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A novel fluorogenic coumarin substrate for monitoring acid phosphatase activity at low pH environment.

Yang D, Li Z, Allan Diwu Y, Fu H, Liao J, Wei C, Diwu Z - Curr Chem Genomics (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Chemical Engineering, Baoji University of Arts & Science, Shaanxi, China.

ABSTRACT
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.

No MeSH data available.


Detection of acid phosphatase activity with CF-MUP and MUP. For the top and bottom curves MUP and CF-MUP were incubated with acid phosphatase buffer at pH 6.0, and the resulting fluorescence signal was recorded with Ex/Em=360 nm/450 nm without pH adjustment. For the middle curve, MUP was incubated with acid phosphatase buffer at pH 6.0, and the enzyme reaction solutions were adjusted to have pH = 10 by 1 M NaOH alkalization. The resulting fluorescence signal was recorded with Ex/Em=360 nm/450 nm.
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Figure 3: Detection of acid phosphatase activity with CF-MUP and MUP. For the top and bottom curves MUP and CF-MUP were incubated with acid phosphatase buffer at pH 6.0, and the resulting fluorescence signal was recorded with Ex/Em=360 nm/450 nm without pH adjustment. For the middle curve, MUP was incubated with acid phosphatase buffer at pH 6.0, and the enzyme reaction solutions were adjusted to have pH = 10 by 1 M NaOH alkalization. The resulting fluorescence signal was recorded with Ex/Em=360 nm/450 nm.

Mentions: The CF-MUP substrate overcomes the problems of non-continuous assay mode and high assay background since its reaction product, CF-MU, has a pKa of ~4.8 (Fig. 2) with the absorption peak at ~360 nm and emission peak at 450 nm, that are available in many detection instruments including microplate readers, fluorescence microscopes and flow cytometers. We found that CF-MUP is about 100 times more sensitive than MUP for the detection of acid phosphatase at pH 5.5 (Fig. 3). This higher sensitivity is resulted from both the higher turn-over rate of CF-MUP by acid phosphatase and lower pKa of CF-MU. The direct comparison of initial hydrolysis CF-MUP and MUP by acid phosphatase at pH 5.5 followed by raising pH to 10 indicated that CF-MUP is still slightly more sensitive than MUP for acid phosphatase detection (see Fig. 3).


A novel fluorogenic coumarin substrate for monitoring acid phosphatase activity at low pH environment.

Yang D, Li Z, Allan Diwu Y, Fu H, Liao J, Wei C, Diwu Z - Curr Chem Genomics (2008)

Detection of acid phosphatase activity with CF-MUP and MUP. For the top and bottom curves MUP and CF-MUP were incubated with acid phosphatase buffer at pH 6.0, and the resulting fluorescence signal was recorded with Ex/Em=360 nm/450 nm without pH adjustment. For the middle curve, MUP was incubated with acid phosphatase buffer at pH 6.0, and the enzyme reaction solutions were adjusted to have pH = 10 by 1 M NaOH alkalization. The resulting fluorescence signal was recorded with Ex/Em=360 nm/450 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803437&req=5

Figure 3: Detection of acid phosphatase activity with CF-MUP and MUP. For the top and bottom curves MUP and CF-MUP were incubated with acid phosphatase buffer at pH 6.0, and the resulting fluorescence signal was recorded with Ex/Em=360 nm/450 nm without pH adjustment. For the middle curve, MUP was incubated with acid phosphatase buffer at pH 6.0, and the enzyme reaction solutions were adjusted to have pH = 10 by 1 M NaOH alkalization. The resulting fluorescence signal was recorded with Ex/Em=360 nm/450 nm.
Mentions: The CF-MUP substrate overcomes the problems of non-continuous assay mode and high assay background since its reaction product, CF-MU, has a pKa of ~4.8 (Fig. 2) with the absorption peak at ~360 nm and emission peak at 450 nm, that are available in many detection instruments including microplate readers, fluorescence microscopes and flow cytometers. We found that CF-MUP is about 100 times more sensitive than MUP for the detection of acid phosphatase at pH 5.5 (Fig. 3). This higher sensitivity is resulted from both the higher turn-over rate of CF-MUP by acid phosphatase and lower pKa of CF-MU. The direct comparison of initial hydrolysis CF-MUP and MUP by acid phosphatase at pH 5.5 followed by raising pH to 10 indicated that CF-MUP is still slightly more sensitive than MUP for acid phosphatase detection (see Fig. 3).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Chemical Engineering, Baoji University of Arts & Science, Shaanxi, China.

ABSTRACT
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.

No MeSH data available.