Comparison of Resorufin Acetate andp-Nitrophenyl Acetate as Substrates for Chymotrypsin

Kitson, Trevor M.

doi:10.1006/bioo.1996.0029

Cited by 14 publications

(8 citation statements)

References 9 publications

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“…Profluorophore 10 has long been recognized as an effective esterase substrate,53 but the utility of this compound is limited by its poor stability in aqueous solution 54. The chemical stability of resorufin derivatives 9 and 10 was similar to that of the Tokyo Green-based substrates, as expected from their analogous structures.…”

Section: Resultsmentioning

confidence: 87%

Synthesis and utility of fluorogenic acetoxymethyl ethers

2011

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Phenolic fluorophores such as fluorescein, Tokyo Green, resorufin, and their derivatives are workhorses of biological science. Acylating the phenolic hydroxyl group(s) in these fluorophores masks their fluorescence. The ensuing ester is a substrate for cellular esterases, which can restore fluorescence. These esters are, however, notoriously unstable to hydrolysis, severely compromising their utility. The acetoxymethyl (AM) group is an esterase-sensitive motif that can mask polar functionalities in small molecules. Here, we report on the use of AM ether groups to mask phenolic fluorophores. The resulting profluorophores have a desirable combination of low background fluorescence, high chemical stability, and high enzymatic reactivity, both in vitro and in cellulo. These simple phenyl ether-based profluorophores could supplement or supplant the use of phenyl esters for imaging biochemical and biological systems.

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Section: Resultsmentioning

confidence: 87%

Synthesis and utility of fluorogenic acetoxymethyl ethers

2011

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“…[25][26][27][28][29][30][31][32][33][34][35] However, to our knowledge, generation of 2 from 1 has been used as an indicator reaction only for studies of esterase activity of cytosolic aldehyde dehydrogenase and chymotrypsin. [20][21][22][23] Thus, this is the first report to show that transformation of 1 to 2 is induced by H 2 O 2 . Although practical application of the present indicator reaction requires prevention of simple hydrolysis of 1 in blank solution, the present results suggested some intriguing points from the standpoint of enzymatic analysis of biological compounds, as follows: 1) H 2 O 2 -induced deacylation of 1 and other acylated 2 will find applications as indicator reactions for enzymatic analysis of various biological compounds based on oxidoreductases with oxygen as an electron acceptor such as GOD, cholesterol oxidase, uricase, etc.…”

Section: Resultsmentioning

confidence: 52%

“…Materials GOD from Aspergillus niger (EC 1.1.3.4) and glucose were used as supplied from Wako Pure Chemical Industries, Ltd. Acetyl resorufin (1) [20][21][22][23] was prepared by reaction of resorufin sodium salt with acetic anhydride in pyridine at room temperature and recrystallized from ethyl acetate. 24) All other chemicals were of reagent grade and were used without further purification.…”

Section: Methodsmentioning

confidence: 99%

Hydrogen Peroxide-Induced Deacetylation of Acetyl Resorufin as a Novel Indicator Reaction for Fluorometric Detection of Glucose Using Only Glucose Oxidase.

Maeda

Matsu-Ura

Nishida

et al. 2001

CHEMICAL & PHARMACEUTICAL BULLETIN

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Spectrophotometric or fluorometric methods with glucose oxidase (GOD)-peroxidase (POD)-chromogen(s) systems are well known as useful tools for determination of glucose. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] Indicator reactions in these methods are based on oxidative formation of a colored or fluorescent substance from a chromogen(s) with H 2 O 2 , generated through GODcatalyzed oxidation of glucose, in the presence of POD. These color reactions are well-characterized chemical processes. However, the POD-dependent indicator reactions are known to be inevitably disturbed by electron donors present in biological samples such as ascorbic acid, uric acid, and bilirubin.3) Major processes of interference by these compounds are as follows: 1) reduction of oxidatively formed colored or fluorescent substance to its original chromogen(s); and 2) competition with a chromogen in reduction of H 2 O 2 . Several methodologies have been developed for elimination of such interference in glucose determination with GOD-POD-chromogen(s) systems.3) However, the most straightforward approach to glucose determination free from such interference is design of a novel indicator reaction without recourse to redox reactions coupled with H 2 O 2 and POD. To our knowledge, only a few methods with non-redox color reactions for glucose determination using only GOD have been reported. These methods utilize transformation of a vanadium(V), 16) titanium(IV) 17) or dinuclear iron(III) 18) complex to its adduct with H 2 O 2 accompanied by a bathochromic shift as a spectrophotometric indicator reaction. As expected, it was demonstrated that glucose determination with vanadium(V) and titanium(IV) complexes was not affected by various substances usually present in serum or added to test solution. 16,17) However, spectrophotometric or fluorometric determination of glucose with high accuracy should use formation of a colored or fluorescent substance from a chromogen rather than a color change of a dye as an indicator reaction.Recently, resorufin (2) was shown to be able to reoxidize a reduced form of GOD at 37°C, being transformed to a colorless dihydro derivative, although the reductive bleaching of the dye is of no use for GOD-based determination of glucose as an indicator reaction. 19) This finding prompted us to examine how resorufin derivatives such as acetyl resorufin (1) would behave in GOD-catalyzed oxidation of glucose, and it was found that 1 behaves in a manner different from 2 in the enzymatic reaction. Here, we report that deacetylation of non-fluorescent 1 to fluorescent 2 is induced by H 2 O 2 generated in GOD-catalyzed oxidation of glucose, and the transformation is promising as an indicator reaction for fluorometric determination of glucose without significant effects of ascorbic acid, uric acid, or bilirubin. ExperimentalMaterials GOD from Aspergillus niger (EC 1.1.3.4) and glucose were used as supplied from Wako Pure Chemical Industries, Ltd. Acetyl resorufin (1) [20][21][22][23] was prepared by reaction of resorufin sod...

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“…As both substrates show a pre-steady-state burst of product release (see below), we know k cat represents the rate of acyl-enzyme hydrolysis, and (in the absence of other factors) this should of course be exactly the same for both substrates since they have a common acyl group. Our experimental values are close but not identical; it is possible that the rate of acyl-enzyme hydrolysis is affected by other substrate molecules binding in chymotrypsin's extended substrate-binding domain (11).…”

Section: Action Of Chymotrypsin On 4-methylumbelliferyl Acetatementioning

confidence: 61%