
Go To Lecture 5 for more details!
©Wilbur H. Campbell, 1995; wcampbel@mtu.edu