Upload
geofrey-sy-baje
View
20
Download
8
Embed Size (px)
DESCRIPTION
Amino Acid Sequencing
Citation preview
AMINO ACID SEQUENCING
• AFTER PURIFICATIO OF PROTEIN AND DETERMINATION OF ITS MASS, NEXT IS SEQUENCING
- DETERMINE AMINO ACID COMPOSITION BY HYDROLYSIS IN 6N HCl AT 110o C FOR
24H. - SEPARATION OF HYDROLYSATES BY
CHROMATOGRAPHY
• IDENTIFICATION OF A.A. BY ELUTION VOLUME • QUANTIFICATION OF A.A. BY REACTING IT WITH
NINHYDRIN• IDENTIFICATION OF N-TERMINAL BY LABELING
IT WITH COMPOUND THAT FORMS STABLE COVALENT BOND. (α-AMINO ACID) USING FNDB (FLUORODINITROBENZENE), DABSYL CHLORIDE AND DANSYL CHLORIDE
• REPEATED DEGRATION BY EDMAN METHOD
REAGENT CLEAVAGE SITE
CYANOGEN BROMIDE CARBOXYL SIDE OF M RESIDUES
0-IODOSOBENZOATE CARBOXYL SIDE OF W RESIDUES
HYDROXYLAMINE N-G BONDS
2-NITRO-5-THIOCYANOBENZOATE AMINO ACIDS OF C RESIDUES
TRYPSIN Carboxyl side of K and R
clostripain Carboxyl side of R residues
Staphylococcal protease Carboxyl side of D and E residues
thrombin Carboxyl side of R
chymotrypsin Carboxyl side of Y, W, F, L, and M
Carboxypeptidase A Amino side of C-terminal a.a. not R, K, and P
PROBLEMS• A solution of peptide of unknown sequence was divided into two
samples. One sample was treated with trypsin and the other with chymotrypsin. The smaller peptides obtained by trypsin treatment had the ff sequence
L-S-Y-A-I-RN-G-M-F-V-K
The smaller peptides obtained by chymotrypsin treatment had the ff sequence:
V-K-L-S-YA-I-R
N-G-M-FDeduce the sequence of the original peptide
• An organism of unknown origin produces a potent inhibitor of nerve conduction that you wish to sequence, aa analysis shows the peptide’s composition to be A5– K1- F1 . Reaction of the intact peptide with FNDB followed by acid hydrolysis liberates free FDNB alanine. Trypsin cleavage gives a tripeptide and a tetrapeptide, with compositions A3 –F1
and K1 –A2 Reaction with the intact peptide with chymotrypsin yields a hexapeptide plus free alanine. What is the inhibitor sequence?
Reaction ConclusionsFDNB/acid hydrolysis (identifies amino-terminal aa
A is at amino terminal
Trypsin cleavage (cleaves on the carboxyl side of K and R)
A-A-K-/-(A3 , F)
Chymotrypsin cleavage (cleaves on the carboxyl side of F, W, and Y)
A-A-K-A-A-F-A