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