Cyanylation of sulfhydryl groups by 2-nitro-5-thiocyanobenzoic acid. High-yield modification and cleavage of peptides at cysteine residues

Degani, Yair; Patchornik, A.

doi:10.1021/bi00698a001

Cited by 242 publications

(159 citation statements)

References 29 publications

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“…The contribution from spontaneous hydrolysis of NTCB was subtracted by addition of NTCB to 1 ml 50 mM potassium phosphate, pH 7.6, inserted into the reference position of the spectrophotometer. The quantitative estimation of the modification was based on A~;412 = 13600 (9).…”

Section: Modifications Of Cpd-ymentioning

confidence: 99%

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Cyanylation of the single sulfhydryl group in carboxypeptidase Y with cyanogen bromide

Breddam

Kanstrup

1987

Carlsberg Res. Commun.

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The unblocked cysteinyl residue (Cys341), located at the binding site of carboxypeptidase Y, does not react with the bulky cyanylating reagent 2-nitro-5-thiocyanatobenzoic acid unless the enzyme is denatured. However, it reacts specifically with small concentrations of ~4CNBr forming the corresponding alkyl thiocyanate. This results in reductions in the kcat/Km values for the hydrolysis of ester and peptide substrates to 5-25% of the values obtained with unmodified enzyme, due both to decreases in the k~a t values and increases in the Km values. The rate of deacylation of the indoleacryloyl acyl enzyme intermediate, formed by reaction of the cyanylated enzyme with indoleacryloyl imidazole, is reduced to 20% of the value obtained with the unmodified enzyme. The results suggest that Cys TM participates in catalysis although it is not essential.

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Section: Modifications Of Cpd-ymentioning

confidence: 99%

“…The substrates FA-Phe-Leu-OH, FA-Phe-Phe-OH, FA-AIa-Phe-OH, FA-Phe-Gly-OH, and FAPhe-OEt and the reagent NTCB were synthesized as previously described (5,8,9).…”

Section: Introductionmentioning

confidence: 99%

Cyanylation of the single sulfhydryl group in carboxypeptidase Y with cyanogen bromide

Breddam

Kanstrup

1987

Carlsberg Res. Commun.

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“…Samples (a)-(d) were each suspended in 40/al buffer A containing 8 M urea (BRL, ultra pure grade), 10 mM dithiothreitol and 1°70 SDS, and incubated at 37°C for 1 h; sample (e) was resuspended in buffer A (40/A). 2/d of 1 M 2-nitro-5-thiocyanobenzoic acid (NTCB, Fluka) [10] in ethanol was then added to samples (a), (c) and (e). After incubation of all samples at 37°C for 30 min, the pH was raised to 9 by addition of 3 M Tris base (-3.5/d), followed by further incubation at 37°C for 20 h. Electrophoresis sample buffer (5 ×) was then added and electrophoresis and autoradiography were performed as described.…”

Section: S-cyanylation Of Affinity-labeled Membranesmentioning

confidence: 99%

“…1, lanes 3,5) implies incomplete modification or cleavage due to side reactions such as/5-elimination [10]. It is worth noting that the cytoplasmic Cys 244 probably reacted with NTCB in the intact membranes to yield F21 ( fig.3B), but not in membranes solubilized in SDS.…”

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confidence: 98%

Chemical characterization of ligand binding site fragments from turkey β‐adrenergic receptor

Eshdat¹,

Chapot²,

Strosberg³

1989

FEBS Letters

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Affinity-labeled ~-adrenergic receptor from turkey erythrocyte membranes was specifically cleaved near cysteine residues after S-cyanylation. Analysis of the labeled polypeptide fragments suggests that iodocyanopindolol diazirine reacted with an amino acid residue which is located in the non-glycosylated region containing the sixth and seventh transmembrane domains of the receptor. However, the possibility cannot be excluded that a second residue, located between the third and fifth transmembrane domains, was also labeled. Since treatment with either hydroxylamine or triethylamine resulted in removal of the affinity label from the protein, the present study suggests that aspartic or glutamic acid residues are present in the adrenergic-binding site which is located in the above-mentioned domains. The procedure for specific chemical cleavage of the afffinity-labeled adrenergic receptor should also be useful for future structural and comparative studies of other adrenergic receptors.

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“…2-Nitro-5-thiocyanatobenzoic acid (NTCB) is used for the break of polypeptide bonds formed by cysteine residues [13]. NTCB cyanates the cysteine thiol group and transforms it into β-thiocyanatoaniline residue which is cyclized into 2-iminothiazolidine carbonic acid with the break of peptide bond under soft conditions [14,15].…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Antimicrobial Activity of Products Obtained via Anionarylation of Acrylic and Metahcrylic Acids Amides and Nitriles by 5-Carboxyphenylene-1,3-Bisdiazonium Salts

Yatsyuk¹,

Pokryshko²,

Yaniv³

et al. 2015

ChChT

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Cyanylation of sulfhydryl groups by 2-nitro-5-thiocyanobenzoic acid. High-yield modification and cleavage of peptides at cysteine residues

Cited by 242 publications

References 29 publications

Cyanylation of the single sulfhydryl group in carboxypeptidase Y with cyanogen bromide

Cyanylation of the single sulfhydryl group in carboxypeptidase Y with cyanogen bromide

Chemical characterization of ligand binding site fragments from turkey β‐adrenergic receptor

Synthesis and Antimicrobial Activity of Products Obtained via Anionarylation of Acrylic and Metahcrylic Acids Amides and Nitriles by 5-Carboxyphenylene-1,3-Bisdiazonium Salts

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