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Method for rapidly determining trypsin and aprotinin combination mechanism

A trypsin, rapid assay technology, used in biochemical equipment and methods, microbial assay/inspection, fluorescence/phosphorescence, etc.

Inactive Publication Date: 2013-08-14
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study of the binding mechanism of trypsin and aprotinin by fluorescence spectroscopy has not been reported yet

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] respectively in the containing 2×10 -6 Add an equal amount of 2.6×10 to the Tris-HCl buffer of mol / L trypsin -6 ~2.34×10 -5 mol / L aprotinin, mix well, put in water bath at 18°C ​​for 30min, in a fluorescence spectrophotometer, use about 280nm as the excitation wavelength, detect the fluorescence intensity of the mixed solution in the emission spectrum of 260-600nm, and measure △λ= Simultaneous fluorescence at 15nm and Δλ=60nm.

Embodiment 2

[0030] respectively in the containing 2×10 -6 Add an equal amount of 2.6×10 to the Tris-HCl buffer of mol / L trypsin -6 ~2.34×10 -5 mol / L aprotinin, mix well, put in water bath at 28°C for 30min, in a fluorescence spectrophotometer, use about 280nm as the excitation wavelength, detect the fluorescence intensity of the mixed solution in the emission spectrum of 260-600nm, and measure △λ= Simultaneous fluorescence at 15nm and Δλ=60nm.

Embodiment 3

[0032] respectively in the containing 2×10 -6 Add an equal amount of 2.6×10 to the Tris-HCl buffer of mol / L trypsin -6 ~2.34×10 -5 mol / L aprotinin, mix well, put in a water bath at 37°C for 30min, in a fluorescence spectrophotometer, use about 280nm as the excitation wavelength, detect the fluorescence intensity of the mixed solution in the emission spectrum of 260-600nm, and measure △λ= Simultaneous fluorescence at 15nm and Δλ=60nm.

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Abstract

The invention discloses a method for rapidly determining a trypsin and aprotinin combination mechanism, which is designed for studying a quenching mechanism of aprotinin to trypsin the binding constant K and the binding-site number n of aprotinin and trypsin by using a fluorescent spectrometry including fluorescence quenching and synchronous fluorescence. The method comprises the following steps: selecting a mixture of trypsin and aprotinin in a suitable proportion; at a temperature of 28-37 DEG C, and under corresponding excitation wavelengths, determining the quenching effects of aprotinin with different concentrations on trypsin; and in combination with a Stern-Volmer curve, further determining the binding constant K and binding-site number n of aprotinin and trypsin, and then obtaining an acting mechanism of aprotinin and trypsin. According to the method, a convenient, accurate and effective method can be provided for studying the mutual action of inhibitors and other micromolecular quenchers as well as proteins from a molecular level.

Description

technical field [0001] The invention relates to a method for rapidly determining the binding mechanism of trypsin and aprotinin Background technique [0002] Trypsin is a kind of protease. In vertebrates, acts as a digestive enzyme. Synthesized in the pancreas as the precursor of the enzyme trypsinogen. It not only acts as a digestive enzyme, but also restricts and decomposes the precursors of other enzymes such as chymotrypsinogen, carboxypeptidase, and phospholipase, and activates them. It is the most specific protease, and it becomes an indispensable tool in determining the amino acid arrangement of proteins. [0003] As a proteolytic enzyme, trypsin can selectively hydrolyze the peptide chain formed by the carboxyl group of lysine or arginine in protein, can digest and dissolve denatured protein, and has no effect on undenatured protein. Therefore, it can make pus, phlegm It can decompose and thin out fluid and blood clots, facilitate drainage and drainage, accelerat...

Claims

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Application Information

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IPC IPC(8): C12Q1/37G01N21/64
Inventor 庄红董姝婷唐宁徐娜
Owner JILIN UNIV
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