Enzymatic Fragmentation

Some of the proteolytic enzymes that are used for specifically fragmenting proteins are listed in Table 7.1. They can be separated into exo- and endopeptidases. An exopeptidase cleaves either the N- or C-terminal amino acid from the peptide chain (section 7.2). An endopeptidase cuts a peptide at specific locations within the chain to generate a number of fragments that are characteristic for each protein. 

Trypsin is the most commonly used endopeptidase, due to its high specificity. It catalyses the cleavage of the peptide chain at the carboxyl side (C-side) of positively charged residues (Arg and Lys), but only if the next residue is not Pro (Fig. 7.10). Tryptic digestion is often used as a stand-alone identification method for known proteins. The characteristic fragment pattern obtained can be compared to a database. This is often sufficient to identify a protein. 

Endopeptidases that are less specific than trypsin may also be used. However, the obtained fragments may be too small and might not exhibit enough overlap with other fragments to enable ordering of the fragments into the right sequence (section 7.8). 

By adjusting the reaction conditions and shortening the reaction time, the enzymes have only a limited access to the polypeptide due to steric hindrance. This approach is known as limited proteolysis. The method is also useful for trypsin digestion of proteins with a high Arg and Lys content. 

---

Brash has also used CD to study eluted proteins and finds large changes in a-helix content of fibrinogen, perhaps due to enzymatic fragmentation produced by the surface-induced activation of plasminogen to plasmin11 18). 

For MALDI-TOF MS several sample preparations are available with different matrices (26). The choice of the matrix and of the sample preparation should be adapted to the molecular mass of the compounds and to the complexity of the samples to analyze. a-Cyano-4-hydroxycinnamic acid (4HCCA) is preferred for peptides between 1 and 15 kDa, and the sandwich sample preparation can be universally used for molecular mass determination of pure peptides and enzymatic fragments or complex mixtures (e.g., crude hemolymph, enzymatic digests). The procedures reported below are the ones used for the discovery of the Drosophila immune-induced peptides (19,27,30). 

Watanabe, M., Watanabe, J., Sonoyama, K., Tanabe, S. 2000. Novel method for producing hypoallergenic wheat flour by enzymatic fragmentation of the constituent allergens and its application to food processing. Biosci Biotechnol Biochem 64 2663-2667. 

Figure 13 shows a simplified model of HSA based on its amino acid sequence (37,38) and results from chemical and enzymatic fragmentation studies (41,42,43,44) and low-angle X-ray scattering (45). Much... 

The synthetic scheme is shown in Fig. 15 (138), which was based on 4 + 1 enzymatic fragment condensation. A -Cbz-Tyr-Gly-Gly-Phe-Leu-NH2 and A -Boc-Tyr-Gly-Gly-Phe-Met-NH2 were prepared in good yield. 

The Pj-22 isoforms were further purified by reverse phase HPLC (separation on the basis of hydrophobicity data not shown) and subjected to amino acid composition analysis. The data are summarized in Table 1. Knowing the relative amounts of each amino acid will define an enzymatic fragmentation strategy that will permit determination of the total Pj-22 sequence. 

A considerable amount of information is available on the reversible, pH-dependent molecular conformational alterations which occur in both bovine and human serum albumins. This work has yielded valuable knowledge regarding the structural organization of the albumin molecule and has been instrumental in devising conditions and procedures for the enzymatic fragmentation of the protein and is therefore pertinent to an immunochemical consideration. 

Li J, ElRassi Z. 2002. High performance liquid chromatography of phenolic choline ester fragments derived hy chemical and enzymatic fragmentation processes Analysis of sinapine in rape seed. Journal of Agricultural and Food Chemistry, 50(6) 1368-1373. 

---