Protein cyanogen bromide cleavage

Fig. 24.8 Release of somatostatin from a hybrid protein by cyanogen bromide cleavage. Somatostatin can be purified free of cyanogen bromide and fragments of /J-galactosidase.

Treatment of proteins with cyanogen bromide results in cleavage of the peptide chain COOH-terminal to methionyl residues with concomitant conversion of the methionine to homoserine lactone which is in equilibrium with homoserine. In certain instances (e.g. -Met-Ser-or -Met-Thr-) some of the methionine is converted to homoserine without peptide bond cleavage (see Schroeder et al. 1969). Homoserine and its lactone are also products of the breakdown of the carboxymethylsulfonium salts of methionine ( 2.5.10). 

Cleavage of the intact protein with cyanogen bromide yields methionine and a pen-tapeptide. Treating the intact hexapeptide with trypsin yields a dipeptide, which contains tyrosine and glutamate, and a tetrapeptide. When the intact hexapeptide is treated with carboxypeptidase A, a tyrosine residue and a pentapeptide are produced. Bearing in mind that the hexapeptide is isolated from a mouse, write its amino acid sequence, using both three-letter and one-letter abbreviations. 

Hapten density, and also the common positions where haptens are bound, can also be estimated by cyanogen bromide or enzymatic cleavage of the protein and either MALDI-MS or separation of the components by reversed-phase ion-pair chromatography and electrospray or electrospray time-of-flight (TOF) analysis. 

Spectra to be analyzed via PMF are derived from a protein sample been treated with an enzyme (e.g., trypsin) or other chemical (cyanogen bromide) with specific cleavage activity. The experimental m/z values for each peptide are converted into peptide masses and compared with the theoretical mass... 

Cytochrome c(l-65) and -(66-104), derived by cyanogen bromide cleavage, form a stable complex in which the 65-66 peptide bond spontaneously slowly re-forms to give a protein with full biological activity.1124 ... 

In the treatment of RNase-A with cyanogen bromide, chain cleavage occurs at Met 13. The peptide comprising residues 1-13, where 13 is now homoserine or homoserine lactone rather than methionine, is designated C-peptide (139). This derivative added to S-protein at a molar ratio of 600 1 gave between 50 and 80% of the maximum activity. 

Another approach has been the preparation of fragments of cytochrome c, in which the heme group is bound to a portion of the protein, either by tryptic digestion or cyanogen bromide cleavage. 

PeptideMass (ExPASy Proteomic tools), which is designed for use in peptide mapping experiments, determines the cleavage products of a protein after exposure to a specific protease or chemical reagent. The enzymes and reagents available for cleavage via PeptideMass are trypsin, chymotrypsin, Lys C, cyanogen bromide, Arg C, Asp N, and Glu C. 

Figure 1 Strategy for cloning a peptide-coding sequence (CDS) as tandem repeats in the vector pET31b. The resulting fusion protein, comprising the ketosteroid isomerase (KSI), peptide repeats, and His-tag, is targeted to inclusion bodies. The fusion protein can be recovered and cleaved, in this case, with cyanogen bromide (CNBr) which acts at the methionine (M) residues allowing further separation of pure peptide from the other fusion components. The cleavage by CNBr results in a C-terminal homoserine lactone (hsl) on each peptide monomer.

Fig. 19. Topography of the NBS cleavage of the six tyrosyl peptide links of native and

Conversion of half-cystine residues in proteins and peptides to the S-methyl derivatives is advantageous in subsequent studies of amino acid sequence. Under the usual conditions of acid hydrolysis ( 2.1), S-methylcysteine is recovered in a 90% yield (Heinrikson 1971). The phenylthiohydantoin of S-methylcysteine is readily identified by routine thin layer chromatography procedures (Rochat et al. 1970). With the increasing use of the sequenator, PTH-S-methylcysteine offers a marked advantage over derivatives such as PTH-cysteic acid, or PTFl-carboxymethylcysteine, which have to be identified by special techniques (Edman 1960, 1970). S-methylcysteinyl residues provide a new point of cleavage for cyanogen bromide (5). 

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