Edman degradation proteolytic digestions

Conventionally, the first attribute known about an enzyme used to be its function, usually in a crude extract. This property was screened for in microbial cultures or in tissue samples. The crude extract was then purified to homogeneity and the protein subjected to biochemical studies to learn of its pH and T profiles, its pi and subunit composition, catalytically important residues, and other properties. Proteolytic digestion of the protein with subsequent Edman degradation led to the primary sequence, but no information on the secondary structures such as a-heli-ces and [5-sheets or the folding in three dimensions of the polypeptide chain. The primary sequence could have been used to deduct the gene sequence but, with the degeneration of the code, several possibilities for certain amino acids occur, which makes prediction of the gene sequence a risk. 

Depending on the number of phosphorylation sites in the phosphoprotein to be analyzed, and the efficiency of proteolytic digestion as few as a 100 Cerenkov cpm of a phosphoprotein may suffice to give a satisfactory analytical peptide map. Ideally, a few hundred counts per minute should be used, and for most phosphoproteins this should be attainable by metabolic labeling of cells with and easily achieved if in vitro phosphorylation with [y- P]ATP is used to label the protein. If phospho-amino acid determination, secondary digestion, or Edman degradation are to be carried out on individual phosphopeptides (Sections I-L), then commensurately more starting radioactivity will be required. 

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