CN100342229C - Rapid determination technique for angiotensin converting enzyme inhibition activity of protein zymolyte - Google Patents
Rapid determination technique for angiotensin converting enzyme inhibition activity of protein zymolyte Download PDFInfo
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- CN100342229C CN100342229C CNB2004100758256A CN200410075825A CN100342229C CN 100342229 C CN100342229 C CN 100342229C CN B2004100758256 A CNB2004100758256 A CN B2004100758256A CN 200410075825 A CN200410075825 A CN 200410075825A CN 100342229 C CN100342229 C CN 100342229C
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Abstract
The present invention relates to a method for rapidly testing the inhibitory activity of an angiotensin converting enzyme of proteolytic products by using the capillary electrophoresis technology, which belongs to the technical field of applied biology. The method of the present invention comprises: measuring the value of IC50 of which inhibits the ACE activity of various proteolytic products with the capillary electrophoresis method; judging the activity of various marine proteolytic products to inhibit the ACE according to the value of IC50; the lower the IC50 is, the stronger the activity of the proteolytic products is to inhibit the ACE, and the higher the content of hypotensive active peptide is to inhibit the ACE. Since the capillary electrophoresis technology is applied to sieve anti-hypertension active peptide from proteolytic products, the present invention has the advantages of good repeatability, high sensitivity and reliable IC50 value measurement. Compared with past chromatography, the present invention greatly shortens the time to measure the value of IC50 and need not use expensive organic solvent as mobile phase.
Description
(1) technical field
The present invention relates to utilize the method for the angiotensin converting enzyme inhibition activity of capillary electrophoresis technique fast measuring protein zymolyte, belong to the Applied Biotechnology field.
(2) background technology
Hypertension is a principal element that threatens adult's health, and its incidence of disease is in rising trend, and the adult in the whole world nearly 20% is subjected to the threat of high blood pressure disease.Hypertension can cause organ damages such as patient's heart and brain kidney, and with glycolipid metabolism disorder and diabetes substantial connection is arranged, entail dangers to life when serious.Therefore the research of antihypertensive therapy medicine more and more causes the attention of Chinese scholars, the peptide that particularly suppresses angiotensin converting enzyme, owing to be natural active peptide, there is not toxic and side effect, has the incomparable superiority of synthetic antihypertensive drugs, and it is present in the processing enzymolysis product of various food proteins more, and bigger using value and economic potential are arranged.
In people's physiology course, the renin angiotensin-enzyme system of boosting interacts and regulates vessel retraction.Feritin enters blood the proangiotensin Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Tyr-Ser-R in the blood plasma is hydrolyzed to angiotensin I (ATI) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, angiotensin I does not have vigor, and angiotensin I is under the effect of angiotensin converting enzyme (ACE), can change into great-hearted Angiotensin II (ATII) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Angiotensin II can be strengthened the convergent force of cardiac muscle, and makes the vascular smooth muscle contraction cause increased blood pressure simultaneously.On the other hand, also have kassinin kinin-kallikrein system also to be subjected to the regulation and control of ACE, it can make the bradykinin with vasorelaxation action change into does not have the slowly-releasing of vigor peptide, and under the effect of ACE, synergy has just caused the rising of blood in human body in pressure just because of two systems.If can suppress the effect of angiotensin converting enzyme, just can realize hypotensive effect.Ace inhibitory peptide is exactly a kind of ACE inhibitor, because ACE is the dipeptides excision enzyme of a specific C end, ace inhibitory peptide generally is the substrate analogue of ACE, like this when ACE effect angiotensin I, ace inhibitory peptide will form competitive the inhibition to it, combines with the ACE specificity, thereby reduces the effect of ACE to angiotensin I, the amount of the Angiotensin II with vessel retraction effect of Chan Shenging has just significantly reduced like this, thereby reaches the purpose that suppresses elevation of blood pressure.
Since 1970, constantly have new antihypertensive active peptide to be studied and to find, comprising chemosynthesis, separation and Extraction from natural materials, more be food proteins through the proteinase effect after, the enzymolysis peptide class fragment of generation.The protein of having researched and developed at present is based on food proteins, as soybean protein, and lactoprotein, livestock skeletal muscle albumen, livetin, ovalbumin etc.The kind of protein is very many, and the amino acid composition and the amino acid-level sequence of each protein are also different.The kind that can be used for the proteinase of enzymolysis has the restriction enzyme site of multiple, different proteinase to be not quite similar.Therefore, with a kind of albumen, use different protease hydrolyzeds, it is active different that the ACE of zymolyte suppresses; The same enzyme, the protein that enzymolysis is different, the ACE enzyme inhibition activity of its zymolyte is also inequality.Based on this, the kind of zymolyte is very many, and therefore, high-flux fast screening has the zymolyte of high ACE enzyme inhibition activity from multiple zymolyte, for further separation, purifying, the new ace inhibitory peptide of evaluation, seems particularly important.The inhibition vigor of in the past identifying the angiotensins peptide for inhibiting often adopts spectrophotometric method or high pressure lipuid chromatography (HPLC) (HPLC), not only time-consuming but also expensive, and Capillary Electrophoresis has fast, highly sensitive, advantages such as amount of samples is few, therefore the method with Capillary Electrophoresis can rapid screening have the activity of angiotensins peptide for inhibiting, promptly has the protein zymolyte of hypotensive activity, will identify that new blood pressure lowering peptide lays the foundation for further separation and purification.
(3) summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of fast measuring technology of angiotensin converting enzyme inhibition activity of protein zymolyte is provided, can from a large amount of proteolysis products, screen rapidly and accurately and be rich in the zymolyte that suppresses angiotensin converting enzyme (ACE) active peptide, be used to screen the protein zymolyte that is rich in blood pressure lowering peptide.
Method of the present invention comprises the IC that measures the inhibition ACE activity of various proteolysis products with the method for Capillary Electrophoresis
50Value is according to IC
50The size of value is judged the activity of the inhibition ACE of various marine protein zymolytes, IC
50Low more, show that the activity of this protein zymolyte inhibition ACE enzyme is strong more, the content of the active antihypertensive peptide of inhibition ACE is high more.Specifically comprise the steps:
(1) enzyme reaction
The series of samples reactant liquor comprises: 10 μ l substrate Hip-His-Leu (1mM), the angiotensin converting enzyme of 0.8mU (ACE) respectively with (0.03～40mg/ml) the 10 μ l proteolysis product of variable concentrations.Above-mentioned example reaction liquid is all used borate buffer (comprise 0.3M NaCl, the pH 8.3) preparation of 100mM.Reactant liquor all at 37 ℃ of reaction 30min, is used 0.1% trifluoroacetic acid TFA (10 μ l) stopped reaction then.Substrate produces independent hippuric acid and His-Leu dipeptides after the ACE effect.
(2) detect
Angiotensin converting enzyme inhibitory reaction product directly detects on capillary electrophoresis apparatus.Capillary electrophoresis apparatus is Beckman Coulter P/ACE MDQ (Fullerton, CA) device is equipped with PDA (photodiode array) detecting device, data acquisition, analysis, system's control P/ACE MDQ software, this software from Beckman Coulter (Fullerton, CA).Blend sample after the stopped reaction directly detects with Capillary Electrophoresis, and last sample pressure is 1psi, and the time is 5 seconds, electrophoretic voltage 20kV, and uv absorption is 228nm, electrophoresis time 5 minutes, electrophoretic buffer are the borate buffer (pH 9.18) of 20mM.Washed kapillary 1 minute with damping fluid after sample of every survey.Substrate and product hippuric acid went out the peak at 2.5 minutes and 3.5 minutes respectively, according to the peak area of computed in software hippuric acid.
(3) ACE suppresses active calculating (IC
50The calculating of value)
Hippuric acid is mixed with variable concentrations 0.2,0.4,0.6,0.8,1.0,1.2,2,4, and 6mM measures the linear relationship of its peak area and hippuric acid concentration by Capillary Electrophoresis.
Peak area=K * Chip+N
ACE vigor (umol/min)=V * Chip ÷ t
V in the above-mentioned formula: reaction volume, t: reaction time, K: according to the curve constant that peak area and hippuric acid concentration are obtained, Chip: hippuric acid concentration, N: the intersection point of hippuric acid concentration curve and Y-axis.
By the angiotensin converting enzyme effect, do not contain any inhibitor in the reaction mixture, the amount of the hippuric acid HA that discharges from substrate Hip-His-Leu is defined as the 100%ACE vigor.The inhibitor serial dilution that will measure ACE inhibition vigor is become variable concentrations, react jointly with substrate and enzyme respectively,, calculate the IC of inhibitor then according to the peak area of the hippuric acid that is produced
50Value.The amount of the marine protein enzymolysis product of the ACE vigor of inhibition 50% is defined as the inhibition vigor of this enzymolysis product.
IC
50The linear-logarithmic method is adopted in the calculating of value, and to Log (hydrolysate concentration mg/ml) mapping, the value that the gained straight line is corresponding with the intersection point of X-axis is M with Log (ACE vigor/(1-ACE vigor)), and 10
MThe IC that suppresses the ACE vigor for enzymolysis product
50Value.
IC with enzymolysis ace inhibitory peptide
50Value is 0.2 μ M-500 μ M, IC
50Be worth more for a short time, illustrate the inhibiting effect of ACE by force more, it is good more to fall the hemepeptide effect.
The albumen that above-mentioned steps (1) is different at first passes through homogenate, carries out enzymolysis with proteinase then, and behind 5 hours enzymolysis, the centrifuging and taking supernatant with the supernatant freeze-drying, is got an amount of freeze-dried powder and is mixed with certain density inhibitor.
Above-mentioned steps (1) is reflected in the little centrifuge tube of 0.2ml carries out, and reaction directly is placed on sample on the kapillary sample bottle support after finishing.
The present invention uses the ace inhibitory peptide that capillary electrophoresis technique is measured zymolyte, can reduce classic method and expend time in, and expends the shortcoming of sample, can realize the ACE inhibition activity of multiple protein resource zymolyte is carried out high-flux fast screening.
The invention has the advantages that capillary electrophoresis technique is applied to screen antihypertensive active peptide from the proteolysis product.Be embodied in 1, the inhibition vigor of utilization capillary electrophoresis technique fast measuring ACE inhibitor, 2, good reproducibility, highly sensitive, IC
50PH-value determination pH is accurate, and 3, compare with chromatography in the past, significantly reduced mensuration IC
50Be worth the used time, and do not need expensive organic solvent as moving phase.4, utilize capillary electrophoresis technique exploitation to have proteolysis product, the vast market space is arranged the blood pressure function.
(4) description of drawings
Fig. 1 is the IC of embodiment 1
50The linear-logarithmic figure of value, horizontal ordinate is Log (Anchovies fish zymolyte mg/ml) ordinate is Log (ACE vigor/(1-ACE vigor)), to Log (hydrolysate concentration mg/ml) mapping, the intersection point of gained straight line and X-axis is-3.39 with Log (ACE vigor/(1-ACE vigor)).
Fig. 2 is the IC of embodiment 2
50The linear-logarithmic figure of value, horizontal ordinate is Log (zein zymolyte) mg/ml) ordinate is Log (ACE vigor/(1-ACE vigor)), to Log (hydrolysate concentration mg/ml) mapping, the intersection point of gained straight line and X-axis is-3.01 with Log (ACE vigor/(1-ACE vigor)).
(5) embodiment
(1) angiotensin converting enzyme reaction conditions
Example reaction liquid comprises: 10 μ l substrate Hip-His-Leu (1mM), the enzymolysis product of the variable concentrations Hai Yang Anchovies flesh of fish of the angiotensin converting enzyme of 0.8mU (ACE) and 10 μ l.Specific as follows:
The enzymolysis product of the sample substrate Hip-His-Leu Xue angiotensin Zhuanization Mei Anchovies flesh of fish
1 10μl，1mM 0.8mU 10μl，39mg/ml
2 10μl，1mM 0.8mU 10μl，9.3mg/ml
3 10μl，1mM 0.8mU 10μl，4.6mg/ml
4 10μl，1mM 0.8mU 10μl，1.8mg/ml
5 10μl，1mM 0.8mU 10μl，0.63mg/ml
6 10μl，1mM 0.8mU 10μl，0.29mg/ml
Contrast 10 μ l, 1mM 0.8mU water 10 μ l
Above-mentioned reactant liquor is all used borate buffer (comprise 0.3M NaCl, the pH 8.3) preparation of 100mM.Simultaneously with not containing enzymolysis product, the reaction system of having only ACE and substrate Hip-His-Leu in contrast.All samples all is loaded in respectively in the little centrifuge tube of 0.2ml, at 37 ℃ of reaction 30min, uses 0.1% trifluoroacetic acid TFA (10 μ l) stopped reaction then, and sample volume is 40 μ l in the centrifuge tube.
The enzymolysis of the Anchovies flesh of fish adopts existing known technology, for example Anchovies is oppressed elder generation through homogenate, carries out enzymolysis with proteinase then, and behind 5 hours enzymolysis, the centrifuging and taking supernatant with the supernatant freeze-drying, is got an amount of freeze-dried powder and is mixed with certain density inhibitor.Other are by prior art.
(2) testing conditions
Above-mentioned reaction product directly detects on capillary electrophoresis apparatus.Last sample pressure is 1psi, and the time is 5 seconds, electrophoretic voltage 20kV, and uv absorption is 228nm, electrophoresis time 5 minutes, electrophoretic buffer are the borate buffer (pH 9.18) of 20mM.Washed kapillary 1 minute with damping fluid after sample of every survey.Capillary electrophoresis apparatus is BeckmanCoulter P/ACE MDQ (Fullerton, CA) device is equipped with PDA (photodiode array) detecting device, data acquisition, analysis, system's control P/ACE MDQ software, this software from Beckman Coulter (Fullerton, CA).
(3) ACE suppresses active calculating (IC
50The calculating of value)
IC
50The linear-logarithmic method is adopted in the calculating of value, and to Log (hydrolysate concentration mg/ml) mapping, the value that the gained straight line is corresponding with the intersection point of X-axis is M with Log (ACE vigor/(1-ACE vigor)), and 10
MThe IC that suppresses the ACE vigor for enzymolysis product
50Value.
Above-mentioned steps (3) is carried out integration by the area to hippuric acid in all samples, and according to formula logarithm mapping (Fig. 1), the intersection point that draws straight line and X-axis is-3.39, so IC then
50Be 0.4mg/ml.Illustrate that this zymolyte is stronger to the inhibiting effect of ACE.These data and the IC that obtains with high pressure lipuid chromatography (HPLC) (HPLC) method
50Be worth close.
Embodiment 3. is with embodiment 1, and that different is as ACE inhibitor is captopril (Captopril).
Captopril is the competitive inhibitor of ACE, and compound concentration is 0.03～3mg/ml, measures its IC
50Be 22nM, match with the result of spectrophotometric method or high-pressure liquid phase chromatogram therapy determining.
The invention is not restricted to the foregoing description.
The Capillary Electrophoresis figure good reproducibility of the embodiment of the invention can calculate ACE various proteolysis products and various ACE inhibitor accurately and suppress active, for quick isolation identification ace inhibitory peptide provides the good detection means.
Claims (3)
1. the rapid assay methods of the angiotensin converting enzyme inhibition activity of protein zymolyte is characterized in that measuring with the method for Capillary Electrophoresis the IC of the angiotensin converting enzyme inhibition activity of various proteolysis products
50Value specifically comprises the steps:
(1) enzyme reaction
The series of samples reactant liquor comprises: 10 μ l substrate Hip-His-Leu 1mM, 0.8mU angiotensin converting enzyme be respectively the 10 μ l proteolysis products of 0.03～40mg/ml with concentration, above-mentioned example reaction liquid all uses pH 8.3 to contain the borate buffer preparation of the 100mM of 0.3MNaCl, reactant liquor is all at 37 ℃ of reaction 30min, use 0.1% trifluoroacetic acid, 10 μ l stopped reactions then, produce independent hippuric acid and His-Leu dipeptides after the effect of substrate menses angiotensin invertase;
(2) detect
Above-mentioned angiotensin converting enzyme inhibitory reaction product directly detects on capillary electrophoresis apparatus, capillary electrophoresis apparatus is a Beckman Coulter P/ACE MDQ device, the PDA detecting device is equipped with, data acquisition, analyze, system's control P/ACE MDQ software, last sample pressure is 1psi, time is 5 seconds, electrophoretic voltage 20kV, uv absorption is 228nm, electrophoresis time 5 minutes, electrophoretic buffer are the borate buffer pH 9.18 of 20mM, wash kapillary 1 minute with damping fluid after sample of every survey, substrate and product hippuric acid went out the peak at 2.5 minutes and 3.5 minutes respectively, according to the peak area of computed in software hippuric acid;
(3) angiotensin converting enzyme inhibition activity IC
50The calculating of value
Hippuric acid is mixed with variable concentrations 0.2,0.4,0.6,0.8,1.0,1.2,2,4, and 6mM measures the linear relationship of its peak area and hippuric acid concentration by Capillary Electrophoresis:
Peak area=K * Chip+N
ACE vigor (umol/min)=V * Chip ÷ t
V in the above-mentioned formula: reaction volume, t: reaction time, K: according to the curve constant that peak area and hippuric acid concentration are obtained, Chip: hippuric acid concentration, N: the intersection point of hippuric acid concentration curve and Y-axis;
By the angiotensin converting enzyme effect, do not contain any inhibitor in the reaction mixture, the amount of the hippuric acid HA that discharges from substrate Hip-His-Leu is defined as 100% angiotensin converting enzyme vigor, the protein zymolyte serial dilution that will measure angiotensin converting enzyme inhibition vigor is become variable concentrations, react jointly with substrate and enzyme respectively, according to the peak area of the hippuric acid that is produced, calculate the IC of protein zymolyte then
50Value; The amount of the marine protein enzymolysis product of the ACE vigor of inhibition 50% is defined as the inhibition vigor of this enzymolysis product;
IC
50The linear-logarithmic method is adopted in the calculating of value, and to Log (proteolysis substrate concentration mg/ml) mapping, the value that the gained straight line is corresponding with the intersection point of X-axis is M with Log (ACE vigor/(1-ACE vigor)), and 10
MThe IC that suppresses the ACE vigor for enzymolysis product
50Value.
2. the rapid assay methods of the angiotensin converting enzyme inhibition activity of protein zymolyte as claimed in claim 1, it is characterized in that, the preparation method of the protein zymolyte of step (1) at first makes albumen through homogenate, carry out enzymolysis with proteinase then, behind 5 hours enzymolysis, the centrifuging and taking supernatant with the supernatant freeze-drying, is got freeze-dried powder and is mixed with described protein zymolyte.
3. the rapid assay methods of the angiotensin converting enzyme inhibition activity of protein zymolyte as claimed in claim 1 is characterized in that, step (1) is reflected in the little centrifuge tube of 0.2ml carries out, and reaction directly is placed on sample on the kapillary sample bottle support after finishing.
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| CA2620403A1 (en) * | 2005-09-02 | 2007-03-08 | Wako Pure Chemical Industries, Ltd. | Complex formation method and separation method |
| CN100434917C (en) * | 2006-01-23 | 2008-11-19 | 湖南师范大学 | Method for quick screening angiotemsin invertase inhibitor combined using high effieient liquid chromatograph and mass spectrum |
| CN100529734C (en) * | 2006-06-13 | 2009-08-19 | 中国科学院上海有机化学研究所 | Acetylcholine esterase inhibitor screening method |
| CN101246140B (en) * | 2007-06-16 | 2012-01-11 | 中国海洋大学 | Novel method for fast measuring collagen hydrolysis degree by biologic sensor |
| CN101246148B (en) * | 2007-11-19 | 2011-05-18 | 中国海洋大学 | Method for detecting external activity of angiotonin enzyme inhibition peptide |
| CN101696233B (en) * | 2009-10-29 | 2012-02-01 | 吉林大学 | Albumin angiotensin converting enzyme inhibition peptide and preparation method thereof |
| CN102288711A (en) * | 2011-05-12 | 2011-12-21 | 扬州大学 | Method for accurately determining in-vitro inhibitory activity of angiotensin-converting enzyme |
| CN115047092B (en) * | 2022-04-07 | 2023-10-20 | 济宁医学院 | Screening method of angiotensin-transferase II inhibitor |
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| JP2003238589A (en) * | 2002-02-08 | 2003-08-27 | National Federation Of Dairy Cooperative Associations | New peptide and use therfor |
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| JP2003238589A (en) * | 2002-02-08 | 2003-08-27 | National Federation Of Dairy Cooperative Associations | New peptide and use therfor |
Non-Patent Citations (3)
| Title |
|---|
| 从小麦胚芽蛋白中分离和鉴定血管紧张素转化酶抑制肽的研究 辛志宏 等，食品科学，第24卷第7期 2003 * |
| 血管紧张素转化酶抑制肽的研究进展 何海伦 等，中国生物工程杂志，第24卷第9期 2004 * |
| 高效毛细管电泳测定血管紧张素转化酶抑制剂captopril的活性 辛志宏 等，药学学报，第38卷第11期 2003 * |
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