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Accurate determination method of activity of procmbius clrkii phenol oxidase

A technology of Procambarus clarkii phenoloxidase and serum phenoloxidase, which is applied to the measurement of color/spectral characteristics and the preparation of test samples, and can solve the problem of inconsistency in measurement time and temperature, accuracy of measurement results, and efficiency Unsatisfactory and other issues

Inactive Publication Date: 2013-12-11
JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Many scholars use ice bath to terminate the reaction when measuring the activity of phenoloxidase to detect, and some people use the method of measuring the concentration of the substrate product of phenoloxidase reaction within a period of time to judge its activity, but many of them are in the measurement time and measurement temperature. All aspects are not uniform, so the accuracy and efficiency of the measurement results are not satisfactory

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] ①Weigh 1g of raw shrimp gill tissue, add 9ml of normal saline, and homogenize with a tissue homogenizer or homogenizer in an ice bath.

[0018] ② The homogenate was centrifuged at 3000r / min at 4°C for 10min, and the supernatant was absorbed and stored in a clean centrifuge tube for testing.

[0019] ③Determination of enzyme activity: Take a clean 10ml graduated test tube, add 3ml of 0.1mol / L potassium phosphate buffer solution with a pH of 5.5, and pretreat in a 35°C water bath for 5min to bring the buffer solution to the preset temperature.

[0020] ④ Add 0.1mL of 0.01mol / L L-DOPA and 0.1mL of the sample to be tested, mix well, quickly measure the initial absorbance value at a wavelength of 490nm, and start the water bath timer at the same time, bath in water for 6 minutes, and measure the absorbance (OD) at the 6th minute value;

[0021] ⑤Take another two test tubes from each group to measure the absorbance of the substrate L-DOPA and the sample at 490nm as OD L and...

Embodiment 2

[0023] ①Preparation of serum phenoloxidase: puncture the carapace with a 5ml disposable syringe, extract hemolymph from the heart, place it in a 2ml centrifuge tube overnight at 4°C.

[0024] ② After the serum is separated, centrifuge at 10000r / min at 4°C for 10min, and store the supernatant in a clean centrifuge tube for testing;

[0025] ③Determination of enzyme activity: Take a clean 10ml graduated test tube, add 3ml of 0.1mol / L potassium phosphate buffer solution with a pH of 5.5, and pretreat in a 35°C water bath for 5min to bring the buffer solution to the preset temperature.

[0026] ④ Add 0.1mL of 0.01mol / L L-DOPA and 0.1mL of the sample to be tested, mix well, quickly measure the initial absorbance value at a wavelength of 490nm, and start the water bath timer at the same time, bath in water for 6 minutes, and measure the absorbance (OD) at the 6th minute value;

[0027] ⑤Take another two test tubes from each group to measure the absorbance of the substrate L-DOPA an...

Embodiment 3

[0029] ①Preparation of serum phenoloxidase: puncture the carapace with a 5ml disposable syringe, extract hemolymph from the heart, place it in a 2ml centrifuge tube overnight at 4°C.

[0030] ② After the serum is separated, centrifuge at 10000r / min at 4°C for 10min, and store the supernatant in a clean centrifuge tube at -20°C for testing;

[0031] ③Before the measurement, take the sample to be tested out of the refrigerator, put it in a 35°C water bath, and wait for the sample to melt before measuring.

[0032] ④ Determination of enzyme activity: Take a clean 10ml graduated test tube, add 3ml of 0.1mol / L potassium phosphate buffer solution with a pH of 5.5, and pretreat in a 35°C water bath for 5min to bring the buffer solution to the preset temperature.

[0033] ⑤ Add 0.1mL of 0.01mol / L L-DOPA and 0.1mL of the sample to be tested, mix well, quickly measure the initial absorbance value at a wavelength of 490nm, and start the water bath timer at the same time, bath in water fo...

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Abstract

An accurate determination method of activity of shrimp and crab phenol oxidase comprises the steps of extracting hemolymph from an organ for determination, preparing gill tissue phenol oxidase, collecting a gill tissue sample from an ice bag rapidly, adding aseptic normal saline, performing ice-bath homogenization, centrifuging homogenate, sucking supernate for storage for determination, adding a potassium phosphate buffer solution, pretreating in a water bath to allow the temperature of the buffer solution to reach preset temperature, adding L-DOPA (levodopa) and the sample to be determined, mixing intensively, rapidly determining initial absorbance when the wavelength is 490nm, simultaneously starting water bath timing, performing the water bath, determining absorbance (OD (optical density)), further taking two test tubes for each group to measure the absorbance of the substrate L-DOPA and the sample at 490nm to be ODL and OD0 as controls, parallelizing each group for three times, and solving a calculation formula: PO = (OD-ODL-OD0) / t by taking 0.001 of average increase of OD at 490nm per minute (t) as an enzyme activity unit (U).

Description

technical field [0001] The invention relates to a method for measuring phenoloxidase activity, in particular to an accurate method for measuring phenoloxidase activity of Procambarus clarkii. Background technique [0002] my country's aquatic crustacean resources are rich and diverse. Crustaceous shrimps and crabs are deeply loved by people because of their delicious taste and high nutritional value. Shrimp and crab are not only important industries in my country's fishery economy, but also one of the main products that earn foreign exchange through export. Procambarus clarkii (Procambarus clarkii), commonly known as crayfish, originated in northern Mexico and southern America, and was introduced to my country in the early 20th century. Procambarus clarkii is a worldwide edible shrimp. The price in my country is constantly rising. It is one of the important shrimp products for export and foreign exchange. It is one of the most popular freshwater large shrimps in the domest...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N1/28
Inventor 王建国吴娟朱光来王权
Owner JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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