Chymotryptic digestion

Figure 8. Fractionation of a-chymotryptic digest of myosin on the SW-3000 column, A 500-nL aliquot of crude digest containing 8.5 mg/mL of protein fsee Methods section) was chromatographed as in Figure 7 at 24° with a flow rate...

Staros, J.V., and Kakkad, B.P. (1983) Cross-linking and chymotryptic digestion of the extracytoplasmic domain of the anion exchange channel in intact human erythrocytes. J. Membr. Biol. 74, 247-254. 

K Narita. Isolation of acetylseryltyrosine from the chymotryptic digests of five strains of tobacco mosaic virus. Biochim Biophys Acta 30, 352, 1958. 

You have a peptide that is a potent inhibitor of nerve conduction and you wish to obtain its primary sequence. Amino acid analysis reveals the composition to be Ala(5) Lys Phe. Reaction of the intact peptide with FDNB releases free DNP-alanine on acid hydrolysis. e-DNP-lysine (but not a-DNP-lysine) is also found. Tryptic digestion gives a tripeptide (composition Lys, Ala (2)) and a tetrapeptide (composition Ala(3), Phe). Chymotryptic digestion of the intact peptide releases a hexapeptide and free alanine. Derive the peptide sequence. 

The direct acylation and methylation of the free 01-NH2 groups of proteins have been proposed to be useful in providing resistance toward proteolytic attacks. Although the basis for this explanation is not always readily apparent from the known specificities of proteases, it may be valid in some cases. Thus, the acetylated N-terminus of a-crystallin, the major protein found in eye lens (17), is presumably important for the protein to survive in an environment rich in leucine aminopeptidase. On the other hand, it is difficult to rationalize that the acetylated N-terminus of bovine pancreatic a-amylase is in any way responsible for the fact that the enzyme is exceedingly stable against tryptic and chymotryptic digestion (18). The function of the acetylation is, in this case, as obscure as is the basis on which a-amylase is selected for acetylation among the many non-acetylated companion pancreatic proteins. 

Tsuge, K., Seto, Y. (2006). Detection of butyrylcholinesterase-nerve gas adducts by liquid chromatography-mass spectro-metric analysis after in gel chymotryptic digestion. J. Chromatogr. B 838 21-30. 

Comparison of the contour plot of a chymotryptic digest of cytochrome-C with that of tile re-deutero-acetylated chymotryptic digest revealed two major and two minor peptides that had unchanged mobility and mass (Fig. 1). Each of these were investigated as the possible amino terminal peptide. 

Figure 3. Contour plot of an LC/MS of a chymotryptic digest of modifled phosphorylase b, printed in black and white mode. Circles indicate the positions of peaks from a contour plot of the le-acetylated digest for comparison. The two significant shared peaks (indicated by arrows) are the N-terminal blocked peptide (at m/z=514 and 13 min elution time) and the leucine enkephalin internal standard (at m/z=SS6 and 21 min elution time).

Comparison of e contour plot from the chymotryptic digest of phosphorylase to that after re-acetylation revealed only two ions shared by both (Fig. 3). One peptide eluted at about 20 minutes with an m/z of 556.6, again representing the leucine enkephalin internal standard. The other shared ion eluted at about 12 minutes with m/z 514.5. Tandem mass spectrometry of this ion gave a CID spectrum (Fig. 4) consistent with the amino terminal four amino acids, including the acetylated (natural abundance) amino terminal serine. This indicates that the chymot sin cleaved at the fourtii residue, a leucine. 

In the course of sequence analysis studies, chymotryptic digests of several proteins and peptides have been examined in detail. These studies have revealed significant chymotryptic cleavage at many bonds often thought to be resistant to chymotrypsin. Table VI lists the types of bonds in nine... 

Figure 4.25. Overlap Peptides. The peptide obtained by chymotryptic digestion overlaps two tryptic peptides, establishing their order.

The limited chymotryptic digestion of the LHC Il-apo-proteins was done with electroeluates of gel pieces from SDS-PAGE, containing about 0.1% SDS. 

Syntheses of human insulin B-chain S-sulfonate, the destripeptide B of human insulin, and the destripeptide B of bovine insulin were described by Katsoyannis and collaborators. Two advances noted which were not suitable for incorporation in the table concerned the complete amino acid sequence of ribonuclease Ti based on chymotryptic digests, and the photochemical modification of Gly-containing proteins such as collagen lysozyme and ribonuclease by photoalkylation with 1-butene. ... 

Fig. 5. Digestion of automodified poly(ADP-ribose) synthetase with a-chymotrypsin and with papain. The enzyme (40 Mg) was automodified with 1.4 idM [adenine- ]- C]NAD (423 dpm/ pmol) and then digested with oi-chymotrypsin or further with papain. The radioactivity was detected by fluorography. a The automodified enzyme b the enzyme after digestion with a-chymotrypsin c the fragment obtained by digestion with papain after chymotryptic digestion...

Fig. 1. Epitope mapping Proteolytic cleavage of poly(ADP-ribose) polymerase. Stained gel (A) and immunoblot (B). The chymotryptic digestion (1) and the chymotrypdc-tr tic digestion (2) were carried out at 25°C in 50 mM Tris pH 8,0.2 M NaCl, 10 mM mercaptoethanol and 10% glycerol. The 30 min chymotryptic digestion was stopped with 1 niM PMSF and the subsequent 10 min tryptic digestion by treatment with 20% TCA. The digests were dissolved in SDS sample buffer and subjected to a 15% acrylamide SDS electrophoresis (6). The immunoblots were revealed witib goat anti-mouse antibody (Amersham). Relative molecular masses of standards (St) and fragments as extrapolated from standards are indicated as kDa.

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