Method for determining adenine content and RIP activity

A determination method, adenine technology, applied in the field of enzyme engineering, can solve the problems of high laboratory conditions, inconvenient testing of a large number of samples, long measurement time, etc., achieve good application prospects, realize analysis automation, and be easy to promote.

Pending Publication Date: 2021-04-30
CHENGDU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the problems of high laboratory conditions, no RNase interference, inconvenient testing of a large number of samples, long measurement time or high cost in the existing RIP activity measurement technology, it is urgent to find a quantifiable, simple, fast, low-cost and effective method. Extended RIP Activity Assay

Method used

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  • Method for determining adenine content and RIP activity
  • Method for determining adenine content and RIP activity
  • Method for determining adenine content and RIP activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: colored liquid of the present invention

[0059] Accurately weigh 40.3mg BCIP, add pH 7.0 0.1M phosphate buffer 10mL, that is 10mM. Just before use, add enzyme and dilute to the required concentration of BCIP, and store at low temperature.

Embodiment 2

[0060] Embodiment 2: Determination of adenine concentration

[0061] The steps of this embodiment are as follows:

[0062] 1) Mix 0.3mL of the solution to be tested containing adenine with 2.7mL of the chromogenic solution, and react at 37°C for 30min;

[0063] 2) Measure the light absorption value at 606nm to calculate the concentration of adenine, the calculation formula is:

[0064] ΔA=k×(A-A 0 ),

[0065] Among them, ΔA is the concentration of adenine, k is the rate of change of absorbance with the concentration of adenine, A is the detected absorbance, and A 0 is the absorbance measured when the concentration of adenine in the solution is 0.

Embodiment 3

[0066] Example 3: RIP activity assay

[0067] The steps of this embodiment are as follows:

[0068] 1) The reaction process between RIP and substrate: Take an appropriately diluted 0.15mL RIP solution and 0.15mL ATP or NAD + (20mM) mixed, under the conditions of 53°C and pH 3.0, accurately incubated for 30min, and cooled in an ice bath.

[0069] 2) Color reaction process: Add 2.7mL of color solution and react at 37°C for 30min. Absorbance at 606 nm was measured to indicate relative activity.

[0070] Activity unit calculation formula:

[0071] U=(ΔA / t)×(V t / Ve) or U / mg=(ΔA / t)×(V t / Ve) / C,

[0072] Among them, U is the activity unit; U / mg is the specific activity unit, ΔA is the adenine concentration (μM), V t is the total reaction volume (mL), V e is the RIP volume (mL); t is the reaction time (min), and C is the RIP content (mg).

[0073] Under the conditions of this experiment, catalyzing the substrate to generate 1 μM adenine per minute is one RIP activity unit (U...

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Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a method for determining adenine content and RIP activity, and aims at overcoming the defects in the existing RIP activity determination method. A developing solution, which consists of xanthine oxidase and BCIP, for adenine test is provided. The invention also provides an adenine test method, which comprises the following steps: reacting a sample to be tested with xanthine oxidase and BCIP, and testing the light absorption value. The invention also provides an RIP activity testing method. The RIP activity testing method comprises the following steps: mixing an RIP solution with ATP or NAD < + >, reacting, testing the adenine concentration in the solution, and calculating the RIP activity. The method can be used for detecting the dynamic synthesis condition of RIP in a biological sample, discovering the existence of new RIP, detecting and tracking the destination of RIP in the separation and purification process and the like.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, in particular to a method for measuring adenine content and RIP activity. Background technique [0002] Ribosome-inactivating protein (RIP) is a kind of toxic protein with N-glycosidase activity, which can inactivate ribosomes in eukaryotic cells and inhibit protein synthesis. It is widely found in angiosperms, bacteria, fungi and in algae. RIP has broad-spectrum anti-tumor, anti-virus, immune regulation and other biological functions, but has little toxicity to normal cells. It has been widely concerned and studied by scientists around the world in recent decades. [0003] A long-standing serious problem in the research and development of RIP—there is no simple and reliable method to measure the activity of RIP. At present, the existing methods roughly include the following: ①Determination of in vitro RIP inhibition of protein biosynthesis in cell-free systems. This method is not o...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N1/38
CPCG01N21/31G01N1/38
Inventor 孟尧孟延发
Owner CHENGDU MEDICAL COLLEGE
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