Fluorogenic substrate

Fluorogenic substrates Extraneous fluorescent material may be present in ordinary distilled water, making filtration necessary. 

BGase is the most often used enzyme if high detectability is required. The hydrolysis of 4-methylumbelliferyl-D-galactopyranoside yields fluorescent 4-MU. The wavelength used for excitation and emission analysis are 360 and 440 or 450 nm, respectively. Although 

Prepare 10 mM sodium phosphate buffer, pH 7.0. containing 100 mM NaCl, 1 mM MgCh, 0.r% NaNjand 0.1% BSA. 

Prepare a 0.1 mM 4-MU-Gal solution in the buffer. This is achieved by diluting 

The stop-buffer , used to terminate the reaction after a predetermined period e.g 60 min) by adding 0.5 volume, consists of 1.0 M glycine-NaOH, pH 10.3. 

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Oxidant production is measured with the fluorogenic substrate para-hydroxyphenylacetic acid (PHPA) in the presence of superoxide dismutase and peroxidase (9). Under these conditions, superoxide is converted to H2O2 by the superoxide dismutase, and two molecules of PHPA are converted to a fluorescent diadduct by H2O2 and peroxidase. Similar assays have been devised using homovanillic acid (16) or scopoletin (17) instead of PHPA. 

Gershkovich AA, Kholodovych W (1996) Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). J Biochem Biophys Methods 33 135-162... 

The introduction of FRET pairs with improved properties allows the generation of probes suitable for the measurement of spatio-temporal activities in living cells and in vivo. One example is the imaging of caspase-3 activation in HeLa cells after inducing apoptosis. Previous fluorogenic substrates, such as DEVD-MCA, could not be used in living cells because the autofluorescence interfered with the low emission wavelength of the coumarin dye MCA [123], With the incorporation of the FRET pair 6-carboxydichlorofluorescein and 5-carboxytetramethylrhodamine into another peptide sequence cleavable by caspase-3, Mizukami et al. measured caspase-3-like activity in HeLa cells stimulated by an inducer of apoptosis [124],... 

Matayoshi, E. D., Wang, G. T., Krafft, G. A. and Erickson, J. (1990). Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247, 954-958. 

Washiya, K., Furuike, T., Nakajima, F., Lee, Y. C. and Nishimura, S. I. (2000). Design of fluorogenic substrates for continuous assay of sialyl-transferase by resonance energy transfer. Anal. Biochem. 283, 39-48. 

Zaikova, T. (2001). Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C. Bioconjug. Chem. 12, 307-313. 

Xing, B., Khanamiryan, A. and Rao, J. (2005). Cell-permeable near-infrared fluorogenic substrates for imaging beta-lactamase activity. J. Am. Chem. Soc. 127, 4158-4159. 

Measurement of blue and green fluorescence of NADH and FAD in living tissues Quantitative fluorescent cytochemistry Using permeable fluorogenic substrates of enzymes, specific inhibitors, and kinetic analysis... 

Enzymes can be linked to immunoassay reagents to amplify detection by the use of fluorogenic substrates. Enzyme-linked fluoroimmunoassays (ELFIAs) are very similar to photometric EIAs in format and workflow. EIAs are widely used, and many commercial ELFIA assays and systems are available/15 The most commonly used enzymes in ELFIAs are horseradish peroxidase, alkaline phosphatase, and fi-D-... 

Figure 21 Hapten 35 and fluorogenic substrates 36-38 used to screen phage displayed Fabs with an aldolase activity.

N. Baggett, M. A. Case, P. R. Darby, and C. J. Cray, Action of almond P-D-glucosidase on fluorogenic substrates derived from 4-substituted 7-hydroxycoumarins, Enzyme Microb. Technol., 15 (1993) 742-748. 

Figure 1. Analytical isoelectric focusing of cellulases from Trichodtrma ree-sei. Detection of CBH I and EG I activities using MeUmbLac, in the absence (A) and presence (B) of 10 mM cellobiose. Lane 1, EG I lane 2, EG I (iso-components) lane 3, CBH I (pi 3.9 component) lane 4, EG I-CBH I mixture). Gels were flooded with the fluorogenic substrate (pH 5.0) and after 5-10 min (room temperature) photographed (Polaroid 57, green filter) on a long wavelength UV-transilluminator (8).

The DDI fluorescent inhibition screen is a high-throughput screen, primarily as a result of reduced analysis time, and can range from a few hundred compounds per week to a thousand per week. The assay uses recombinant microsomes prepared from insect cells infected with recombinant baculovirus containing a human CYP enzyme and individual fluorogenic substrates. In this instance the substrates are not specific and hence cannot be used in a mixed enzyme system such as HLM. In addition, this assay is more prone than the conventional inhibition screen (Section 8.3.2) to NCE interferences within the assay (i.e., inherently fluorescent NCEs, fluorescent quenching by the NCE). 

Figure 5.8 Fluorogenic substrates to screen for lipases and esterases (upper...

Hydrogen peroxide causes oxidation of resorufin (121a) or its acetylated derivative, Amplex Red (121b), according to equation 41. The process takes place in Tris buffer at pH 7.4 and the product 122 is determined by FLD (Aex = 563 nm, An = 587 nm). Care has to be paid that the fluorogenic substrate is in excess, lest 122 undergoes further oxidation to a nonfluorescent compound. This assay was proposed for measurement of the... 

All enzymes are measured at acidic pH with artificial fluorogenic substrates, except for arylsulfatase A, which is measured with a chromogenic substrate. The fluorogenic substrates are derivatives of 4-methylumbelliferone or 6-hexadecanoylamino-... 

N-(hexadecanoyl)amino-4-nitrophenylphosphorylcholine is not recommended and the assay with the fluorogenic substrate 6-hexadecanoylamino-4-methylumbel-liferylphosphorylcholine should be used with caution [54]. Assay using natural radioactive substrate is probably still the most reliable and will be described below in addition to assay using a fluorogenic substrate. 

Reagents and Chemicals (Assay with Fluorogenic Substrate)... 

The TNF-a-induced DNA fragmentation may have been caused by caspase-activated deoxyribonuclease (CAD) in PC-12 cells. In non-apoptotic cells, CAD is present as an inactive complex with the inhibitor jOvd p3,24]. During apoptosis, caspase-3 inactivates ICAD, leaving CAD free to function as a nuclease [25]. Therefore, we used die fluorogenic substrate, Ac-DEVD-MCA, to determine whether the caspase-3 in PC-12 cells was activated by treatment with TNF-a and/or crocin. As shown in... 

Naphthol AS-acetate (3-acetoxynaphthoic acid anilide] [1163-67-3] M 305.3, m 152°, 160°. Recrystd from hot MeOH and dried in vacuo over P2O5. It is slightly soluble in AcOH, EtOH, CHCI3 or CgHg. It is a fluorogenic substrate for albumin esterase activity. [Chen and Scott Analyt Letters 17 857 1984], At kgx 320nm it had fluorescence at 500nm. [Brass and Sommer B 61 1000 1928]. 

J.O. Capobianco, C.G. Lerner, and R.C. Goldman, Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase, Anal. Biochem., 204, 96, 1992. 

In practice it is often more convenient to measure the release of a phenol from an aryl phosphomonoester. Standard serum phosphatase methods employ phenyl phosphate (188), p-nitrophenyl phosphate (189), phenolphthalein monophosphate (140), or thymolphthalein monophosphate (141) where the phenol released can be determined spectrophoto-metrically [only the Bodansky method (13) uses a Pi determination]. A number of fluorogenic substrates have been used for phosphatase studies, e.g., jS-naphthyl phosphate (30, 148), 4-methylumbelliferyl phosphate (143), and 3-O-methylfluorescein phosphate (144) The main advantage here is the much greater sensitivity of fluorescence as compared with spectrophotometric assays as little as 1 pmole of 4-methyl-umbelliferone can be detected in continuous assay. 

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