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Method for extracting and purifying high ferro myohemoglobin reductase from myocardium

A technology of ferromyoglobin and reductase, applied in the field of extraction and purification of ferromyoglobin reductase, can solve the problems of slow research progress and difficult MetMbR biomaterials, etc.

Inactive Publication Date: 2008-10-29
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it is difficult to prepare ideal and purified MetMbR biological materials, and the research on experimental conditions in this area is slow, and there are no MetMbR biological products on the market

Method used

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  • Method for extracting and purifying high ferro myohemoglobin reductase from myocardium
  • Method for extracting and purifying high ferro myohemoglobin reductase from myocardium
  • Method for extracting and purifying high ferro myohemoglobin reductase from myocardium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Preparation of metmyoglobin reductase

[0033] Material

[0034] Fresh pig heart, anion exchange chromatography material (DEAE-Sepharose Fast Flow, Pharmacia), cation exchange chromatography material (CM-Sephadex Fast Flow, Pharmacia), blue agarose FastFlow affinity chromatography gel (Affi-Gel Blue Fast Flow, Pharmacia), (porcine cardiac myoglobin (self-made in this laboratory), EDTA (Amresco company), NADH (purity 98%, Amresco company), phosphate, K 4 Fe(CN) 6 etc. are of domestic analytical grade, dialysis bag (14000CoMW), polyethylene glycol, Coomassie brilliant blue kit, and other reagents are of analytical grade.

[0035] main instrument

[0036] Spectrumlab54 UV-visible spectrophotometer (Shanghai), 722 spectrophotometer (Shanghai), FJ-200 high-speed dispersion homogenizer (Shanghai), PHS-3C precision pH meter (Shanghai), JB22 constant temperature magnetic stirrer, super constant temperature Water bath (Shanghai), biochemical temperature refrige...

Embodiment 2

[0049] Embodiment 2: Qualitative and quantitative of metmyoglobin reductase

[0050] The crude extraction sample that (1)-(5) step obtains in embodiment 1, through ammonium sulfate salting-out sample, through anion-cation exchange chromatography sample and affinity chromatography processing sample carry out SDS-PAGE and PAGE electrophoresis test, electrophoresis picture see Figure 4 . Use CS-9000 gel analysis software (λ=595nm; W×H: 0.05×2.0mm) to carry out band scanning quantitative analysis on the gel after SDS-PAGE electrophoresis, and the results are shown in Image 6 . from Image 6 It can be seen from the figure that there are more protein types in the crude extraction sample and after 40%-70% ammonium sulfate segmental salting out, and the proteins cannot be separated well; after anion-cation exchange chromatography, it can be roughly seen 2 bands, the bands are also relatively clear, and the concentration of the band corresponding to MetMbR is the highest. After s...

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Abstract

The invention relates to a process for extracting and purifying protease, in particular to a method for extracting and purifying metmyoglobin reductase from cardiac muscle. The method comprises the following steps: obtaining crude extractive liquid from cardiac muscle of an animal, salting out, and separating and purifying metmyoglobin reductase from pig heart by chromatography adopting DEAE-Fast Flow and CM-Sephadex FF ion exchange medium and blue sepharose FF medium. The method has the advantages of high product purity, simple process, low cost, etc., and can fill the blank in bio-reagent preparation and achieve higher economic value.

Description

technical field [0001] The invention relates to a process for extracting and purifying protease, in particular to a method for extracting and purifying metmyoglobin reductase from myocardium. Background technique [0002] Meat color is an important economic trait of meat production, which is concerned by scientists, producers and consumers. The bright red and lustrous color of meat indicates that the meat is fresh and of good texture. It is related to myoglobin (Mb) and its reductase system in muscle ( Reductase) are closely related. Mb exists in three forms in the body, deoxymyoglobin, purple in color; oxymyoglobin (MbO 2 ), which is bright red; both can be oxidized to metmyoglobin (MetMb), which is dark brown. There is a redox system in which the three are converted to each other in the muscle, which are converted to each other under the action of enzymes, and maintain dynamic balance and stable meat color. After the muscle is isolated, with the gradual loss of metmyogl...

Claims

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Application Information

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IPC IPC(8): C12N9/02
Inventor 金邦荃王玮
Owner NANJING NORMAL UNIVERSITY
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