CN102928362A - Ninhydrin detection method as improvement to free amino quantitative method in solid-phase polypeptide synthesis - Google Patents
Ninhydrin detection method as improvement to free amino quantitative method in solid-phase polypeptide synthesis Download PDFInfo
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- CN102928362A CN102928362A CN2012103991153A CN201210399115A CN102928362A CN 102928362 A CN102928362 A CN 102928362A CN 2012103991153 A CN2012103991153 A CN 2012103991153A CN 201210399115 A CN201210399115 A CN 201210399115A CN 102928362 A CN102928362 A CN 102928362A
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Abstract
The invention provides a ninhydrin detection method as improvement to a free amino quantitative method in solid-phase polypeptide synthesis. Synthesized Rink_Amide_MBHA (4-Methyl-benzhydrylamine) resin is used as a standard sample and is subjected to continuous weight increment weighing to obtain a group of standard samples; and light absorption values are read under OD570 through a ninhydrin reagent reaction to acquire a standard curve; and free amino quantitative detection analysis can be performed on unknown peptide resin according to the standard curve.
Description
Technical field
The present invention relates to the quantitative detection of amino residue, especially adapt to new solid-phase polypeptide synthetic in, dispose through ninhydrin reagent, record light absorption value, make typical curve, be used for judging the method for the remaining amino content of unknown resin peptide.
Background technology
In the process of the synthetic polypeptide of solid phase method, free amino quantity is a kind of direct embodiment of synthetic completeness, and still present neither one accurately method can obtain remaining amino concrete quantity, with confidence level and the accuracy of verifying its result; Therefore production standard sample and typical curve are used for the free amino of unknown resin peptide is carried out quantitative test, and important more practical value is arranged.
Conventional triketohydrindene hydrate detection method is synthesized-the quantitative detection of NH2 residue for solid-phase polypeptide; the general light absorption value substitution formula that will react rear resin that all adopts; determine synthetic ratio by calculating; this result is subject to the reaction time; heating-up temperature; the factor affecting such as heat time heating time; and owing to there being batch difference of reagent preparation; thereby make the result have larger floating; and because synthetic constantly carrying out; amino acid whose increase; also can so that resin peptide weight change; so that the accuracy in result's judgement is had a greatly reduced quality; appearance for fear of above-mentioned situation; this project is improved ninhydrin reaction reagent, can draw at short notice a stable result, and use slough synthetic after the protecting group with resin as standard items; set up a typical curve; all reactions are all carried out under identical conditions, thereby guaranteed result's confidence level, can more reliable free amine group be carried out quantitatively.
Literature search discloses: mostly conventional triketohydrindene hydrate detection method is to come the condensation completeness is judged by the variation of color; The method is used 2 kinds of reagent, reagent A: get 0.66mg/ml potassium cyanide aqueous solution 2ml and join in the 98ml pyridine solution, add 4g/ml phenol ethanolic solution 10ml, mixing again; Reagent B: take by weighing the 500mg triketohydrindene hydrate and be dissolved in the 10ml absolute ethyl alcohol; When synthesizing the completeness detection, get the resin peptide 3mg after condensation reaction is finished, use absolute ethyl alcohol to clean 3 times, after the vacuum drying, add reagent A 0.15ml, reagent B 0.05ml, 100 ℃ were heated 10 minutes, see that change color judges the condensation completeness, if color is incomplete for blueness or green just are judged to be condensation, it is complete that displaing yellow is judged to be condensation.
The present invention's design is the remaining amino quantitative analysis method during improved solid-phase polypeptide synthesizes, and as standard items, the production standard curve carries out a judgement, the subject matter of its solution, the one, synthetic resin establishment of standard to synthetic ratio with synthetic resin; The 2nd, to determining of the quantitative sensing range of free amine group; The 3rd, the determining of the stability of light absorption value; The 4th, reactant liquor does not add potassium cyanide after improving, and reduces the danger of reactant liquor preparation, and this quantivative approach has important application and popularization value.
Summary of the invention
The object of the invention is to: the remaining amino quantitative analysis method during improved solid-phase polypeptide is synthetic, the light absorption value production standard curve of establishing criteria product resin can obtain the remaining amino content in the unknown resin peptide sample, has realized accurate quantitative analysis.
The object of the present invention is achieved like this: the free amine group quantivative approach during the triketohydrindene hydrate detection method is synthetic to solid-phase polypeptide is improved, with the Rink_Amide_MBHA resin identical with synthetic reaction as standard model, the production standard curve, sample to the unknown carries out the amino quantitatively detection of remnants, and its step is as follows:
Its 1 sample preparation:
Take by weighing Rink_Amide_MBHA resin 2g, calculate resin molal quantity A; With the amino acid with the Fmoc protecting group, every seed amino acid is got the 2A mole; With the Rink_Amide_MBHA resin of 1A mole and the amino acid with the Fmoc protecting group of 2A mole, place successively the container of the n-formyl sarcolysine base pyrrolidone solution that contains 8% carbodiimides, reacted 2 hours, obtain resin peptide; Through 25% piperidine solution 26ml, processed 30 minutes again, obtain removing the resin peptide after the protecting group, for subsequent use;
The wherein preparation of 25% piperidine solution: 25ml piperidines+75ml n-formyl sarcolysine base pyrrolidone mixing gets;
The computing formula of resin molal quantity wherein:
In the formula: A is the molal quantity of Rink_Amide_MBHA resin;
M is the grams of Rink_Amide_MBHA resin;
SD is that the Rink_Amide_MBHA resin replaces value;
Its 2 detecting step:
1) will remove except the resin peptide 15mg after the protecting group, and use absolute methanol solution 20ml that it is cleaned 3 times, dispose its temperature 45 C, vacuum tightness 93.3～98.6KPa through vacuum drier; The dry end takes by weighing 1.0mg, 1.5mg, 2.0mg, 2.5mg, 3.0mg; Add respectively 300ul Buffer A: and 75ul Buffer B, evenly mixed, with 100 ℃ of heating water bath 15min, and place 15-20 ℃ of water to be cooled to room temperature; The reacted mixed liquor of getting 25ul joins in 60% ethanolic solution of 1.0ml, mixed evenly after, under OD570, carry out light absorption value and read, and the production standard curve;
2) take by weighing and treat test sample 2.0-3.0mg, add Buffer A:300ul; Buffer B:75ul, mixing, 100 ℃ of heating water bath 15min, and with cold water cooling, the reacted mixed liquor of getting 25ul joins in 60% ethanolic solution of 1.0ml, mixed evenly after, under OD570, measure light absorption value;
The wherein preparation of Buffer A: 5ml pyridine+1.185g phenol+5ml ethanol mixing;
The wherein preparation of Buffer B: 500mg triketohydrindene hydrate+10ml ethanol;
3) owing to using synthetic Rink Amide_MBHA resin as standard items, thus when calculating, need and will deduct owing to the molecular weight that condensation increases, to guarantee the accuracy of result of calculation;
Remove the formula that affects of resin peptide increment:
X in the formula: the original peptide weight after the amino acid molecular amount that removal increases;
W: resin peptide theoretical molecular;
M: the actual weigh of resin peptide;
The SD:Rink_Amide_MBHA resin replaces value;
Wherein the theoretical molecular of 4mer resin peptide is: 541.73;
The Rink_Amide_MBHA resin replaces value: 0.38mmol/g.
The mechanism of action of the present invention: be that amino acid and triketohydrindene hydrate hydrate are under mild acid conditions during common heating, the oxidized deamination of amino acid, decarboxylation, and the triketohydrindene hydrate hydrate is reduced, its reduzate can add the amino that thermal decomposition produces with amino acid and be combined, become the bluish violet compound with another molecule triketohydrindene hydrate condensation again, number according to solution free ammonia base, its color also has the depth and changes, can pass through the spectrophotometer measurement light absorption value, according to the difference of light absorption value, can judge the amino content in the reaction.
This method adopts the Rink_Amide_MBHA resin identical with synthetic reaction as standard model, the production standard curve, it is more accurate to judge for the amino residual content in the solid phase building-up process, can judge synthetic expected results more intuitively, shows technical progress.
Embodiment
The present invention further illustrates in conjunction with the embodiments.
The implementation step of this method:
(1) preparation of reaction reagent:
Buffer A:5ml pyridine+1.185g phenol+5ml ethanol mixing;
Buffer B:500mg triketohydrindene hydrate+10ml ethanol;
25% piperidine solution: 25ml piperidine solution+75ml n-formyl sarcolysine base pyrrolidone mixing;
(2) sample preparation:
1) solid phase building-up process: take by weighing Rink_Amide_MBHA resin 2g, and calculate required resin molal quantity A according to the computing formula of resin molal quantity, select the amino acid with the Fmoc protecting group, every seed amino acid is got the 2A mole, use the Rink_Amide_MBHA resin of 1A mole and the amino acid with the Fmoc protecting group of 2A mole, reaction is 2 hours under the n-formyl sarcolysine base pyrrolidone solution effect that contains 8% carbodiimides, obtain resin peptide, processed 30 minutes through 25% piperidine solution again, wherein every mole of resin peptide adds 25% piperidine solution 26ml, must remove the resin peptide behind the Deprotection, for subsequent use;
The computing formula of resin molal quantity:
In the formula: A is the molal quantity of Rink_Amide_MBHA resin;
M is the grams of Rink_Amide_MBHA resin;
SD is that the Rink_Amide_MBHA resin replaces value;
2) standard items preparation: use the Rink_Amide_MBHA resin identical with synthetic reaction as standard model, processed 30 minutes through 25% piperidine solution (every mole of resin adds 26ml), use absolute methanol solution (per 100 milligrams of resins use the 5ml absolute methanol solution) to clean 3 times, use afterwards vacuum drier to carry out drying (temperature 45 C, vacuum tightness 95KPa).
3) sample preparation: get the resin peptide 8mg that sloughs after the protecting group, use absolute methanol solution 15ml that it is cleaned 3 times, use afterwards vacuum drier to carry out drying (temperature 45 C, vacuum tightness 93.3KPa).
4) add Buffer A:300ul; Buffer B:75ul, mixing, 100 ℃ of heating water bath 15min, and cool off 15min with cold water, extract reaction solution afterwards the ethanolic solution mixing of 25ul+1.0ml 60%; Buffer A:5ml pyridine+1.185g phenol+5ml ethanol mixing wherein; Buffer B:500mg triketohydrindene hydrate+10ml ethanol;
5) carrying out light absorption value under OD570 reads.
(3) testing process:
1) standard items after processing is taken a sample, accurately take by weighing the resin of 1.0mg, 1.5mg, 2.0mg, 2.5mg, 3.0mg;
2) sample after processing is taken a sample, accurately take by weighing 3.0mg, and record weight;
3) every pipe standards product and sample all add Buffer A:300ul; Buffer B:75ul, mixing, 100 ℃ of heating water bath 15min, and cool off 15min with cold water, the reacted mixed liquor of getting 25ul joins in 60% ethanolic solution of 1.0ml, mixed evenly after, under OD570, carry out light absorption value and read, and the production standard curve, its result sees Table 1.
Table 1
| STD-1 | STD-2 | STD-3 | STD-4 | STD-5 | |
| The weighing grams | 1.0mg | 1.5mg | 2.0mg | 2.5mg | 3.0mg |
| Day-1 | 0.1430 | 0.1897 | 0.2422 | 0.3021 | 0.3759 |
| Day-2 | 0.1405 | 0.1902 | 0.2402 | 0.3045 | 0.3782 |
| Day-3 | 0.1441 | 0.1923 | 0.2461 | 0.3055 | 0.3742 |
| Mean value | 0.1425 | 0.1907 | 0.2428 | 0.3040 | 0.3761 |
| Std.Dev | 0.0018 | 0.0014 | 0.0030 | 0.0017 | 0.0020 |
| RSD% | 1.2943 | 0.7233 | 1.2356 | 0.5747 | 0.5338 |
Drawing linear formula by above-mentioned data is: Y=0.1161X+0.0191, R
2=0.9931, the RSD% value of the linear light absorption value of standard items is all less than 2, the R that it is linear
2Value is greater than 0.99, and the result has repeatability and accuracy.
4) 1 part of resin peptide behind the normal synthetic 4mer Deprotection is got in experiment; through accurate weighing; linear formula Y=0.1161X+0.0191 according to standard items; can be in the hope of its theoretical light absorption value; but owing to the weight of resin peptide in building-up process increases to some extent, thus when calculating, need and will deduct owing to the molecular weight that condensation increases, to guarantee the accuracy of result of calculation; so these molecular weight that need to will increase in computational analysis are removed, to guarantee the accuracy of result of calculation.
Remove the formula that affects of resin peptide increment:
X in the formula: remove the later weight of increment;
W: resin peptide theoretical molecular;
M: the actual weigh of resin peptide;
The SD:Rink_Amide_MBHA resin replaces value;
Wherein the theoretical molecular of 4mer resin peptide is: 541.73;
The Rink_Amide_MBHA resin replaces value: 0.38mmol/g.
5) obtain the productive rate of synthetic peptide according to the ratio of actual light absorption value and theoretical light absorption value, see Table 2.
Table 2
Learnt by upper table data: measured synthetic yield is more stable, its average synthetic yield be 95.96%, RSD% value less than 2, show that the result has preferably repeatability and stable.
6) final finished calculates synthetic yield, sees Table 3.
Table 3
| Load weight resin before synthetic | 50g |
| Total resin weight after synthetic | 66.38g |
| FMOC protecting group quality | 222.2 |
| Resin replacement amount (producer provides) (mM/gram) | 0.38 |
| Synthetic peptide molecular mass with blocking group | 663.72 |
| Actual synthetic peptide weight (gram) | 12.15 |
| Theoretical synthetic peptide weight (gram) | 12.61 |
| Synthetic yield | 96.35% |
Learnt by upper table data: synthetic peptide productive rate and we that linear formula calculates expect that the result that will reach conforms to, and can assess actual synthetic yield.
In sum: use the Rink_Amide_MBHA resin identical with synthetic reaction as standard model, calculate the method for synthetic yield, can be more simple and efficient calculate final synthetic yield, in building-up process, just final synthetic yield there is a clearer and more definite judgement, has made things convenient for the monitoring of operating personnel to product quality.
The present invention test is selected synthetic with resin (Rink Amide_MBHA Resin) available from Tianjin Nankai Hecheng S﹠T Co., Ltd., article No.: HCRAm04-1-1; Piperidines is available from Sigma, article No.: 110-89-4; N-formyl sarcolysine base pyrrolidone closes safe chemical industry company limited, article No.: 872504 available from the Zhangjiagang; Triketohydrindene hydrate is available from Sigma, article No.: N4876; Ethanol is available from large reagent forever, article No.: xk13-011-00011; Pyridine is available from Tianjin Fengchuan Chemical Reagent Science ﹠ Technology Co., Ltd., article No.:: xk13-201-00115; Phenol is available from Sigma, article No.: P5566-100g; The amino acid with the Fmoc protecting group that uses is available from the biochemical (Shanghai) Co., Ltd. of gill.
Claims (2)
1. the free amine group quantivative approach during the triketohydrindene hydrate detection method is synthesized solid-phase polypeptide is improved, it is characterized in that: use the Rink_Amide_MBHA resin identical with synthetic reaction as standard model, the production standard curve carries out to the sample of the unknown that remnants are amino quantitatively to be detected, and its step is as follows:
Its 1 sample preparation:
Take by weighing Rink_Amide_MBHA resin 2g, calculate resin molal quantity A; With the amino acid with the Fmoc protecting group, every seed amino acid is got the 2A mole; With the Rink_Amide_MBHA resin of 1A mole and the amino acid with the Fmoc protecting group of 2A mole, place successively the container of the n-formyl sarcolysine base pyrrolidone solution that contains 8% carbodiimides, reacted 2 hours, obtain resin peptide; Through 25% piperidine solution 26ml, processed 30 minutes again, obtain removing the resin peptide after the protecting group, for subsequent use;
The wherein preparation of 25% piperidine solution: 25ml piperidines+75ml n-formyl sarcolysine base pyrrolidone mixing gets;
The computing formula of resin molal quantity wherein:
In the formula: A is the molal quantity of Rink_Amide_MBHA resin;
M is the grams of Rink_Amide_MBHA resin;
SD is that the Rink_Amide_MBHA resin replaces value;
Its 2 detecting step:
1) will remove except the resin peptide 15mg after the protecting group, and use absolute methanol solution 20ml that it is cleaned 3 times, dispose its temperature 45 C, vacuum tightness 93.3～98.6KPa through vacuum drier; The dry end takes by weighing 1.0mg, 1.5mg, 2.0mg, 2.5mg, 3.0mg; Add respectively 300ul Buffer A: and 75ul BufferB, evenly mixed, with 100 ℃ of heating water bath 15min, and place 15-20 ℃ of water to be cooled to room temperature; The reacted mixed liquor of getting 25ul joins in 60% ethanolic solution of 1.0ml, mixed evenly after, under OD570, carry out light absorption value and read, and the production standard curve;
2) take by weighing and treat test sample 2.0-3.0mg, add Buffer A:300ul; Buffer B:75ul, mixing, 100 ℃ of heating water bath 15min, and with cold water cooling, the reacted mixed liquor of getting 25ul joins in 60% ethanolic solution of 1.0ml, mixed evenly after, under OD570, measure light absorption value;
The wherein preparation of Buffer A: 5ml pyridine+1.185g phenol+5ml ethanol mixing;
The wherein preparation of Buffer B: 500mg triketohydrindene hydrate+10ml ethanol;
3) owing to using synthetic Rink Amide_MBHA resin as standard items, thus when calculating, need and will deduct owing to the molecular weight that condensation increases, to guarantee the accuracy of result of calculation;
Remove the formula that affects of resin peptide increment:
X in the formula: the original peptide weight after the amino acid molecular amount that removal increases;
W: resin peptide theoretical molecular;
M: the actual weigh of resin peptide;
The SD:Rink_Amide_MBHA resin replaces value;
Wherein the theoretical molecular of 4mer resin peptide is: 541.73;
The Rink_Amide_MBHA resin replaces value: 0.38mmol/g.
2. according to the method for inspection claimed in claim 1, it is characterized in that: select synthetic be Rink Amide_MBHA Resin with resin, available from Tianjin Nankai Hecheng S﹠T Co., Ltd., article No.: HCRAm04-1-1; Piperidines is available from Sigma, article No.: 110-89-4; N-formyl sarcolysine base pyrrolidone closes safe chemical industry company limited, article No.: 872504 available from the Zhangjiagang; Triketohydrindene hydrate is available from Sigma, article No.: N4876; Pyridine is available from Tianjin Fengchuan Chemical Reagent Science ﹠ Technology Co., Ltd., article No.: xk13-201-00115; With the amino acid of Fmoc protecting group, available from the Shanghai company limited of gill biochemistry.
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| CN103408720A (en) * | 2013-08-14 | 2013-11-27 | 合肥市科天化工有限公司 | Method for chain re-extension of waterborne polyurethane |
| CN106353308A (en) * | 2016-08-24 | 2017-01-25 | 程玉慧 | Preparation method of para hydroxybenzene alanine urine detection reagent |
| CN106442359A (en) * | 2016-10-11 | 2017-02-22 | 湖南科伦制药有限公司 | Method for detecting total amino acid content in periplaneta americana solution |
| CN106706530A (en) * | 2016-12-28 | 2017-05-24 | 佛山市海科知识产权交易有限公司 | Method for determining free amino acid in allium chinensis |
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| CN2828803Y (en) * | 2005-08-27 | 2006-10-18 | 张静 | Kit for quantity testing phenylalanine concentration on filtering paper dry blood piece |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103408720A (en) * | 2013-08-14 | 2013-11-27 | 合肥市科天化工有限公司 | Method for chain re-extension of waterborne polyurethane |
| CN106353308A (en) * | 2016-08-24 | 2017-01-25 | 程玉慧 | Preparation method of para hydroxybenzene alanine urine detection reagent |
| CN106442359A (en) * | 2016-10-11 | 2017-02-22 | 湖南科伦制药有限公司 | Method for detecting total amino acid content in periplaneta americana solution |
| CN106706530A (en) * | 2016-12-28 | 2017-05-24 | 佛山市海科知识产权交易有限公司 | Method for determining free amino acid in allium chinensis |
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Application publication date: 20130213 |