Method for measuring purity of papain by fast protein liquid chromatogram

A technology of papain and liquid chromatography, which is applied in the field of determining the purity of papain by fast protein liquid chromatography, can solve the problems of limited concentration range of papain, cumbersome operation steps, measurement errors, etc., and achieve credible results, simple operation, time-consuming effect

Inactive Publication Date: 2009-10-28
DONGHUA UNIV
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Problems solved by technology

[0003] At present, the most commonly used method for measuring the purity of papain is to calculate the specific enzyme activity of the sample after measuring the enzyme activity and protein content of papain in the sample respectively. The disadvantage of this method is that there are certain errors in the determination of enzyme activity and protein content. The concentration range of papain that can be measured is limited, and the operation steps are cumbersome and the repeatability is poor, especially when the concentration of papain is too high or too low, large measurement errors will occur

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  • Method for measuring purity of papain by fast protein liquid chromatogram
  • Method for measuring purity of papain by fast protein liquid chromatogram

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Embodiment 1

[0026] Utilize fast protein liquid chromatography (FPLC) to measure the purity of papain, concrete steps are as follows:

[0027] (1) Prepare mobile phase: take sodium acetate and prepare 0.05mol / L sodium acetate solution and sodium chloride, measure deionized water, and prepare mobile phase A and B solutions in proportion.

[0028] Mobile phase A solution: weigh 4.102g of sodium acetate to prepare a 0.05mol / L pH 4.5 acetic acid-sodium acetate buffer solution with a volume of 1L;

[0029] Mobile phase B solution: weigh 4.102g sodium acetate and 58.44g sodium chloride to prepare a 0.05mol / L pH4.5 acetic acid-sodium acetate-sodium chloride buffer solution with a volume of 1L;

[0030] (2) Preparation of reference substance solution: Accurately weigh 500 mg of refined papain, put it into a 50ml volumetric flask, add mobile phase A solution to the 50ml scale line to constant volume, shake well, until papain is fully dissolved, and prepare papain reference substance solution;

[...

Embodiment 2

[0035] Utilize fast protein liquid chromatography (FPLC) to measure the purity of papain, concrete steps are as follows:

[0036] (1) Prepare mobile phase: take sodium acetate and prepare 0.05mol / L sodium acetate solution and sodium chloride, measure deionized water, and prepare mobile phase A and B solutions in proportion.

[0037] Mobile phase A solution: weigh 4.102g of sodium acetate to prepare a 0.05mol / L pH 4.5 acetic acid-sodium acetate buffer solution with a volume of 1L;

[0038] Mobile phase B solution: weigh 4.102g sodium acetate and 116.88g sodium chloride to prepare a 0.05mol / L pH4.5 acetic acid-sodium acetate-sodium chloride buffer solution with a volume of 1L;

[0039] (2) Preparation of reference substance solution: Accurately weigh 500 mg of refined papain, put it into a 50ml volumetric flask, add mobile phase A solution to the 50ml scale line to constant volume, shake well, until papain is fully dissolved, and prepare papain reference substance solution;

...

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Abstract

The invention relates to a method for measuring purity of papain by fast protein liquid chromatogram, comprising the steps of: preparing mobile phase solution A: acetic acid-sodium acetate buffer solution and mobile phase solution B: acetic acid-sodium acetate-sodium chloride buffer solution; placing refined papain and rough-wrought papain respectively into a flask with a volume of 50 ml; adding the mobile phase solution A till scale mark of 50 ml for constant volume and agitating till the papain is dissolved completely; and finally performing chromatographic separation by using an ion exchange column chromatography of the fast protein liquid chromatogram (FPLC), and calculating the purity of the papain by a formula for calculating the peak area ratio. The measuring method is fast and simple in operation, little in time consumption, reliable in result and high in repeatability, and provides basis for measuring the purity of papain precisely, thereby having important learning value andpractical application significance.

Description

technical field [0001] The invention belongs to the field of measuring the purity of papain, in particular to a method for measuring the purity of papain by using fast protein liquid chromatography. Background technique [0002] Papain (EC3.4.22.2) is a kind of sulfhydryl protease, which widely exists in the root, stem, leaf and fruit of papaya (Carica papaya), and is most abundant in immature milk. Widely used in food industry, such as clarification of beer and tenderization of meat, as well as leather, textile, daily chemical and pharmaceutical industries. In recent years, scientific research has found that papain has great practical value in the pharmaceutical industry, and drugs containing papain can have anti-cancer, anti-tumor and lymphatic leukemia effects. Since the pharmaceutical industry has relatively high requirements for the purity of papain, how to establish a method for quickly, efficiently and accurately measuring the purity of papain is a hot topic of resea...

Claims

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Application Information

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IPC IPC(8): C12Q1/37G01N30/02
Inventor 朱利民苏赛男聂华丽
Owner DONGHUA UNIV
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