CN104198631B - Method for improving hydrogen and deuterium atomic exchanging efficiency of protein in nuclear magnetism reagent - Google Patents
Method for improving hydrogen and deuterium atomic exchanging efficiency of protein in nuclear magnetism reagent Download PDFInfo
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- CN104198631B CN104198631B CN201410394622.7A CN201410394622A CN104198631B CN 104198631 B CN104198631 B CN 104198631B CN 201410394622 A CN201410394622 A CN 201410394622A CN 104198631 B CN104198631 B CN 104198631B
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Abstract
The invention provides a method for improving hydrogen and deuterium atomic exchanging efficiency of protein in nuclear magnetism reagent by pressurizing inert gas and radiating isotopes under an anaerobic condition. The to-be-detected protein is dissolved in the nuclear magnetism reagent, and inert gas is introduced into a solution system so as to increase the pressure to 0.15MPa to 1.0MPa. The radioactive source selects radioactive isotopes such as cobalt-60 and caesium-137, and the dosage is 5kGy to 500kGy. The detected protein basically contains all protein types, and the exchanging rate is detected in a mass-spectrography.
Description
Technical field
The present invention relates to a kind of pressurize using under anaerobic state, isotopic radiation provides additional energy, in order to improve albumen
The method of matter hydrogen D-atom exchange efficiency in deuterated reagent.The invention belongs to chemical reaction heat mechanics field.
Background technology
Albumen in the raw typically all has the foldable structure compacting.Constitute in the amino acid backbone of protein
Alpha-position hydrogen atom is due to folding or helical conformation, and is in different locus.Under anaerobic, radiosiotope
The alpha-position Hydrogen transfer of amino acid fragment can be caused, forms alpha-carbon free radical center by radiation.Meanwhile, necessarily several
In rate, if the α-side chain terminal of amino acid fragment also contains hydrogen atom, likewise, can also shape in described alpha-position side chain terminal
Become alpha-position side chain free radical center.Deuterated reagent under radiation condition, also can form D-atom free radical under anaerobic, can
With amino acid fragment free radical (alpha-carbon free radical or α-side chain free radical) bonding (the sp3 rail for example being provided by carbon atom
Road bonding).This process, can regard the hydrogen D-atom exchange process of protein main chain as, and this is a kind of radical reaction, with
Traditional amide hydrogen atom exchanges and there is essential distinction.
In amino acid backbone, the effective site of proton exchange effect is determined by the accurate construction of albumen.Described effective site
Hydrogen atom on the alpha -carbon atom that adjoined with amide n is referred mainly on protein main chain, or the alpha-position side chain of amino acid fragment.
When protein is dissolved in d2After o and some other deuterated reagent, under the conditions of radical reaction, the friendship of hydrogen D-atom will be there is
Change.When albumen is under tight folded state, only fewer number of α-hydrogen atom or aterminal hydrogen atom can contact solvent simultaneously
Participate in proton exchange.Described exchange efficiency is in protein folding space depending on α-hydrogen atom or whether participates in protein
Between interaction.The hydrogen deuterium exchange speed of protein difference sequence fragment can be determined by detection, thus judging albumen
In matter structure which position take part in conformation change having formed active center may and potential protein-protein mutual
Action site.This proton-exchange reaction of α-hydrogen atom can serve as judging, characterizes the probe application of protein structure in enzyme
The center of living, the judgement of drug target.
The isotopic Exchange of protein main chain can be characterized by one-dimensional or two-dimentional nuclear-magnetism and be measured, but researchers are more
Tend to use mass spectrography, by the ratio of molecular weight increase and theoretical value, directly calculate the hydrogen exchange rate of deuterium.For macromolecule
Proteins react system, how to be tested using MALDI-TOF-MS (maldi-tof), right
In general small molecular weight protein fragment such as enzyme etc., ion trap mass spectrometry and triple level Four bar mass spectrum all can be used to measure.
The reaction system that on above-mentioned protein main chain, hydrogen D-atom free radical exchanges, is typically mating with protein example
Ph scope, in anaerobic environment, allows system voluntarily to react, until it reaches the thermodynamical equilibrium that hydrogen D-atom exchanges.This traditional
Proton exchange is allowed to naturally tend to the reaction method balancing, although can be on the premise of keeping protein example property constant, in advance
Survey protein structure and active site, but response speed is slow (＞ 30min), exchange rate low (＜ 80%).One kind is efficiently quick
Hydrogen D-atom exchange method, has important research and development meaning and is in long-term technology improvement.
The application of isotopic radiation technology speculates scientific theory in molecular level, or even atom, atomic nucleus level
Dynamically examined, enriched research meanses.Isotopic radiation technology radioactive breeding, deinsectization sterilization, food fresh keeping and
The aspects such as hydraulic engineering have all created huge economic benefit and social benefit.In chemical reaction field, radiation ray with
The interaction of material, says to radioactive source, and it is a kind of energy transmission and energy loss process；For exposure system, it
It is a kind of reaction of the physical property to extraneous energy and absorption process.For protein, accepting the situation of isotopic radiation
Under, protein structure accepts energy and becomes easier to change conformation.Simultaneously extra pressure, on the one hand makes protein more readily soluble
Solution, in reagent, makes protein conformation more towards unfolding；Another aspect malleation makes free radical center thermodynamically be more easy to occur；This
Outward, noble gas pressure process can exclude oxygen by promotion system, maintains anaerobic environment.Isotopic radiation is combined to albumen with gas pressurized
In the deuterated solution system of matter, the impact of hydrogen deuterium exchange efficiency does not have been reported that.
Content of the invention
The invention provides a kind of utilize under anaerobic state, it is filled with noble gas pressurization, isotopic radiation improves protein in deuterium
Method for hydrogen D-atom exchange efficiency in reagent.In the present invention, proteolytic to be detected need not in deuterated reagent
Step protein crystal afterwards.Described deuterated reagent is the mixed system of a kind of deuterated reagent refering in particular to or several deuterated reagent.Deuterium
For the ph value of solution, depending on the dissolubility of albumen to be detected.
In order that reaction system reaches anaerobic environment, it is filled with argon in the deuterated solution of described protein and is more than 20 minutes,
It is filled with n to the deuterated solution of described protein again2O gas is more than 15 minutes, and the deuterated solution oxygen content of described protein is less than
300ppm.
While solubilising protein, apply gas pressure to the deuterated liquid level of solution of protein.Described applying gas pressure
Method is to be filled with the hermetic container containing protein solution with noble gases.Protein solution system pressure is 0.15-
1.0mpa, preferably 0.3-0.5mpa.Described noble gases are one of helium, argon, nitrogen or two of which gas
Mixed gas, preferably argon.
The detection object of the present invention is included for albumin, globulin, histone, protamine, alcohol soluble protein, glutelin, hard
Protide, nucleoprotein, glycoprotein, lipoprotein, chromoprotein, metalloprotein, phosphoprotein one or several.For different inspections
Survey object, selected deuterated reagent is heavy water, deuterated acetic acid, deuterated acetone, deuterated acetonitrile, deuterated methanol, deuterochloroform, deuterium
For dichloromethane, deuterated normal hexane, deuterated normal octane, deuterated pentane, deuterated oxolane, deuterated toluene, deuterated dimethylbenzene,
One of deuterated benzoic acid or wherein several mixed solutions.During detection albumen, one of selection gist of deuterated solvent is
Testing protein can be allowed to be completely dissolved；One of selection gist of deuterated solvent is not cause testing protein degeneration.
The radioactive source of the present invention come from radiosiotope americium -241, barium -133, cadmium-109, californium -252, cobalt -57, cobalt -
60th, caesium -137, europium -152, Iron-55, gadolinium -153, krypton -85, sodium -22, nickel -63, promethium -147, ruthenium -106, selenium -75, Strontium-90, ytterbium -
169th, one of Iridium-192 source or several.It is preferably cobalt -60 and caesium -137.Radiosiotope using dosage is 1 × 10-4-1
×104Kgy, preferably 5-500kgy.During detection albumen, one of selection gist of radiosiotope using dosage is to lead
Cause testing protein degeneration.
Under heretofore described pressurized conditions, isotopic radiation improves protein hydrogen D-atom in deuterated reagent and exchanges effect
The method of rate, more than 99% in 2min, described exchange efficiency is detected hydrogen D-atom conversion ratio with mass spectrography.Wherein, molecular weight
Protein example more than 5,000 is tested with MALDI-TOF-MS (maldi-tof), point
Son amount is less than 5,000 protein example ion trap mass spectrometry or triple level Four bar mass spectroscopy.Exchange rate equation below
Calculate, e=[(m-m0%)/(m100%-m0%)] × 100%；Wherein e is hydrogen D-atom exchange rate, and m is instant sampling gained albumen
Quality sample molecular mass, m0%For protein example initial molecular weight, m100%For protein example alpha-position hydrogen is former or alpha-position side chain
Aterminal hydrogen atom replaced by D-atom completely after molecular weight.
Brief description
Specific embodiment
With specific embodiment, technical scheme is described, but protection scope of the present invention is not restricted to this.
Embodiment 1
In being connected with the manometric pressure glass reaction container of digital display, bovine hemoglobin is dissolved in heavy water, and (99.9% is former
Sub- d), (98% atom d) and deuterated methanol are (in the mixed solution (volume ratio is 95: 2: 3) of 99.5% atom d) for deuterated acetic acid.
It is filled with argon 30 minutes, then be filled with n to the deuterated solution of described protein2O gas 20 minutes, the deuterated solution of described protein is oxygen-containing
Amount=200ppm.Solution concentration is 0.1mol/l, to system applying argon gas so that system pressure reaches 0.15mpa.Radioactive source be cobalt-
60, dosage is 5kgy.Reaction 2min, gained test specimens are characterized with MALDI-TOF-MS.By public affairs
Formula e=[(m-m0%)/(m100%-m0%)] × 100% be calculated, hydrogen D-atom exchange rate is 99.2%.
Embodiment 2
In being connected with the pressure glass reaction container of manometric tetrafluoro interface, Kallidin I is dissolved in heavy water, and (99.9% is former
It is sub- that d) (in the mixed solution (volume ratio is 99: 1) of 98% atom d), solution concentration is 0.2mol/l with deuterated acetic acid.It is filled with argon
Gas 25 minutes, then it is filled with n to the deuterated solution of described protein2O gas 25 minutes, the deuterated solution oxygen content of described protein=
150ppm.To system applying argon gas so that system pressure reaches 0.4mpa.Radioactive source is caesium -137, and dosage is 500kgy.Reaction
2min, gained test specimens are characterized with ion trap mass spectrometry.By formula e=[(m-m0%)/(m100%-m0%)] × 100% be calculated,
Hydrogen D-atom exchange rate is 99.5%.
Claims (8)
1. a kind of method improving protein hydrogen D-atom exchange efficiency in deuterated reagent is it is characterised in that protein is deuterated
Solution is under anaerobic state, and described deuterated solution is the heavy water of 99.9% atom d, 98% atom d that volume ratio is 95: 2: 3
Deuterated acetic acid, the mixed solution of deuterated methanol of 99.5% atom d or volume ratio be 99: 1 the heavy water of 99.9% atom d,
The deuterated acetic acid mixed solution of 98% atom d；To system applying argon gas so that system pressure reaches 0.15mpa or 0.4mpa, pass through
Radioisotopic radiation provides additional energy, promotes the exchange of the hydrogen atom on protein main chain and D-atom.
2. method according to claim 1 is it is characterised in that described protein deuterated solution oxygen content is less than 300ppm.
3. method according to claim 2 is more than 20 it is characterised in that being filled with argon in the deuterated solution of described protein
Minute, then it is filled with n to the deuterated solution of described protein2O gas is more than 15 minutes.
4. method according to claim 1 is it is characterised in that use non-metallic conduit to the deuterated solution containing protein
Container in be filled with argon.
5. method according to claim 1 it is characterised in that described radiosiotope be americium -241, barium -133, cadmium -
109th, californium -252, cobalt -57, cobalt -60, caesium -137, europium -152, Iron-55, gadolinium -153, krypton -85, sodium -22, nickel -63, promethium -147,
One of ruthenium -106, selenium -75, Strontium-90, ytterbium -169, Iridium-192 source or several.
6. method according to claim 1 is it is characterised in that radiosiotope radiation dose is 1 × 10-4-1×
104kgy.
7. method according to claim 6 is it is characterised in that radiosiotope radiation dose is 5-500kgy.
8. method according to claim 1 is it is characterised in that described protein is albumin, globulin, histone, essence
Albumen, alcohol soluble protein, glutelin, scleroprotein class, nucleoprotein, glycoprotein, lipoprotein, chromoprotein, metalloprotein, phosphoprotein
In one or several.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102574753A (en) * | 2009-10-26 | 2012-07-11 | E.I.内穆尔杜邦公司 | Method for preparing deuterated aromatic compounds |
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| EP2419918A4 (en) * | 2009-04-14 | 2016-12-21 | Univ Northeastern | Rapid gas-phase isotopic labeling for enhanced detection of protein conformations |
| WO2012112448A1 (en) * | 2011-02-16 | 2012-08-23 | Waters Technologies Corporation | Immobilized enzymatic reactor |
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| CN102574753A (en) * | 2009-10-26 | 2012-07-11 | E.I.内穆尔杜邦公司 | Method for preparing deuterated aromatic compounds |
Non-Patent Citations (3)
| Title |
|---|
| Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS;John R.Engen;《Analytical Chemistry》;20091001;第81卷(第19期);第7870～7875页 * |
| Identification of the sites of hydroxyl radical reaction with peptides by hydrogen/deuterium exchange: prevalence of reactions with the side chains;Michael B.Goshe等;《Biochemistry》;20000127;第39卷;第1762页右栏第2-3段，第1765页右栏第1段 * |
| Pressure effects on protein secondary structure and hydrogen deuterium exchange in chymotrypsinogen: a Fourier transform infrared spectroscopic study;P.T.T. Wong等;《Biochimica et Biophysica Acta》;19881231;第956卷;摘要，第4页右栏第2-19行 * |
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