Cleavage Of Tryptophanyl Peptide Bonds Research Articles

High-yield cleavage of tryptophanyl peptide bonds by o-iodosobenzoic acid.

A new procedure to cleave tryptophanyl peptide bonds in high yield is reported. The method involves treatment of the S-alkylated protein with o-iodosobenzoic acid. The procedure is highly selective for tryptophan and does not modify tyrosine or histidine, but may convert methionine to its sulfoxide derivative. The yields in the cleavage are 70--100%. Tryptophanyl bonds to alanine, glycine, serine, threonine, glutamine, arginine, and S-(pyridylethyl)cysteine are split in nearly quantitative yield, while those preceding isoleucine or valine are split in approximately 70% yield in the proteins examined in this work. The chemical mechanism for tryptophanyl bond cleavage has not been defined, but it is likely that oxidation of the indole ring occurs during the reaction with o-iodosobenzoic acid. Some problems with the quality of commercial preparations of the reagent are discussed.

Cleavage of tryptophanyl peptide bonds in cytochrome b5 by cyanogen bromide.

Quantitative cleavage of peptide bonds adjacent to tryptophanyl and methionyl residues in the polar moiety of cytochrome b5 was effected using cyanogen bromide in the presence of heptafluorobutyric and formic acids. Application of this method to native cytochrome b5 resulted in cleavage at tryptophanyl and methionyl residues in the polar and membranous segments in high yield. Amino acid analysis of peptides isolated from such digests indicated that tyrosine was modified and the derivative eluted in a position preceding lysine; however, the color constant with ninhydrin remained unchanged. Hydriodic acid hydrolysis of the phenylthiohydantoin derivative of the modified tyrosine regenerated the parent amino acid. Peptides containing the altered tyrosine remained susceptible to chymotryptic cleavage at this residue. Cleavage of methionyl bonds could be prevented by methylene blue-sensitized photooxidation prior to cyanogen bromide/anhydrous heptafluorobutyric acid treatment. The conditions employed for photooxidation were selective for methionyl residues, and the yield and specificity of tryptophanyl cleavage by the cyanogen bromide/anhydrous heptafluorobutyric acid method was unaffected.

Use of N-chlorosuccinimide/urea for the selective cleavage of tryptophanyl peptide bonds in proteins. Cytochrome c.

The conditions and utility of the N-chlorosuccinimide/urea (NCS/urea) reagent for the selective cleavage of tryptophanyl peptide bonds in proteins is demonstrated with cytochrome c. At low concentrations of NCS/urea the oxidation of thioether side chains in cytochrome c is the predominant reaction. Methionyl residues are oxidized to sulfoxide and the heme-thioether bridge is partially cleaved. At 10-fold excess of NCS/urea reagent, cleavage of the tryptophanyl peptide bond is optimal at approximately 50% yield in several species of cytochrome c studied. Analytical data on isolated horse cytochrome c peptide fragments demonstrate lack of modification and cleavage at tyrosyl and histidyl residues. However, at high concentrations of NCS/urea reagent (30-fold) unexpected conversions of methionine to sulfone and cysteine to cysteic acid in intact proteins are observed. This is in contradistinction to the absence of sulfone in NCS/urea-reacted amino acid mixtures. The mechanisms of halogenation and cleavage by N-bromosuccinimide, N-iodosuccinimide, and N-chlorosuccinimide are discussed. It is porposed that the selectivity with respect to halogenation by N-chlorosuccinimide is due to the insignificant participation of molecular chlorine in the NCS/urea reaction. A mechanism of halogenation and cleavage by NCS at tryptophan is also offered.

Selective chemical cleavage of tryptophanyl peptide bonds by oxidative chlorination with N-chlorosuccinimide

Tryptophanyl peptide bonds are selectively cleaved by N-chlorosuccinimide (NCS) under acidic conditions. All other peptide bonds are resistant to cleabage by this reagent. Optimal conditions for cleavage are: 2 equiv of NCS, pH 4-5, or 50-80% acetic acid for 30 min at room temperature. Under these conditions methionine residues are oxidized to methionine sulfoxides and cysteine. Other amino acids are not modified. The cleavage reaction was studied with several peptides containing tryptophan residueas successfully applied to several proteins. In alpha-lactalbumin, Kunitz trypsin inhibitor ,and apomyoglobin, selective cleavage of the expected tryptophanyl peptide bonds was obtained in 19-58% yield. The glucagon molecule was fragmented into two peptides in 32% yield.

Oxidative Cleavage of Tryptophanyl Peptide Bonds during Chemical- and Peroxidase-catalyzed Iodinations

Abstract Tryptophanyl peptide bonds are oxidatively cleaved by active that is generated with H2O2, iodide, and a peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7). Complete oxidation of tryptophan derivatives and peptides to the oxindole occurs within 2 min at pH 3 to 5 with either lactoperoxidase or horseradish peroxidase, and a 2.5-fold molar excess of iodide plus H2O2. Under identical conditions, simple tryptophan peptides are cleaved 30 to 40% in 10 min at pH 5.0, and the rate of cleavage is parallel to, but less than, the rate of oxidation. All three constituents (iodide, H2O2, and peroxidase) are required for oxidation and fission to proceed. Iodinating reagents yield similar results, except that they oxidize tryptophan derivatives and peptides (Z-Trp-X, X-Trp-X) over the pH range of 2 to 11. Fission of the tryptophan peptide proceeds in acid media with a maximum at pH 5.0, and no cleavage occurs in alkaline media. A 2.5-fold molar excess of positive halogen reagents, ICl, iodide oxidized with chloramine-T, and N-iodosuccinimide completely oxidize tryptophan peptides at pH 5.0 within 1 min, whereas oxidation by I2 and I3- is 2 and 8 times slower, respectively. Chloramine-T per se oxidizes tryptophan peptides at about the same rate as I2 below pH 6.5, but is unreactive above this pH. All of the iodinating reagents induce complete oxidation within 1 min at pH 9.5. Thirty to forty percent of a tryptophan peptide (e.g. Z-Trp-Gly or Z-Trp-Leu) is cleaved in 10 min at pH 5 by a 2.5- to 10-fold molar excess of all the reagents, except for I3- which promotes half as much cleavage. With all of the iodinating reagents, the rate of cleavage is parallel to, but less than, the rate of oxidation. Iodimetric titrations and pH profiles suggest that the oxidation and oxidative cleavage of tryptophan peptides during iodination proceeds via the mechanism proposed for brominating agents (Patchornik, A., Lawson, W. B., Gross, E., and Witkop, B. (1960) J. Amer. Chem. Soc. 82, 5923). Two equivalents of I+ or I2 convert the indole nucleus to the oxindole, which undergoes spontaneous hydrolysis after cyclization to the iminolactone. Unlike brominating reagents, a third iodine equivalent does not form a stable iodooxindole derivative with several different tryptophan peptide models or oxindole itself. Two exceptions are Z-Trp-Gly and Z-Trp, which are probably iodinated by ICI on the benzyloxycarbonyl moiety. Free tryptophan is oxidized by 2.6 to 2.9 eq of iodine (ICl or I2), but it does not become iodinated in labeling experiments with 131I2 or 131ICl.

Oxidation and oxidative cleavage of tryptophanyl peptide bonds during iodination

Tryptophan peptides are oxidized to the oxindole by various chemical and peroxidase catalyzed iodination procedures in the pH range 4.0–7.5. This oxidation results in significant cleavage of tryptophanyl peptide bonds at pH 4–5. Thus, destruction of some biologically active peptides and proteins during iodination may be due to the modification of essential tryptophan residues.

Selective chemical cleavage of tryptophanyl peptide bonds in peptides and proteins

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSelective chemical cleavage of tryptophanyl peptide bonds in peptides and proteinsYigal Burstein and Abraham PatchornikCite this: Biochemistry 1972, 11, 25, 4641–4650Publication Date (Print):December 1, 1972Publication History Published online1 May 2002Published inissue 1 December 1972https://doi.org/10.1021/bi00775a001RIGHTS & PERMISSIONSArticle Views126Altmetric-Citations29LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (966 KB) Get e-Alerts Get e-Alerts

Nonenzymatic cleavage of tryptophyl peptide bonds in peptides and proteins: I. Cleavage of C-tryptophyl peptide bonds in model peptides through their conversion into N′-formylkynurenine derivatives

Procedures for effecting specific cleavage of tryptophanyl peptide bonds of proteins have been intensively sought in the past decade. The reagents tested in such procedures include periodate, ozone, and in particular brominating agents. N-Bromosuccinimide (NBS) was the first reagent proposed for the cleavage of tryptophanyl peptide bonds, but various side reactions of this extremely reactive reagent with other amino acid side chains, as well as poor yields in peptide bond fission, hampered practical applications of the reagent. 2,4,6-Tribromo-4-methylcyclohexadienone is found to be less reactive than NBS, giving similar yields of cleavage, but extensive modification of tyrosine, histidine, and sulfur amino acids is observed in this case. Positive halogen reagents, such as ICl, iodide oxidized with Chloramine-T, N-iodosuccinimide, and active iodine generated with H2O2, iodide, and a peroxidase, also give moderate yields of cleavage. A mixture of dimethyl sulfoxide (DMSO) and concentrated aqueous HBr has been recently used as a brominating agent to effect selective cleavage of tryptophanyl peptide bonds in peptides and proteins.