CN1888908B

CN1888908B - Timing method for measuring micro-protein

Info

Publication number: CN1888908B
Application number: CN2006101065827A
Authority: CN
Inventors: 李建平; 熊金平
Original assignee: Guilin University of Technology
Current assignee: Guilin University of Technology
Priority date: 2006-07-15
Filing date: 2006-07-15
Publication date: 2010-07-07
Anticipated expiration: 2026-07-15
Also published as: CN1888908A

Abstract

A timing method for determining micro-protein content, that is: the Potassium Bromate can oxidize acid Chrome blue K to discolour in the 0.2mol/L vitriol liquor, forms Landolt reaction, the protein produces the sensitive inhibitory effect for the indication reaction and extends the fading time, establishes the determining micro-protein dynamics analysis method according to the above. The longest absorption wavelength is 526 nm (nanometer), the density of the protein with the range of 0-90 mug/10mL assumes nicer linearity relation.

Description

A kind of chronometry of measuring trace protein

Technical field

The present invention relates to a kind of method of utilizing dynamic analysis fast measuring trace protein, can apply to the mensuration of protein content in the food such as soybean and yoghurt.

Background technology

Protein is one of important composition composition of biosome, is the material base of biological phenomena.Protein and nutrition, eucaryotic cell structure, enzyme, hormone, virus, immunity, substance operation, heredity etc. are closely related, and the assay determination of protein is a most important work in biological chemistry and other related discipline and the product quality inspection.In recent years, along with fulfiling ahead of schedule of the Human Genome Project, bioassay technique enters the back era gene, and the research of biomacromolecule (particularly protein and nucleic acid) detection method becomes the field, forward position of life science.Because the importance of protein in life science, the research of quantitative analysis of protein method just becomes a focus.Protein measuring has Kjeldahl, biuret method, Lowry method .Bradford method etc.It is simple that spectrophotometric protein determination has equipment, and characteristics such as accurately accurate and visual result have been widely used in protein measuring in biological chemistry and the clinical chemistry.But most of analytical photometries are not too complicated, are exactly that sensitivity is not high, and employed instrument and equipment is also mostly to be to cost an arm and a leg.

Summary of the invention

The invention reside in provide a kind of highly sensitive, can carry out method for measuring, i.e. chronometry to the protein content of Gamma Magnitude.

Conceive as follows: we find that potassium bromate can fade by the oxidation acid chrome blue K, this reaction has significant inhibitory effect and trace of albumin is verified, between catalyst system and catalyzing and the on-catalytic system bigger aberration is arranged, set up a kind of chronometry of measuring trace protein on this basis.

The present invention relates to Landolt (Landolt) reaction, belong to redox reaction.There to be certain reaction to be its feature latent period, essence is a kind of chronometry, i.e. the fixed concentration method.Normally use oxygenant oxidation indicant, the concentration of indicant slowly reduces, and when the concentration of reacting to indicant is depleted to certain value, measures the needed time.At this moment the reciprocal proportional relation in catalyst concentration and reaction time.

Concrete steps are as follows:

One, the preparation of detectable:

(1) acid chrome blue K solution: take by weighing the blue K0.05g of solid acid chromium, the adding distil water constant volume is in the volumetric flask of 250mL, and concentration is 0.02%.

(2) potassium bromate solution: take by weighing solid potassium bromate 2.5g, the adding distil water constant volume is in the volumetric flask of 250mL, and concentration is 10.0g/L.

(3) bovine serum albumin (BSA) solution: take by weighing the 0.005g bovine serum albumin and be dissolved in the 0.5%NaCl solution of 100mL, prepare to such an extent that concentration is the protein solution of 50 μ g/mL; Get 10mL constant volume in the volumetric flask of 50mL during use again, getting concentration is the protein solution of 10 μ g/mL.The protein solution for preparing places refrigerator to preserve.

(4) sulfuric acid solution: the concentrated sulphuric acid that pipettes 55.6mL with transfer pipet goes to constant volume in the 500mL volumetric flask in 400mL distilled water, and concentration is 2mol/L.

(5) Coomassie brilliant blue solution: take by weighing 100mg Coomassie brilliant blue G-250, be dissolved in 50mL 90% ethanol, add the phosphatase 11 00ml of 85% (W/V), use the distilled water constant volume at last, get the Coomassie brilliant blue solution of 0.1g/L to 1000mL.

Experiment is redistilled water with distilled water.

Two, detection method

Get the 10mL color-comparison tube of two scale unanimities, the acid chrome blue K solution that in an arm wherein, adds 2.0mL 0.02%, 1.0mL the protein standard solution of 10 μ g/mL, 1.0mL 2.0mol/L sulfuric acid solution, arrive near scale with distilled water diluting, the potassium bromate solution that adds 0.7mL 10.0g/L then fast, and be diluted with water to scale, shake up, put into 7200 type visible spectrophotometers rapidly, sentencing distilled water in wavelength 526nm is reference, with the cuvette of 1cm, measure its absorbance, record A-t curve, the measurement system is to the required time that fades rapidly (add in addition solution mix fully after to the set time 10s between beginning to measure), as catalyst system and catalyzing, absorbance is designated as A, and the reaction time is t; The acid chrome blue K that in another arm, adds 2.0mL0.02%, 1.0mL the 2.0mol/L sulfuric acid solution is as the on-catalytic system, arrive near scale with distilled water diluting, add 0.7mL 10.0g/L potassium bromate solution then fast, be diluted with water to scale, shake up, the system of measuring equally is to the required time that fades rapidly (add in addition solution mix fully after to the set time 10s between beginning to measure), as the on-catalytic system, absorbance is designated as A ₀, the reaction time is t ₀Computing time 1/t reciprocal ₀And 1/t, and further calculate Δ (1/t)=(1/t ₀)-(1/t).The solution absorbance that contains variable concentrations protein is situation of change example such as Fig. 1 in time.

Three, the drafting of standard working curve:

The acid chromium blue k that in two 10mL color comparison tubes, adds 2.0mL 0.02% respectively, the sulfuric acid of 1.0mL 2.0mol/L, the potassium bromate of 0.7mL 10.0g/L adds the different protein of measuring again respectively.Distilled water diluting is reference to scale with distilled water, measures in the 526nm place.When absorbance reaches 0.800, write down reaction time t ₀And t.Protein consumption and time relationship such as Fig. 2, protein are good linear dependence with Δ (1/t) in 0～90 μ g/10mL: Δ (1/t)=-0.0264C+0.3292, (R=0.9987).

Four, testing protein Determination on content

The acid chrome blue K that in two 10ml color comparison tubes, adds 2.0ml 0.02% respectively, the sulfuric acid of 1.0mL 2.0mol/L, the potassium bromate of 0.7mL 10.0g/L, wherein one does not add, as the on-catalytic system.Another adds a certain amount of protein, and as catalyst system and catalyzing, distilled water diluting is reference to scale with distilled water, measures in the 526nm place.When absorbance reaches 0.800, write down reaction time t ₀And t.Calculate Δ (1/t).And with its substitution Δ (1/t)=-0.0264C+0.3292.Calculate C.The i.e. amount of contained protein in this 10mL pipe as can be known.

The present invention has overcome prior art and had too many shortcomings such as complexity, and is highly sensitive, be easy to robotization for the detection of Gamma Magnitude protein content.

Description of drawings

Fig. 1 is the solution absorbance situation of change figure in time that contains variable concentrations protein.

Fig. 2 is protein consumption and time chart.

Embodiment

Embodiment 1

With the soybean impurity elimination of buying on the market, wear into fine powder, accurately take by weighing 0.01g and cross 60 purpose bean powderes, add the buffer solution mixing of 10mL, pH=1.30, adding 1%NaCl solution 15mL again stirs, after extracting 1.5h, filter, divide the washing residue 10 times with 200mL1%NaCl solution, filtrate is settled to 1000mL, get an amount of solution high speed centrifugation 10min, get clear liquid 1mL, measure protein content in the leaching liquor by following detection method:

Get the 10mL color-comparison tube of two scale unanimities, the acid chrome blue K solution that in an arm wherein, adds 2.0mL 0.02%, 1.0mL Protein Extraction clear liquid, 1.0mL 2.0mol/L sulfuric acid solution, arrive near scale with distilled water diluting, the potassium bromate solution that adds 0.7mL 10.0g/L then fast, and be diluted with water to scale, shake up, put into 7200 type visible spectrophotometers rapidly, sentencing distilled water in wavelength 526nm is reference, with the cuvette of 1cm, measure its absorbance, record A-t curve, the measurement system is to the required time that fades rapidly (add in addition solution mix fully after to the set time 10s between beginning to measure), as catalyst system and catalyzing, absorbance is designated as A, and the reaction time is t; The acid chrome blue K that in another arm, adds 2.0mL 0.02%, 1.0mL the 2.0mol/L sulfuric acid solution is as the on-catalytic system, arrive near scale with distilled water diluting, add 0.7mL 10.0g/L potassium bromate solution then fast, be diluted with water to scale, shake up, the system of measuring equally is to the required time that fades rapidly (add in addition solution mix fully after to the set time 10s between beginning to measure), as the on-catalytic system, absorbance is designated as A ₀, the reaction time is t ₀Computing time 1/t reciprocal ₀And 1/t, and further calculate Δ (1/t)=(1/t ₀)-(1/t).

Embodiment 2

Purchase milk, sample is put in the water-bath, temperature is 20-30 ℃ and slowly shakes mixing 3-4min under bubble-tight situation, stirs fatty separating emulsions and oil reservoir.Stir gently, the obvious separation of milk is put in the water-bath then, make temperature rise to 35-40 ℃ and slowly mix to help fat to separate.Should adjust temperature to 20 ℃ insulation 1min when fat is separated rapidly, draw solution 1mL is put in the centrifuge tube and mixes.Centrifuging 2min. gets the volumetric flask that 1mL places 100mL, and the NaCl with 0.5% is diluted to scale.Press the detection method of embodiment 1 and measure protein content in the leaching liquor.

The measurement of reference value is according to the Bradford detection method.Measuring method is as follows, and the range protein solution of above-mentioned preparation is respectively got 0.1mL, adds 1mL Coomassie brilliant blue CBG-250 working fluid, and vibration shakes up, and measures the absorbance A at wavelength 595nm place behind 2min ₅₉₅Value, measurement should be finished in 1h.Result such as table 1.

Table 1 sample analysis result

Claims

1. a chronometry of measuring trace protein is characterized in that detectable adopts acid chrome blue K solution, potassium bromate solution; Measuring method adopts chronometry.