Digestion, cyanogen bromide

Radiolabeled 3-demethyl-3-chloroacetylthiocolchicine with a l4C label in the chloroacetyl moiety (DCTC) was found to be a potent inhibitor of tubulin polymerization and of colchicine binding to tubulin. The reaction was 80-90% inhibited in the presence of saturating, amounts of known antitubulin compounds such as podophyllotoxin, combretastatin A-4, and colchicine itself. The tubulin /3 subunit was labeled 5-6 times faster than the a subunit. Cyanogen bromide digestion of the /3 subunit which had reacted covalently with DCTC indicated that at least three positions in /3-tubulin had reacted with DCTC. Purification and amino acid sequencing of these peptides are in progress (138). 

Fig. 4 Cyanogen Bromide Digest of Sonic Hedgehog Protein.

Insoluble peptides produeed from tryptic or cyanogen bromide-digested proteins were effectively analyzed by SEC with SDS in the mobile phase to separate families of peptides that could subsequently be analyzed by gel electrophoresis or another technique [54]. Complexes between heat shock proteins and unfolded substrate proteins have also been monitored successfully using SEC [55]. The effect of ATP and ADP on the complexation could be readily ascertained with this technique. 

Location of the Modified Residues. In order to determine the location and number of amino acid residues reacted with ozone, the ozonized samples were allowed to undergo cyanogen bromide cleavage and trypsin digestions. 

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. 

Harris et al. [8] has described methods for the determination of cyanide in these materials based on either spectrophotometry using p-phenylene diamine pyridine or gas chromatographically following conversion of cyanide to cyanogen bromide. Cyanide is extracted from the sample by digestion with phosphoric acid. Recoveries were in the range 96-99% (spectrophotometric method) and 90-96% (gas chromatographic method). 

Fig. 1 Summary of the data used to establish the complete amino acid sequence of Er-1 mating pheromone. The peptides have been designated and numbered according to the type of digest ana the theoretical order in which they appear in the sequence. Designations are CNBr, cyanogen bromide T, trypsin V8, . aureus V8 protease CT, chymotrypsin. Peptiaes indicated by two numbers connected with a hyphen result from partial cleavage. Residues directly identified by automated Edman degradation and carboxypeptidase Y digestion (CP-Y) are marked by right and left arrows, respectively, residues identified by amino acid composition are indicated by dashed lines. Taken from ref. 13 and reproduced by permission of the American Society of ...

Fig. 2. Schematic linear sequence diagram of fiagment alignment illustrating the major fiagments produced by eidier limited chymotryptic proteolysis or digestion with cyanogen bromide. The two homologous cGMP-biding sites are indicated by the black rectangles. Reprinted from (52).

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