TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase
We introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled O-phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrat...
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MDPI AG
2017-08-01
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| Series: | Sensors |
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| Online Access: | https://www.mdpi.com/1424-8220/17/8/1877 |
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| author | Akio Shiba Emiko Kinoshita-Kikuta Eiji Kinoshita Tohru Koike |
| author_facet | Akio Shiba Emiko Kinoshita-Kikuta Eiji Kinoshita Tohru Koike |
| author_sort | Akio Shiba |
| collection | DOAJ |
| description | We introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled O-phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrate. TAMRA-Phos-tag specifically captured TAMRA-PEA to form a 1:1 complex at pH 7.4; the intensity of the fluorescence peak of the complex at 580 nm (λex = 523 nm) was significantly reduced to 32% of the average value for the two individual components as a result of the mutual approach of the TAMRA moieties. As TAMRA-PEA was dephosphorylated by AP, the resulting TAMRA-labeled ethanolamine dissociated and the fluorescence increased in a manner dependent on the AP dose and the time. In the second assay, pyrophosphate (PP), a natural AP-substrate, was used as a bridging ligand to form a dimeric TAMRA-Phos-tag complex. The dimerization reduced the fluorescence intensity to 49% of that in the absence of PP. As pyrophosphate was hydrolyzed to two orthophosphate moieties by AP, the 580-nm fluorescence recovered in a time-dependent manner. By examining the initial slope of this time-dependent fluorescence recovery, we succeeded in evaluating the 50% inhibitory concentrations of orthovanadate toward two AP isozymes under near-physiological conditions. |
| first_indexed | 2024-04-11T11:05:11Z |
| format | Article |
| id | doaj.art-6fcb20d8f40e41f3a26cb9bff7cbd5db |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-04-11T11:05:11Z |
| publishDate | 2017-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-6fcb20d8f40e41f3a26cb9bff7cbd5db2022-12-22T04:28:24ZengMDPI AGSensors1424-82202017-08-01178187710.3390/s17081877s17081877TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline PhosphataseAkio Shiba0Emiko Kinoshita-Kikuta1Eiji Kinoshita2Tohru Koike3Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, JapanDepartment of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, JapanDepartment of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, JapanDepartment of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, JapanWe introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled O-phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrate. TAMRA-Phos-tag specifically captured TAMRA-PEA to form a 1:1 complex at pH 7.4; the intensity of the fluorescence peak of the complex at 580 nm (λex = 523 nm) was significantly reduced to 32% of the average value for the two individual components as a result of the mutual approach of the TAMRA moieties. As TAMRA-PEA was dephosphorylated by AP, the resulting TAMRA-labeled ethanolamine dissociated and the fluorescence increased in a manner dependent on the AP dose and the time. In the second assay, pyrophosphate (PP), a natural AP-substrate, was used as a bridging ligand to form a dimeric TAMRA-Phos-tag complex. The dimerization reduced the fluorescence intensity to 49% of that in the absence of PP. As pyrophosphate was hydrolyzed to two orthophosphate moieties by AP, the 580-nm fluorescence recovered in a time-dependent manner. By examining the initial slope of this time-dependent fluorescence recovery, we succeeded in evaluating the 50% inhibitory concentrations of orthovanadate toward two AP isozymes under near-physiological conditions.https://www.mdpi.com/1424-8220/17/8/1877alkaline phosphatasefluorometric analysisfluorescence quenchingtetramethylrhodamineO-phosphorylethanolaminepyrophosphate |
| spellingShingle | Akio Shiba Emiko Kinoshita-Kikuta Eiji Kinoshita Tohru Koike TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase Sensors alkaline phosphatase fluorometric analysis fluorescence quenching tetramethylrhodamine O-phosphorylethanolamine pyrophosphate |
| title | TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase |
| title_full | TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase |
| title_fullStr | TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase |
| title_full_unstemmed | TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase |
| title_short | TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase |
| title_sort | tamra tamra fluorescence quenching systems for the activity assay of alkaline phosphatase |
| topic | alkaline phosphatase fluorometric analysis fluorescence quenching tetramethylrhodamine O-phosphorylethanolamine pyrophosphate |
| url | https://www.mdpi.com/1424-8220/17/8/1877 |
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