CN100535124C - Method for measuring polypeptide in minute quantitics and content of protein - Google Patents
Method for measuring polypeptide in minute quantitics and content of protein Download PDFInfo
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- CN100535124C CN100535124C CNB2005100124240A CN200510012424A CN100535124C CN 100535124 C CN100535124 C CN 100535124C CN B2005100124240 A CNB2005100124240 A CN B2005100124240A CN 200510012424 A CN200510012424 A CN 200510012424A CN 100535124 C CN100535124 C CN 100535124C
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Abstract
The invention relates to the measuration of micro-polypeptide and protein content. The principle of the invention is: the polypeptide protein is digested by sulfuric acid and produces the ammonium sulfate that is decomposed by NaOH to release the ammonia; add the Nai series reagent; use the optical splitter to test the ammonium salt and converse it into the nitrogen content. The invention is composed of the preparation of the testing reagent, the testing method and the calculation of the protein content. The invention overcomes the failures of the prior art.
Description
Technical field
The present invention relates to the mensuration of a peptide species and protein content, particularly the measuring method of trace (being low to moderate several micrograms) polypeptide and protein content.
Background technology
Protein determination is one of analytical procedure the most frequently used, the most basic in the biochemical research.Protein content determination has two class methods at present, and a class is to utilize proteinic physicochemical property, and promptly mensuration such as specific refractory power, proportion, uv-absorbing are learnt; Another kind of is to utilize the chemical gauging protein content, i.e. nitriding, biuret method (Biuret method), Folin-phenol reagent process (Lowry method), ultraviolet absorption method and Xylene Brilliant Cyanine G method (Bradford method).
Wherein Bradford method and the sensitivity of Lowry method are the highest, and is than 10～20 times of ultraviolet absorption method sensitivities, sensitiveer more than 100 times than Biuret method.It should be noted that, except that nitriding, these back four kinds of methods all participate in polypeptide or protein molecule in chemical reaction as a reaction member, and the sequence of polypeptide protein molecule, structure, character are varied, even measure with these four kinds of methods with a kind of protein soln, they also might draw four kinds of different results, so can not all be applicable to any type of protein under any condition.
In these methods, though the nitriding more complicated is more accurate, often the protein of measuring with nitriding is as the standard protein of additive method, and it is that itrogenous organic substance promptly decomposes generation ammonia (digestion) with sample and vitriol oil heat altogether, ammonia again with effect of sulfuric acid, become sulfate of ammoniac.Make it to decompose through highly basic alkalization and emit ammonia, borrow steam that ammonia is steamed to acid solution, the degree that is neutralized according to this acid solution can calculate the nitrogen content of sample.Because each polypeptide and protein all have its constant nitrogen content, can try to achieve polypeptide and Protein content by the mensuration of total nitrogen.But the minimum detectable polypeptide protein of Kjeldahl determination also has only 0.3mg, and for some genetically engineered of Gamma Magnitude or the polypeptide and the protein in other source, this method is also inapplicable.
Summary of the invention
The object of the present invention is to provide the measuring method of a kind of micro-polypeptide and protein content, adopt nitriding of the present invention to carry out accurate assay polypeptide and protein under the micro-example situation are only arranged.
Overall technology design of the present invention is:
The polypeptide protein quality sample generates ammonium sulfate after sulfuric acid digestion, ammonium sulfate is decomposed by sodium hydroxide emits ammonia, adds Nessler's reagent, uses the spectrophotometric determination ammonium salt, and converts nitrogen content to.
Protein+H
2SO
4→ (NH
4)
2SO
4
(NH
4)
2SO
4+2NaOH→Na
2?SO
4+2NH
4OH
This measuring method is made up of following steps:
The measuring method of a kind of micro-polypeptide and protein content is characterized in that it is made up of following steps:
The preparation of A, detection reagent:
A, take by weighing the 0.2-0.5g sodium selenate, 0.1-0.2g copper sulfate adds the 1.5-2.5N sulfuric acid dissolution and becomes 500ml as digestive pharmaceutical; Vitriol oil 14-42ml adds in the 100-150ml water, and the dilution of cooling back is 1.5-2.5N sulfuric acid; Sodium hydroxide 20-60g is dissolved in water to 500ml and obtains 1-3N sodium hydroxide;
The preparation of b, Nessler's reagent:
The 20-30g potassiumiodide is added water 20-30ml dissolving make liquor kalii iodide; Mercury perchloride 8-14g heating is dissolved in 160ml water and makes liquor hydrargyri perchloridi; 60-80g potassium hydroxide is added water to the 350ml dissolving make potassium hydroxide solution; Then above-mentioned liquor hydrargyri perchloridi is poured in the liquor kalii iodide, till the red precipitate that generates no longer dissolves; This mixed solution is poured in the potassium hydroxide solution, mixing adds water to 500ml again, the static supernatant of getting;
The preparation of c, standard ammoniumsulphate soln (nitrogen content 0.02-0.06mg/ml):
After getting ammonium sulfate and being dried to constant weight, take by weighing 0.4720-1.4159g and add water to the 100ml dissolving, shake up, obtain nitrogen content 1-3mg/ml standardized solution, refrigeration; Get above-mentioned standardized solution 1ml, be diluted with water to 50ml and shake up;
B, detection method:
A, add Digestive system 1ml in the testing sample of nitrogenous 8～10 μ g, two of granulated glass spherees in the stink cupboard internal heating, make to be heated liquid and to keep slight boiling condition; Digest be light green (3-5 hour) to solution after, strengthen firepower and continue to digest to solution clarification (1-2 hour); After the cooling, add the dilution of 10ml water, add Nessler's reagent 0.5ml more successively, 2N sodium hydroxide shakes up, and places 10-25 minute, in 390-410nm and 460-480nm wavelength colorimetric;
B, standard ammonium sulfate curve: measure 5 parts of standard ammoniumsulphate solns (nitrogen content is 0.02-0.06mg/ml) in flask, every part is 0.1-0.5ml, and moisturizing is to 0.5ml respectively, add Digestive system 1ml, two of granulated glass spherees digest simultaneously with testing sample, press a method operation in the B step;
C, calculate total nitrogen, thereby calculate protein content.
The concrete processing condition and the processing parameter of each step of the present invention are:
Return law of the straight line is adopted in the calculating of total nitrogen in the described C step.
In the preparation of Nessler's reagent 60-80g potassium hydroxide is added water to 350ml, slowly be dissolved in the brown bottle.
In the B step, put on the adjustable firepower electric furnace in the stink cupboard and heat adding testing sample behind Digestive system and the granulated glass sphere or standard ammoniumsulphate soln.
In the preparation process of standard ammoniumsulphate soln, drying temperature is 100 ℃.
Among the step a of detection reagent preparation, the density of the vitriol oil is 1.84kg/L.
The technical progress that the present invention obtains is:
Detection reagent and instrument are easily purchased in this detection method; This detection method has solved existing method and has not been suitable in the micro-example problem of polypeptide and protein being carried out accurate assay, can accurately detect some genetically engineered of Gamma Magnitude or the polypeptide and the protein in other source.
Embodiment
The present invention will be further described below in conjunction with embodiment:
The preparation of A, detection reagent:
A, take by weighing the 0.3g sodium selenate, 0.1g copper sulfate adds the 2N sulfuric acid dissolution and becomes 500ml as digestive pharmaceutical.The vitriol oil (density 1.84kg/L) 28ml adds in the 150ml water, and the cooling back is diluted to 500ml with water becomes 2N sulfuric acid.Sodium hydroxide 40g is dissolved in water to 500ml and obtains 2N sodium hydroxide.
The preparation of b, Nessler's reagent:
The 25g potassiumiodide is added water 26ml dissolving, and mercury perchloride 11.0g heating is dissolved in 160ml water, and 69g potassium hydroxide adds water and slowly is dissolved in the 500ml scale brown bottle, adds water to 350ml; Then above-mentioned mercury perchloride saturated solution is poured in the liquor kalii iodide, till the red precipitate that generates no longer dissolves.This mixed solution is poured in the potassium hydroxide solution, mixing adds water to 500ml again, static for some time, gets supernatant and uses.
The preparation of c, standard ammoniumsulphate soln (nitrogen content 0.04mg/ml):
Get ammonium sulfate and be dried to constant weight in 100 ℃, the precision 0.9439g that weighs uses water dissolution, carefully washes in the 100ml volumetric flask, and adds water to scale, shakes up, and obtains nitrogen content 2mg/ml standardized solution, preserves in the refrigerator standby.Get standard ammonium sulfate stock solution 1ml, place the 50ml volumetric flask, add water to scale, shake up and get final product.
B, detection method
A, get testing sample (nitrogenous 9 μ g) and place kjeldahl flask or Boiling tube, add Digestive system 1ml, two of granulated glass spherees, put on the adjustable firepower electric furnace in the stink cupboard and heat, make liquid in pipe keep slight boiling condition, digest to solution and be light green (3-5 hour), strengthen firepower, continue to digest to solution clarification (1-2 hour), take off cooling after, add the dilution of 10ml water, add Nessler's reagent 0.5ml more successively, 2N sodium hydroxide shakes up, placed 15 minutes, in 400nm and 470nm wavelength colorimetric.
B, standard ammonium sulfate curve: precision is measured standard ammoniumsulphate soln (nitrogen content is 0.04mg/ml) 0.1,0.2,0.3,0.4 and 0.5ml in kjeldahl flask, supply water to 0.5ml, add Digestive system 1ml, two of granulated glass spherees, digest simultaneously with testing sample, operate with method.
C, employing return law of the straight line are obtained total nitrogen, calculate protein content again.
Claims (6)
1, the measuring method of a kind of micro-polypeptide and protein content is characterized in that it is made up of following steps:
The preparation of A, detection reagent:
A, take by weighing the 0.2-0.5g sodium selenate, 0.1-0.2g copper sulfate adds the 1.5-2.5N sulfuric acid dissolution and becomes 500ml as Digestive system; Vitriol oil 14-42ml adds in the 100-150ml water, and the dilution of cooling back is 1.5-2.5N sulfuric acid; Sodium hydroxide 20-60g is dissolved in water to 500ml and obtains 1-3N sodium hydroxide;
The preparation of b, Nessler's reagent:
The 20-30g potassiumiodide is added water 20-30ml dissolving make liquor kalii iodide; Mercury perchloride 8-14g heating is dissolved in 160ml water and makes liquor hydrargyri perchloridi; 60-80g potassium hydroxide is added water to the 350ml dissolving make potassium hydroxide solution; Then above-mentioned liquor hydrargyri perchloridi is poured in the liquor kalii iodide, till the red precipitate that generates no longer dissolves; This mixed solution is poured in the potassium hydroxide solution, mixing adds water to 500ml again, the static supernatant of getting;
C, nitrogen content are the preparation of the standard ammoniumsulphate soln of 0.02-0.06mg/ml:
After getting ammonium sulfate and being dried to constant weight, take by weighing 0.4720-1.4159g and add water to the 100ml dissolving, shake up, obtain nitrogen content 1-3mg/ml standardized solution, refrigeration; Get above-mentioned standardized solution 1ml, be diluted with water to 50ml and shake up;
B, detection method:
A, add Digestive system 1ml in the testing sample of nitrogenous 8～10 μ g, two of granulated glass spherees in the stink cupboard internal heating, make to be heated liquid and to keep slight boiling condition; Digest be light green to solution in 3-5 hour after, strengthen firepower and continue digestion 1-2 hour to solution and clarify; After the cooling, add the dilution of 10ml water, add Nessler's reagent 0.5ml more successively, 2N sodium hydroxide shakes up, and places 10-25 minute, in 390-410nm and 460-480nm wavelength colorimetric;
B, standard ammonium sulfate curve: 5 parts of the standard ammoniumsulphate solns of measuring nitrogen content and be 0.02-0.06mg/ml are in flask, and every part is 0.1-0.5ml, and moisturizing is to 0.5ml respectively, add Digestive system 1ml, two of granulated glass spherees digest simultaneously with testing sample, press a method operation in the B step;
C, calculate total nitrogen, thereby calculate protein content.
2, the measuring method of a kind of micro-polypeptide according to claim 1 and protein content is characterized in that return law of the straight line is adopted in the calculating of total nitrogen in the described C step.
3, the measuring method of a kind of micro-polypeptide according to claim 1 and protein content is characterized in that in the preparation of described Nessler's reagent 60-80g potassium hydroxide being added water to 350ml, slowly is dissolved in the brown bottle.
4, the measuring method of a kind of micro-polypeptide according to claim 1 and protein content, it is characterized in that in the described B step, put on the adjustable firepower electric furnace in the stink cupboard and heat adding testing sample behind Digestive system and the granulated glass sphere or standard ammoniumsulphate soln.
5, the measuring method of a kind of micro-polypeptide according to claim 1 and protein content is characterized in that in the preparation process of described standard ammoniumsulphate soln, and drying temperature is 100 ℃.
6, the measuring method of a kind of micro-polypeptide according to claim 1 and protein content is characterized in that the density of the vitriol oil is 1.84kg/L among the step a of described detection reagent preparation.
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| CN1888908B (en) * | 2006-07-15 | 2010-07-07 | 桂林工学院 | Timing method for measuring micro-protein |
| CN106596428A (en) * | 2016-12-15 | 2017-04-26 | 南京市儿童医院 | Kit for determining vitality of PEG-asparaginase in patient body with acute leukemia |
| CN112595685A (en) * | 2020-12-29 | 2021-04-02 | 中农颖泰林州生物科园有限公司 | A product containing cecropin and method for detecting cecropin content in the product |
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Non-Patent Citations (6)
| Title |
|---|
| 提高凯氏定氮法蛋白质测定速度的研究. 陆晓滨，李敬龙，董贝磊，马美范.中国调味品，第1期. 2003 |
| 提高凯氏定氮法蛋白质测定速度的研究. 陆晓滨，李敬龙，董贝磊，马美范.中国调味品，第1期. 2003 * |
| 浅谈凯氏定氮法. 何进，梁运祥.陕西粮油科技，第21卷第3期. 1996 |
| 浅谈凯氏定氮法. 何进，梁运祥.陕西粮油科技，第21卷第3期. 1996 * |
| 试析食品中蛋白质含量的测定方法－凯氏定氮法. 刘期成.城市技术监督. 2000 |
| 试析食品中蛋白质含量的测定方法－凯氏定氮法. 刘期成.城市技术监督. 2000 * |
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