Pepsin fragmentation specificity

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

To localize the rate of deuterium buildup to specific amides, the analyte protein is fragmented into a collection of peptides using combinations of endo- and exoproteases. Due to the low pH of the quench conditions in which the protein and peptide samples are maintained after deuterium labeling, acid-reactive proteases such as pepsin must be employed. Studies with combinations of acid-reactive en-doproteinases and carboxypeptidases have been employed to achieve greater sequence coverage and higher amide resolution [42, 45]. 

Figure 4.5. Schematic representation of enzyme specific cieavage of immunogiobuiin G (igG) by pepsin and papain. Treatment of igG with pepsin produces two unique fragments. Fab, with two antigen binding sites and Fj without binding sites. Treatment of igG with papain generates two Fab and one F fragments.

Because of its specificity for basic residues, trypsin converts a protein into a relatively small number of tryptic peptides which may be separated and characterized. Trypsin acts primarily on denatura ted proteins, and to obtain good results the disulfide bridges must be broken first. Chymotrypsin is less specific than trypsin and pepsin is even less specific (Table 3-2). Nevertheless, they can be used to cut a peptide chain into smaller fragments whose sequences can be determined. To establish the complete amino acid sequence... 

To establish the amino acid sequence unequivocally it is necessary to have peptides with overlapping sequences. This may be accomplished by determining the sequence of fragments obtained from treating a second aliquot of the protein with chymotrypsin. If these fragments are then treated with trypsin as a check, peptides identical to those obtained previously by successive treatment with trypsin and chymotrypsin are obtained. Other proteolytic enzymes, such as pepsin, subtilisin, and papain, with wider specificity than trypsin and chymotrypsin have proved useful in sequencing of some proteins. 

Next to trypsin chymotrypsin is the most preferred proteolytic enzyme in sequencing. Its specificity is less absolute than that of trypsin. Primarily the bonds that follow phenylalanine, tyrosine and tryptophan are cleaved, but measurable hydrolysis takes place next to leucine and methionine residues as well. It is advisable, therefore, to determine in preliminary experiments the conditions (enzyme-substrate ratio, time, temperature) best suited for the formation of a few and well separable fragments. Occasionally also less specific enzymes, such as pepsin, papain or thermolysin find application in structure elucidation. For the hydrolysis of specific bonds new microbial proteases can be isolated. There are known prolidases and also enzymes which hydrolyze solely the bond which follows a pyroglutamyl residue and so on. 

Figure 6.2 Pepsin (top) and protease type XIII (bottom) cleavage preferences for myoglobin, IPMS, RACE, and KIT proteins. The preference ratio Is the number of fragments with the specified C-terminal amino acid divided by the total number of that specific amino acid In the protein. Reproduced with permission from [31]. 2008, American Chemical Society...

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