Method for measuring hydrolytic enzyme activity

Abstract

The invention discloses a method for measuring hydrolytic enzyme activity. The method comprises the following steps: incubating a detection solution containing a substrate, Fe<3+> and bathophenanthrolinedisulfonic acid disodium salt with a hydrolase solution to be measured; making the substrate react with hydrolase to generate a reduction product; reducing the Fe<3+> into Fe<2+> by the reduction product; combining the Fe<2+> and the bathophenanthrolinedisulfonic acid disodium salt to form a Fe<2+>-bathophenanthroline compound, wherein the detection solution turns from colorless to crimson, and has a very strong absorption peak at 535nm. The higher the hydrolytic enzyme activity in a sample is, the deeper the color of the detection solution becomes. The method has high detection sensitivity for the hydrolytic enzyme activity, high selectivity, high interference resistance, good reproducibility and short detection time, is easy and convenient to operate, and can be applied to rapid and high-sensitivity detection of the hydrolytic enzyme activity in a clinical sample; the detection cost is lowered greatly.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and relates to a method for measuring hydrolase activity, in particular to a method for measuring hydrolase activity based on colorimetric analysis. Background technique [0002] Hydrolase is a general term for a class of enzymes that catalyze hydrolysis reactions, and widely exists in various animals, plants and microorganisms. Simple, rapid and sensitive determination of hydrolase activity is of great significance for early diagnosis of diseases, observation of curative effect and monitoring of prognosis. [0003] At present, fluorescence analysis and colorimetric analysis are mostly used for the determination of hydrolytic enzyme activity. Among them, colorimetric analysis, also known as absorptiometry, is based on the change of the color of the solution, that is, the selective absorption of light by the solution, to quantitatively analyze the content of the substance. It has the ...

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Problems solved by technology

Taking β-galactosidase as an example, there are two types of colorimetric analysis methods currently available for its activity determination: (1) p-nitrophenyl-β-D-galactopyranoside (or o-nitro Phenyl-β-D-galactopyranoside) is the chromogenic substrate, because β-galactosidase can hydrolyze it to generate p-nitrophenol (or o-nitrophenol), the latter solution is in alkaline It is yellow under certain conditions and has a maximum absorption peak at 405nm (or 420nm), thereby realizing the colorimetric analysis of β-galactosidase activity, but there are defects such as poor selectivity, low sensitivity, narrow detection range and insufficient anti-interference ability ; Moreover, due to the poor stability of the substrate, it is easy to cause "false positive" results (1.Maruhn D.(1976).Rapid colorimetric assay ofβ-galactosidase and N-acetyl-β-glucosaminidase in human urine[J].ClinicaChimica Acta, 73(3):453-461; 2. Gary, R.K., Kindell, S.M.(2005). Quantitative assay of senescence-associated β-galactosidase activity in mammalian cellextracts. Analytical Biochemistry, 343(2), 329-334.); (2 ) uses gold nanoparticles as probes, based on the property that the color of the solution changes due to the change of the aggregation state of gold nanoparticles, and realizes the colorimetric analysis of the activity of β-galactosidase, but the preparation process of the probe is complicated, the detection period is long, and the cost High and poor anti-interference ability and other defects (Zeng Z., etal. (2012). Simple and real-time colorimetric assay for glycosidases activity using functionalized gold nanoparticles and its application for inhibitorscreening. Analytical Chemistry, 84 (21), 9089-9095; Chen J., et al. (2016). Colorimetric Detection of Escherichia coli Based on the Enzyme-Induced Metallization of Gold Nanorods. Small, 12(18), 2469-2475.)

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