THE USE OF BENZOPHENONE AS A PHOTOAFFINITY LABEL, LABELING IN <i>p</i>‐BENZOYLPHENYLACETYL CHYMOTRYPSIN AT UNIT EFFICIENCY

Campbell, Peter M.; Gioannini, Theresa L.

doi:10.1111/j.1751-1097.1979.tb07787.x

Cited by 20 publications

(7 citation statements)

References 66 publications

(14 reference statements)

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“…Because of the small amounts of enzyme used, no attempt was made to increase the degree of inhibition by isolating unlabeled enzyme and rephotolyzing in the presence of azidowarfarin. The observed 40% maximum (Figure 4) inhibition is comparable to other similar systems in which only a single photolysis reaction is used to inactivate the enzyme (Campbell & Gioannini, 1979). Fifth, the parent ligand, acenocoumarin, afforded full protection against labeling by the photoaffinity inhibitor.…”

Section: Discussionsupporting

confidence: 61%

Photoaffinity inhibition of rat liver NAD(P)H dehydrogenase by 3-(.alpha.-acetonyl-p-azidobenzyl)-4-hydroxycoumarin

Almeda¹,

Bing²,

Laura³

et al. 1981

Biochemistry

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NAD(P)H dehydrogenase was purified in four steps from a homogenate of rat liver. The final step was affinity chromatography on Sepharose coupled to 3,3'-(m-hydroxybenzylidene)bis(4-hydroxycoumarin). The purified enzyme was inhibited competitively with respect to NADH by 3-(alpha-acetonyl-p-nitrobenzyl)-4-hydroxycoumarin (acenocoumarin) (Ki = 1.7 microM). The acenocoumarin was converted into an azide which was used to photoaffinity inhibit the enzyme. Following photolysis in the presence of the azide, the enzyme was inactivated in proportion to the concentration of azide present during irradiation. A maximum of 35-40% inhibition could be achieved by a single irradiation at 254 nm for 1.5 min. This inhibition was noncompetitive with respect to NADH. The inactivation was shown to be specific as acenocoumarin afforded complete protection against inactivation, irradiation was required to achieve inactivation, and the enzyme was unaffected by irradiation alone.

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Section: Discussionsupporting

confidence: 61%

Photoaffinity inhibition of rat liver NAD(P)H dehydrogenase by 3-(.alpha.-acetonyl-p-azidobenzyl)-4-hydroxycoumarin

Almeda¹,

Bing²,

Laura³

et al. 1981

Biochemistry

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“…There are reports of a benzophenone-containing chymotrypsin inhibitor that photolabels the active site and leaves the labeled enzyme with attenuated lytic activity. , On the basis of this precedent, the apparent photoactivation may be no more than a photolabeling of the active site. To answer this question, two labeling experiments were performed.…”

Section: Resultsmentioning

confidence: 99%

2-Aroylbenzoyl Serine Proteases: Photoreversible Inhibition or Photoaffinity Labeling?

Jones

Porter

1999

J. Am. Chem. Soc.

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Phenyl esters of 2-benzoylbenzoates were determined to be inhibitors of the serine protease enzymes chymotrypsin and thrombin. Thus, p-guanidinophenyl 2-benzoylbenzoate (1b) inhibited thrombin while the corresponding p-nitrophenyl ester (1a) inhibited chymotrypsin activity. Other p-nitrophenyl esters were prepared that display activity as chymotrypsin inhibitors, three having methoxy group substitution on the benzoyl ring: 2-methoxy (2a), 2,5-dimethoxy (3a), and 2,4,5-trimethoxybenzoyl (4). After incubation with 1b, an acyl thrombin was isolated that showed no lytic activity but which slowly regained activity in pH 7.4 buffer. Irradiation of this acyl enzyme with 366 nm light led to an enzyme that showed no gain of lytic activity over time. Incubation of chymotrypsin with 1a − 3a and 4 led to acyl enzymes which showed no activity but which regained activity slowly. Irradiation of these inactive acyl enzymes with 366 nm light led to a rapid increase in enzyme activity. The formation of acylchymotrypsins that can be photochemically deacylated is suggested by these data. Experiments that relate to the mechanism of enzyme acylation and the subsequent photochemistry of the acylenzymes are reported.

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“…For instance, the C-H bond energies for a series of stabilized radicals formed by amino acid side chains (tertiary, benzylic, and α radicals to the heteroatoms or carbonyl groups) allows for the potential labelling of all amino acid side chains through hydrogen abstraction reactions [4]. The 1,2-diradical reacts preferentially with C-H bonds over water, which affords it a higher photo-linking efficiency compared to other photo-crosslinkers such as diazirines [14,15].…”

Section: Benzophenonesmentioning

confidence: 99%

Recent Advances in Chemical Biology Using Benzophenones and Diazirines as Radical Precursors

Hassan

Olaoye

2020

Molecules

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The use of light-activated chemical probes to study biological interactions was first discovered in the 1960s, and has since found many applications in studying diseases and gaining deeper insight into various cellular mechanisms involving protein–protein, protein–nucleic acid, protein–ligand (drug, probe), and protein–co-factor interactions, among others. This technique, often referred to as photoaffinity labelling, uses radical precursors that react almost instantaneously to yield spatial and temporal information about the nature of the interaction and the interacting partner(s). This review focuses on the recent advances in chemical biology in the use of benzophenones and diazirines, two of the most commonly known light-activatable radical precursors, with a focus on the last three years, and is intended to provide a solid understanding of their chemical and biological principles and their applications.

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THE USE OF BENZOPHENONE AS A PHOTOAFFINITY LABEL, LABELING IN p‐BENZOYLPHENYLACETYL CHYMOTRYPSIN AT UNIT EFFICIENCY

Cited by 20 publications

References 66 publications

Photoaffinity inhibition of rat liver NAD(P)H dehydrogenase by 3-(.alpha.-acetonyl-p-azidobenzyl)-4-hydroxycoumarin

Photoaffinity inhibition of rat liver NAD(P)H dehydrogenase by 3-(.alpha.-acetonyl-p-azidobenzyl)-4-hydroxycoumarin

2-Aroylbenzoyl Serine Proteases: Photoreversible Inhibition or Photoaffinity Labeling?

Recent Advances in Chemical Biology Using Benzophenones and Diazirines as Radical Precursors

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