A Novel Fluorogenic Coumarin Substrate for Monitoring Acid Phosphatase Activity at Low pH Environment
نویسندگان
چکیده
This article described the synthesis and application of 6-chloro-8-fluoro-4-methylumbelliferone phosphate (CF-MUP) in analyzing acid phosphatase activity. Compared to the existing MUP, the new coumarin phosphate, CF-MUP, demonstrateed much higher sensitivity and was more robust for detecting the activity of acid phosphatase than the classic substrate 4-methylumbelliferone phosphate (MUP). The product of enzyme reaction, 6-chloro-8-fluoro-4-methylumbelliferone (CF-MU) possesses strong fluorescence at approximately 450 nm with low pKa (4.7), high fluorescence quantum yield and pH independence in the physiological pH range. This new fluorescence dye, CF-MU, is a convenient tool for assays with buffer pH between 4.5 and 8.
منابع مشابه
A phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases.
Mca-Gly-Asp-Ala-Glu-Tyr(PO(3)H(2))-Ala- Ala-Lys(DNP)-Arg-NH(2), where Mca is (7-methoxycoumarin-4-yl)acetyl and DNP is 2,4-dinitrophenyl, was synthesized as a fluorogenic substrate for protein tyrosine phosphatases (PTPs). In the peptide, the fluorescent Mca group is quenched efficiently by the DNP group. Although the fluorescence intensity of the substrate was practically unchanged upon PTP-ca...
متن کاملA single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate.
Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic as...
متن کاملPurification and Characterization of a Novel Thermostable and Acid Stable α-Amylase from Bacillus Sp. Iranian S1
This study reports the purification and biochemical characterization of thermostable and acidic-pH-stable α-amylase from Bacillus sp. Iranian S1 isolated from the desert soil (Gandom-e-Beryan in Lut desert, Iran). Amylase production was found to be growth associated. Maximum enzyme production was in exponential phase with activity 2.93 U ml-1 at 50°C and pH 5. The enzyme was purified by isoprop...
متن کاملAn optimized continuous-monitoring procedure for semiautomated determination of serum acid phosphatase activity.
A continuous-monitoring method for measuring acid phosphatase activity with alpha-naphthyl phosphate as the substrate was critically evaluated and modified. Using partially purified prostatic acid phosphatase, we show that certain conditions for the assay must be satisfied to ensure linearity. These conditions include maintaining the pH between 5.6 and 5.9 and the addition of detergent to susta...
متن کاملProfiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays.
A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The...
متن کامل