Protein sequencing enzymatic methods

It is not practical to sequence more than about 50 amino acid residues on a single protein in this way and larger proteins need to be broken down into polypeptides with more appropriate lengths to allow complete sequencing to be carried out. This shortening of the polypeptide chain may be carried out using chemical or enzymatic methods, cf. hydrolysis. 

The overall accuracy of amino acid sequencing generally declines as the length of the polypeptide increases. The very large polypeptides found in proteins must be broken down into smaller pieces to be sequenced efficiently. There are several steps in this process. First, the protein is cleaved into a set of specific fragments by chemical or enzymatic methods. If any disulfide bonds... 

In comparison with the marked advancement in sequence determination of the amino terminal region of protein, no good methods have been developed for the determination of the amino acid sequence of the carboxyl terminal region, although several enzymatic and chemical procedures are available. Exoproteinases such as carboxypeptidases A, B, C and Y, have been employed for enzymatic determination. These enzymes catalyze sequential liberation of an amino acid from a carboxyl terminal. However, the reaction efficiency, i.e. velocity and specificity, is not always consistent for all amino acids consisiting of a... 

Peptide cleavage, partial hydrolysis of peptides and proteins by chemical ( end group analysis) or enzymatic methods (-> proteolysis) for sequence analysis. Limited peptide cleavage (limited proteolysis) is important for various metabolic processes. 

The complete analysis and sequence determination of proteins and nucleic acids relies on the splitting of these high molecular weight compounds into smaller fragments, using chemical and/or enzymatic methods. The smaller peptide or oligonucleotide fragments so produced are then analysed by various chemical or physical methods. 

The sequencing of DNA can be accomplished by using both chemical (introduced by Gilbert with his student Maxam) and enzymatic methods, especially the Sanger dideoxy-nucleotide protocol. In both approaches, and in analogy to protein analysis (Section 26-5), the unwieldy DNA chain is first cleaved at specific points by enzymes called restriction endonucleases. 

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