A Highly Reactive Colored Reagent with Selectivity for the Tryptophan Residue in Proteins. 2-Hydroxy-5-nitrobenzyl Bromide<sup>1</sup>

Hr, Horton; De, Koshland

doi:10.1021/ja01083a033

Cited by 197 publications

(52 citation statements)

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“…Cystatin was modified by reaction with the tryptophan-specific reagent dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide (Koshland et al, 1964;Horton & Koshland, 1965;Horton & Tucker, 1970) at pH 4.5, resulting in covalent attachment of the HNB group to the only tryptophan residue of the protein, Trp-104. The molar ratio of the bound HNB group to cystatin in preparations ofthe modified inhibitor purified by affinity chromatography was 1.12 + 0.03 (mean + S.D., n = 4), consistent with complete modification of the tryptophan residue.…”

Section: Resultsmentioning

confidence: 99%

Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site

Nycander

Björk

1990

Biochemical Journal

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The single tryptophan residue of chicken cystatin was modified with a 2-hydroxy-5-nitrobenzyl group. The change of the absorption spectrum of this group on binding of the modified cystatin to papain indicated a decreased environmental polarity of the probe. The modified inhibitor had about a 105-fold lower affinity for papain than had intact cystatin, this being due to a higher dissociation rate constant. These results show that Trp-104 of cystatin is located in or near the proteinase-binding site of the inhibitor, in agreement with a model proposed from computer docking experiments.

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Section: Resultsmentioning

confidence: 99%

Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site

Nycander

Björk

1990

Biochemical Journal

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“…Reactions with the chromophoric reagents, 2-hydroxy-5-nitrobenzyl bromide and 2-chloromercuri-4-nitrophenol yielded two yellow-colored protein derivatives having each about 2 mol bound label/mol cyanoprotein. They showed typical visible Determined at pH 12 by absorption at 405 nm using A M-' cm-' for 2-hydroxy-5-nitrobenzyl-cysteine [11,20].…”

Section: Modfication Of the Mono-s-cyano Derivative With Various Thiomentioning

confidence: 99%

Preparation and Properties of S‐Cyano Derivatives of Creatine Kinase

Terrossian

Kassab

1976

European Journal of Biochemistry

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The two reactive thiol groups of rabbit muscle creatine kinase were stoichiometrically reacted with 5,5′‐dithio‐bis(2‐nitrobenzoic acid). In the resulting inactive mixed disulfide derivative they were subsequently substituted with [14C]cyanide, the smallest uncharged thiol‐blocking group. The modified enzyme contained 1.6 mol label/mol protein and showed by Ellman's titration and amino acid analysis a concomitant loss of about 0.8‐0.9‐Si‐i group per subunit. This mono‐Scyano derivative of creatine kinase was found 73% as active as the native unniodified protein. It was still able to react in the native state with a variety of thiol reagents with the further blocking of another pair of thiol groups; their substitution once more with cyanide resulted ire the his‐Scyano derivative of creatine kinase, which lost 2 thiol groups per subunit and had about 50% of the original catalytic activity. It is concluded that the four cyanylated thiol groups are not required for the catalytic activity of creatine kinase and the cyanoprotein derivatives described are shown to be useful tools for some interesting investigations related to this enzyme.

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“…These reagents are highly specific for tryptophan, cysteine being the only other susceptible residue (Horton & Koshland, 1965). Absorption peaks at 420 nm were obtained as the solution turned yellow (shown for one concentration of reagent in Fig.…”

Section: Reagentmentioning

confidence: 99%

Chemical modification of Bacillus thuringiensis activated -endotoxin and its effect on toxicity and binding to Manduca sexta midgut membranes

Cumming

1994

Microbiology

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The effect of chemical modification of aromatic and basic residues of the Bacillus thuringiensis insecticidal CrylA(c) toxin on toxicity and receptor binding was studied. Modification of four or more of the 43 arginine residues resulted in abolition of toxicity towards Manduca sexfa and Pieris brassicae in vivo and a Choristoneura fumiferana cell line in witro. Modification of seven or more of the 27 tyrosine residues resulted in reduction of toxicity. Upon modification of most of the 10 tryptophan residues, toxicity was reduced. Modification of histidine residues had no effect on toxicity. A quantitative binding assay was developed and optimized to compare the binding of derivatives with native toxin t o M. sexta brush border membrane vesicles. The reduction or abolition of toxicity observed upon selective modification of tyrosine or arginine residues was reflected in the binding abilities of the derivatives. However a non-toxic derivative, modified at tryptophan residues, retained its ability to bind vesicles. These results suggest that tyrosine and arginine residues are involved in the binding interaction of toxin with receptor but tryptophan residues are involved in some subsequent step.

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A Highly Reactive Colored Reagent with Selectivity for the Tryptophan Residue in Proteins. 2-Hydroxy-5-nitrobenzyl Bromide¹

Cited by 197 publications

References 0 publications

Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site

Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site

Preparation and Properties of S‐Cyano Derivatives of Creatine Kinase

Chemical modification of Bacillus thuringiensis activated -endotoxin and its effect on toxicity and binding to Manduca sexta midgut membranes

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A Highly Reactive Colored Reagent with Selectivity for the Tryptophan Residue in Proteins. 2-Hydroxy-5-nitrobenzyl Bromide1

Cited by 197 publications

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Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site

Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site

Preparation and Properties of S‐Cyano Derivatives of Creatine Kinase

Chemical modification of Bacillus thuringiensis activated -endotoxin and its effect on toxicity and binding to Manduca sexta midgut membranes

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A Highly Reactive Colored Reagent with Selectivity for the Tryptophan Residue in Proteins. 2-Hydroxy-5-nitrobenzyl Bromide¹