Protein selective cleavage

Selective cleavage of peptides and proteins is an important procedure in biochemistry and molecular biology. The half-life for the uncatalyzed hydrolysis of amide bonds is 350 500 years at room temperature and pH 4 8. Clearly, efficient methods of cleavage are needed. Despite their great catalytic power and selectivity to sequence, proteinases have some disadvantages. Peptides 420,423,424,426 an(j proteins428 429 can be hydrolytically cleaved near histidine and methionine residues with several palladium(II) aqua complexes, often with catalytic turnover. 

Kostic, N. M. Catalysis of selective cleavage of peptides and proteins with palladium(II) complexes, American Chemical Society, Washington, D. C. In Book of Abstracts, 212th ACS National Meeting, Orlando, FL, August 25-29, 1996. 

Withop, 13. Nonenzymatic methods for the preferential and selective cleavage and modification of proteins. Advances in Protein tlhemistry 16, 221 321 (1961). 

Nonenzymatic Methods for the Preferential and Selective Cleavage and Modification of Proteins B. WiTKOP... 

Selective Cleavage and Modification of Peptides and Proteins T. F. Spande, B, Witkop, Y. Decani, AND A. PaTCHORNIK... 

For trypsinogen activation, enteropeptidase (enterokinase, EC 3.4.21.9) [23] is a key enzyme for mammalian protein digestion. The selective cleavage site of trypsinogen by enteropeptidase initiates Lys6-Ile7 bond. Then trypsin (EC 3.4.21.4) activates other zymogens. Thus, the formation of trypsin by enteropeptidase is the master activation step. 

The sequencing strategy follows the order 1) preparation of pure protein, 2) selective cleavage of peptide bond 3) isolation of cleaved peptide fragments, 4) analysis of primary structure on amino acid sequencer, 5) determination of entire primary structure. Although this order has not changed since our primary structure determination work on AspAT carried out in early 1970, many improvements have been made, e.g. preparation of micro scale amount of sample and introduction of high sensitivity analytical instruments. These improvements have shortened analysis time and lowered the sample amount of required. 

When a protein is cleaved into manageable peptides and the sequence of each peptide is determined, the next problem is that of ordering the peptides themselves in the correct sequence. For this, at least two sets of peptide sequences from different selective cleavage methods are required. 

NONENZYMATIC METHODS FOR THE PREFERENTIAL AND SELECTIVE CLEAVAGE AND MODIFICATION OF PROTEINS... 

Where peptide chemistry can make a contribution toward the proof of protein structure is in the application and continued formulation of degrada-tive techniques. Since fragmentation of the protein molecule to smaller peptides is probably the key reaction in degradative processes, the search for specific reagents for the selective cleavage of various peptide bonds and the standardization of existing techniques is of prime importance (Katsoyannis, 1961). 

At the present exploratory level the cleavage of histidine peptides has to await further refinement in order to be applicable to proteins and to qualify as a selective cleavage. 

Certainly, all the reactions utilized for the selective cleavage of proteins are conceptually not new and, in retrospect, surprisingly simple. This preview may serve a useful purpose in delineating, perhaps for the first time, the criteria and requirements that are essential to selective cleavage. In principle, all selective cleavages are, or must be, variations of a key theme,... 

Recently, 2-(2-nitrophenylsulfenyI)-3-methyl-3-bromoindolenine (BNPS-skatole) has replaced N-bromosuccinimide, which had been used frequently in earlier studies. At low reagent to protein tryptophan ratios, in 50% aqueous acetic acid, BNPS-skatole reacts selectively with tryptophan residues converting these to the oxindole derivative. Methionine is concommitantly converted to the sulfoxide. At high concentrations of reagent, slow selective cleavage (to the extent of 15-60%) of the peptide bonds involving tryptophanyl residues is... 

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. In Nature (London) 340 680-685 Laugwitz KL, Spicher K, Schultz G et al. (1994) Identification of receptor-activated G proteins selective immunoprecipitation of photolabeled G-protein a subunits. In Methods Enzymol. 237 283-294 Meyer T, Hilz H (1986) Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistent hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides. In Ear. J. Biochem. 155 157-165... 

Fig. 4.68. Schematic drawing illustrating the selective cleavage of a peptide bond in a protein, which ctm result in altered enzymatic properties.

Site-Selective Cleavage of a Broad Range of Proteins by Cu(II) Complex Combined With an Aldehyde Group / 110... 

Although site-selective cleavage of a protein or amino acid derivatives with intermolecular catalysts is achieved by using Pd(II) complexes, the Pd(II) complexes require highly acidic conditions and, in some cases, acetone medium. The Pd(II) complexes may be useful for practical applications that allow such conditions. 

Having cleaved a protein into peptides of manageable length for Edman analysis, and determined the seqnence of each peptide, next we need to order the peptides correctly. For this, at least two sets of peptide seqnences, from different selective cleavage reactions, are reqnired. 

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