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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. (-AMINO ACID) USING FNDB (FLUORODINITROBENZENE), DABSYL CHLORIDE AND DANSYL CHLORIDE REPEATED DEGRATION BY EDMAN METHOD

REAGENT CYANOGEN BROMIDE


0-IODOSOBENZOATE HYDROXYLAMINE 2-NITRO-5-THIOCYANOBENZOATE TRYPSIN clostripain Staphylococcal protease thrombin chymotrypsin Carboxypeptidase A

CLEAVAGE SITE CARBOXYL SIDE OF M RESIDUES


CARBOXYL SIDE OF W RESIDUES N-G BONDS AMINO ACIDS OF C RESIDUES Carboxyl side of K and R Carboxyl side of R residues Carboxyl side of D and E residues Carboxyl side of R Carboxyl side of Y, W, F, L, and M 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-R N-G-M-F-V-K The smaller peptides obtained by chymotrypsin treatment had the ff sequence: V-K-L-S-Y A-I-R N-G-M-F Deduce 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 peptides 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 FDNB/acid hydrolysis (identifies amino-terminal aa Trypsin cleavage (cleaves on the carboxyl side of K and R) Chymotrypsin cleavage (cleaves on the carboxyl side of F, W, and Y)

Conclusions A is at amino terminal

A-A-K-/-(A3 , F)

A-A-K-A-A-F-A

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