Gee K R, Sun W C, Bhalgat M K, Upson R H, Klaubert D H, Latham K A, Haugland R P
Molecular Probes, Incorporated, 4849 Pitchford Avenue, Eugene, Oregon 97402, USA.
Anal Biochem. 1999 Aug 15;273(1):41-8. doi: 10.1006/abio.1999.4202.
Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.
基于4-甲基伞形酮（4-MU）的荧光底物已被广泛用于检测磷酸酶和糖苷酶活性。然而，这些底物的一个缺点是，由于4-MU的pK(a)约为8，反应产物的最大荧光需要碱性pH值。在对基于4-MU氟化衍生物的五种磷酸酶底物进行初步筛选时，所有底物的pK(a)值均低于4-MU，我们发现一种底物6,8-二氟-4-甲基伞形酮磷酸酯（DiFMUP）在检测酸性磷酸酶活性方面有很大改进。在酸性磷酸酶反应的最佳pH值（5.0）下测量时，DiFMUP产生的荧光信号比4-甲基伞形酮磷酸酯（MUP）高10倍以上。在pH 7时检测蛋白磷酸酶1活性，DiFMUP也优于MUP，在pH 10时检测碱性磷酸酶活性，其灵敏度与MUP相同。还基于6,8-二氟-4-甲基伞形酮制备了一种β-半乳糖苷酶底物。发现这种底物6,8-二氟-4-甲基伞形酮β-D-吡喃半乳糖苷（DiFMUG）在pH 7时检测β-半乳糖苷酶活性比常用底物4-甲基伞形酮β-D-吡喃半乳糖苷（MUG）敏感得多。DiFMUP和DiFMUG分别在中性和酸性pH下连续检测磷酸酶和β-半乳糖苷酶活性方面应该有很大的用途。
