WO2001070240A1 - Use of alkylammonium salts as protein crystallization reagents - Google Patents
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- WO2001070240A1 WO2001070240A1 PCT/US2001/008425 US0108425W WO0170240A1 WO 2001070240 A1 WO2001070240 A1 WO 2001070240A1 US 0108425 W US0108425 W US 0108425W WO 0170240 A1 WO0170240 A1 WO 0170240A1
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- salt
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- protein
- salt product
- alkylammonium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/28—Compounds containing heavy metals
- A61K31/295—Iron group metal compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
-
- C—CHEMISTRY; METALLURGY
- C30—CRYSTAL GROWTH
- C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
- C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
- C30B29/54—Organic compounds
- C30B29/58—Macromolecular compounds
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- C—CHEMISTRY; METALLURGY
- C30—CRYSTAL GROWTH
- C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
- C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
Definitions
- the present invention relates the use of alkylammonium salts in protein crystallography.
- Ethylammonium nitrate a low-melting organic salt
- EAN Ethylammonium nitrate
- EAN has water-like characteristics including a capacity for hydrogen bonding and the promotion of micelle formation by some surfactants (Evans, D.F., Yamauchi, A., Roman, R. & Casassa, E.Z. (1982) J. Colloids Interface Sci. 88, 89-96).
- the present invention relates to a method for preparing a stable protein solvent and crystallization reagent. At least one alkylammonium salt having hydrophobic and ionic characteristics and having the ability to hydrogen bond is combined with a suitable compound to form a precipitate or crystal of the protein.
- the alkylammonium salt has the formula
- Another aspect of the present invention relates to a method for purifying an alkylammonium salt. A suitable amount of an acid is cooled.
- a suitable amount of an alkyl amine is added to the cooled acid to form an alkylammonium salt product.
- the salt product is stirred for a suitable length of time over charcoal pellets.
- the salt product is gravity filtered.
- the charcoal is rinsed with a suitable alcohol to remove any residual salt product.
- the salt product is lyophilized for a suitable length of time.
- the alkylammonium salts are especially useful solvents and crystallization reagents and are especially useful as precipitating agent in protein crystallization.
- the present invention relates to the use of alkylammonium salts as useful solvents for applications in protein chemistry. According to the present invention, proteins treated with an alkylammonium salt are stable. Alkylammonium salts that are contemplated to be within the scope of the present invention have the structure:
- EAN which is an organic salt and has ionic character in addition to the hydrophobic character contributed by the ethyl substituent, is used to treat the protein lysozyme. Lysozyme, when treated with neat EAN and heated to 333°K for 6 h, retains 45% activity after such treatment, compared with a 2% retention of activity when treated similarly with DMSO.
- Ethylamine was purchased from Aldrich.
- Lysozyme (L-7773) was purchased from Sigma.
- Nitric acid, sodium acetate and glacial acetic acid were obtained from JT Baker.
- Lysozyme crystallized from EAN has two crystal forms.
- EAN was synthesized from nitric acid and ethylamine in the following manner. 150 g of 70% nitric acid was added to a 500 ml round-bottom flask. The acid was stirred on ice to allow it to cool. An excess of 70% ethylamine was added drop wise with stirring to the nitric acid. The EAN was stirred overnight with charcoal pellets and then gravity filtered, rinsing the charcoal with methanol to wash off any residual EAN. The product was then lyophilized for one week. The final product was characterized by 1 H NMR.
- Hen egg-white lysozyme was dissolved to 50 mg ml "1 in 100 xrxM acetate buffer pH 4.5.
- Hanging-drop crystallization trays were set up with a 500 ⁇ l reservoir solution that contained 100 m/W acetate buffer pH 4.5-5.6 and 300-500 m EAN.
- the 10 ⁇ hanging drop contained 5 ⁇ of protein solution and 5 ⁇ of reservoir solution.
- Monoclinic crystals grew at pH 4.5-4.6 over an EAN concentration range of 300-500 mM.
- the tetragonal form grows at pH 5.4-5.6 at an EAN concentration of 300-400 mM.
- Crystals were mounted in a glass capillary and room-temperature X-ray diffraction data were collected to 1.6 A resolution on a Rigaku rotating-anode generator equipped with an R-AXIS IV two image-plate detector system. Diffraction intensities were integrated with DENZO and scaled using SCALEPACK (Otwinowski, Z. and Minor, W. (1997), Methods Enzymol., 276, 307-326).
- Example 2 Hydrophobic compounds, such as ferrocene which is normally insoluble in water, are found to be soluble in solutions containing as little as 500 mM EAN. EAN has many applications in protein crystallography. Hydrophobic ligands, which are generally difficult to introduce into a protein crystal dissolve in EAN solutions and are more easily delivered into protein crystals grown with EAN. Example 3 Also, some compounds, especially those with exposed hydrophobic regions and that do not respond to traditional precipitating agents, such as peripheral or integral membrane proteins, have improved solubility in the presence of EAN. Additionally, EAN is a useful additive for improving the monodispersity of the compounds with multiple aggregation states and for increasing crystallizability of the compound solution.
- EAN is a useful additive for improving the monodispersity of the compounds with multiple aggregation states and for increasing crystallizability of the compound solution.
Abstract
A method of preparing a stable solvent and crystallization reagent is described where at least one alkyl ammonium salt having hydrophobic and ionic characteristics and having the ability to hydrogen bond with a suitable compound is combined with the protein to form a precipitate of crystals of the compound. The alkylammonium salt is purified by cooling a suitable amount of an acid; adding a suitable amount of an alkyl amine to the cooled acid to form an alkylammonium salt product; stirring the salt product for a suitable length of time over charcoal pellets; gravity filtering the salt product; rinsing the charcoal with a suitable alcohol to remove any residual salt product; and lyophilizing the salt product for a suitable length of time.
Description
DESCRIPTION USE OF ALKYLAMMONIUM SALTS AS PROTEIN CRYSTALLIZATION REAGENTS Technical Field The present invention relates the use of alkylammonium salts in protein crystallography.
Background of the Invention
Ethylammonium nitrate (EAN), a low-melting organic salt, is a liquid at room temperature and has a freezing point around 287 °K. (Sudgen, S. and Wilkens, H. (1929), J. Chem. Soc, pp. 1291-1298). EAN has water-like characteristics including a capacity for hydrogen bonding and the promotion of micelle formation by some surfactants (Evans, D.F., Yamauchi, A., Roman, R. & Casassa, E.Z. (1982) J. Colloids Interface Sci. 88, 89-96).
Increasingly, large amounts of many types of protein are used in research. The methods currently used to isolate or crystallize the proteins have been inefficient and involve multiple steps.
Therefore, there is a need for an efficient protein crystallization reagent.
There is also a need for a method to increase the crystallizability of a protein solution.
There is a further need for an efficient method to purify alkyl ammonium salts which can be used in a method for making an efficient protein crystallization reagent.
Summary of the Invention
The present invention relates to a method for preparing a stable protein solvent and crystallization reagent. At least one alkylammonium salt having hydrophobic and ionic characteristics and having the ability
to hydrogen bond is combined with a suitable compound to form a precipitate or crystal of the protein.
In preferred embodiments, the alkylammonium salt has the formula
R NH3+ X- where R is CH3, CH2CH3, CH2CH2CH3,
CH2CH2CH2CH2CH3, -CH(CH3)2,
CH2CH(CH3)2, or any five carbon (or smaller) branched or linear chain and where X = NO3-(nitrate), SO4 2 (sulfate), H2PO4-(phosρhate), Cl (chloride),
CH3COO"(acetate), Br (bromide), l (iodide), CIO4-(perchlorate), SCN"
(thiocyanate) or other suitable anionic group. Another aspect of the present invention relates to a method for purifying an alkylammonium salt. A suitable amount of an acid is cooled.
A suitable amount of an alkyl amine is added to the cooled acid to form an alkylammonium salt product. The salt product is stirred for a suitable length of time over charcoal pellets. The salt product is gravity filtered. The charcoal is rinsed with a suitable alcohol to remove any residual salt product. Finally, the salt product is lyophilized for a suitable length of time.
The alkylammonium salts are especially useful solvents and crystallization reagents and are especially useful as precipitating agent in protein crystallization.
Description of the Invention
The present invention relates to the use of alkylammonium salts as useful solvents for applications in protein chemistry. According to the present invention, proteins treated with an alkylammonium salt are stable. Alkylammonium salts that are contemplated to be within the scope of the present invention have the structure:
-NIV X-
where R = CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, CH2CH2CH2CH2CH3, -CH(CH3)2, -CH(CH3)CH2CH3, CH2CH(CH3)2, and the like, preferably any five carbon (or smaller) branched or linear chain, and where X = NO3-(nitrate), SO4 2 (sulfate), H2PO4-(phosphate), Cl (chloride), CH3COO"(acetate), Br (bromide), l (iodide), CIO4-(perchlorate), SCN- (thiocyanate) or other suitable anionic group.
In one embodiment, EAN which is an organic salt and has ionic character in addition to the hydrophobic character contributed by the ethyl substituent, is used to treat the protein lysozyme. Lysozyme, when treated with neat EAN and heated to 333°K for 6 h, retains 45% activity after such treatment, compared with a 2% retention of activity when treated similarly with DMSO.
Example 1 Materials and Methods
Reagents
Ethylamine was purchased from Aldrich. Lysozyme (L-7773) was purchased from Sigma. Nitric acid, sodium acetate and glacial acetic acid were obtained from JT Baker. Lysozyme crystallized from EAN has two crystal forms.
Throughout the pH range examined, large clumps of crystals resembling starbursts were frequently observed. Single rectangular crystals grew and diffraction data were collected and processed. These crystals were determined to be monoclinic with unit-cell parameters a = 28.015, b = 62.75, c = 52.883 A.0 = 90.667 as shown in Table 1 .
EAN Synthesis
EAN was synthesized from nitric acid and ethylamine in the following manner. 150 g of 70% nitric acid was added to a 500 ml round-bottom flask. The acid was stirred on ice to allow it to cool. An
excess of 70% ethylamine was added drop wise with stirring to the nitric acid. The EAN was stirred overnight with charcoal pellets and then gravity filtered, rinsing the charcoal with methanol to wash off any residual EAN. The product was then lyophilized for one week. The final product was characterized by 1H NMR.
Crystallization
Hen egg-white lysozyme was dissolved to 50 mg ml"1 in 100 xrxM acetate buffer pH 4.5. Hanging-drop crystallization trays were set up with a 500 μl reservoir solution that contained 100 m/W acetate buffer pH 4.5-5.6 and 300-500 m EAN. The 10 μ\ hanging drop contained 5 μ\ of protein solution and 5 μ\ of reservoir solution. Monoclinic crystals grew at pH 4.5-4.6 over an EAN concentration range of 300-500 mM. The tetragonal form grows at pH 5.4-5.6 at an EAN concentration of 300-400 mM.
X-ray Characterization
Crystals were mounted in a glass capillary and room-temperature X-ray diffraction data were collected to 1.6 A resolution on a Rigaku rotating-anode generator equipped with an R-AXIS IV two image-plate detector system. Diffraction intensities were integrated with DENZO and scaled using SCALEPACK (Otwinowski, Z. and Minor, W. (1997), Methods Enzymol., 276, 307-326).
Table 1
Monoclinic diffraction data statistics
Resolution l/σ(l) R-ymi CO. TiDleteness No. of unique iAi (%) reflections
99-2.91 24.2 0.029 86.7 4086
2.91-2.31 25.0 0.041 95.3 4426
2.31 -2.02 20.0 0.052 93.3 4298
2.02-1.83 15.4 0.071 90.7 4200
1.83-1.70 9.2 0.125 89.1 4117
1.70-1.60 5.9 0.195 77.9 3598
All hkl 22.5 0.038 88.8 24725
Rsym = ^-h l( I ( iklWhkl I )/∑-hk/hkl-
Diffraction data along the h axis were not measured so systematic absences could not be examined. Therefore, the P2, space group was confirmed by molecular replacement using PDB entry 1 b2k. Cycles of rigid-body, positional and ?-value refinement with X-PLOR (Brϋnger, A.T. (1992), X-PLOR, Version 3.1 A Systems for X-ray Crystallography and NAIR, Yale University, Connecticut, USA) without the addition of water molecules resulted in a final R value of 28.2%. Lysozyme crystallizes in the second crystal form at pH 5.4-5.6 in the tetragonal space group P -32Λ2 (Fig. 1b) with unit-cell parameters a = b = 79.054, c = 38.027 A, as shown in Table 2.
Table 2
Tetragonal diffraction data statistics
Resolution l/σ(l) Bsyml Completeness No. of unique
<Al (%) reflections
99-2.91 30.5 0.050 90.7 2653
2.91-2.31 31.8 0.067 100.0 2755
2.31-2.02 26.4 0.080 100.0 2717
2.02-1 .83 21.1 0.098 100.0 2691
1.83-1.70 12.9 0.138 100.0 2697
1 .70-1 .60 7.7 0.202 98.0 261 1
AH hkl 28.5 0.053 98.0 16124
This data was also subjected to refinement in X-PLOR using PDB entry 1 1 za and the R value converged at 26.8%. These crystallographic refinements, together with the fact that lysozyme retains activity after treatment with EAN, indicates that lysozyme maintains its native structure in the presence of low levels of EAN and is neither inactivated nor denatured.
Example 2 Hydrophobic compounds, such as ferrocene which is normally insoluble in water, are found to be soluble in solutions containing as little as 500 mM EAN. EAN has many applications in protein crystallography. Hydrophobic ligands, which are generally difficult to introduce into a protein crystal dissolve in EAN solutions and are more easily delivered into protein crystals grown with EAN.
Example 3 Also, some compounds, especially those with exposed hydrophobic regions and that do not respond to traditional precipitating agents, such as peripheral or integral membrane proteins, have improved solubility in the presence of EAN. Additionally, EAN is a useful additive for improving the monodispersity of the compounds with multiple aggregation states and for increasing crystallizability of the compound solution.
Example 4
The addition of as little as 5% EAN to the protein solution makes previously polydisperse human replication protein A samples monodisperse as determined by dynamic light scattering using a Protein Solutions DynaPro-801 instrument. Although the invention has been described with respect to particular embodiments and methods, it will be appreciated that various changes and modifications can be made without departing from the invention.
The above detailed description of the present invention is given for explanatory purposes. It will be apparent to those skilled in the art that numerous changes and modifications can be made without departing from the scope of the invention. Accordingly, the whole of the foregoing description is to be construed in an illustrative and not a limited sense, the scope of the invention being defined solely by the appended claims.
Claims
1. A method of preparing a stable solvent and crystallization reagent comprising combining at least one alkyl ammonium salt having hydrophobic and ionic characteristics and having the ability to hydrogen bond with a suitable compound to form a precipitate of the compound.
2. The method of claim 1 , in which the alkylammonium salt comprises
R NHX X-
where R is CH3, CH2CH3, CH2CH2CH3, . CH2CH2CH2CH3, CH2CH2CH2CH2CH3, -CH(CH3)2, -CH(CH3)CH2CH3, CH2CH(CH3)2, or any five carbon (or smaller) branched or linear chain and where X = NO3- (nitrate), SO4 2 (sulfate), H2PO4-(phosphate), Cl (chloride), CH3COO- (acetate), Br'(bromide), l (iodide), CIO4-(perchlorate), SCN (thiocyanate) or other suitable anionic group.
3. The method of claim 1 , in which the precipitate comprises crystals of a protein.
4. The method of claim 2, in which the alkylammonium salt is ethyl ammonium nitrate (EAN).
5. The method of claim 3, in which the protein is lysozyme.
6. The method of claim 1 , in which the compound comprises a hydrophobic compound.
7. The method of claim 1 , in which the compound is ferrocene.
8. The method of claim 1 , in which the compound is human replication protein A.
9. A method for purifying an alkylammonium salt comprising cooling a suitable amount of an acid; adding a suitable amount of an alkyl amine to the cooled acid to form an alkylammonium salt product; stirring the salt product for a suitable length of time over charcoal pellets; gravity filtering the salt product; rinsing the charcoal with a suitable alcohol to remove any residual salt product; and lyophilizing the salt product for a suitable length of time.
10. The method of claim 9, in which the alkylammonium salt comprises
R NH3 + X-
where R is CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, CH2CH2CH2CH2CH3, -CH(CH3)2, -CH(CH3)CH2CH3, CH2CH(CH3)2, or any five carbon (or smaller) branched or linear chain and where X = NO3- (nitrate), SO4 (sulfate), H2PO4-(phosphate), Cl (chloride), CH3COO_ (acetate), Br(bromide), Idodide), CIO4-(perchlorate), SCN (thiocyanate) or other suitable anionic group.
11. The method of claim 9, in which the alkylammonium salt is ethyl ammonium nitrate (EAN).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US19085400P | 2000-03-20 | 2000-03-20 | |
| US60/190,854 | 2000-03-20 |
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| WO2001070240A1 true WO2001070240A1 (en) | 2001-09-27 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112469825A (en) * | 2018-09-11 | 2021-03-09 | 诺维信公司 | Stable granules for feed compositions |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4197315A (en) * | 1977-08-30 | 1980-04-08 | Mcneil Laboratories, Inc. | (2-Amino-2-oxoethoxy)acetic acid compounds, compositions and methods |
| US4377654A (en) * | 1981-11-16 | 1983-03-22 | Polaroid Corporation | Polymerization processes and polymer compositions |
| US5296588A (en) * | 1991-12-31 | 1994-03-22 | Lever Brothers Company, Division Of Conopco, Inc. | Process of preparing N-substituted aldonamides |
| US5416170A (en) * | 1992-05-27 | 1995-05-16 | Agency Of Defense Development | Conductive polymeric composite with high electrical activation density and method for preparing the same |
| US5492723A (en) * | 1990-05-22 | 1996-02-20 | Sepragen Corporation | Adsorbent medium |
| US6024855A (en) * | 1997-08-15 | 2000-02-15 | Sachem, Inc. | Electrosynthesis of hydroxylammonium salts and hydroxylamine using a mediator |
-
2001
- 2001-03-16 WO PCT/US2001/008425 patent/WO2001070240A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4197315A (en) * | 1977-08-30 | 1980-04-08 | Mcneil Laboratories, Inc. | (2-Amino-2-oxoethoxy)acetic acid compounds, compositions and methods |
| US4377654A (en) * | 1981-11-16 | 1983-03-22 | Polaroid Corporation | Polymerization processes and polymer compositions |
| US5492723A (en) * | 1990-05-22 | 1996-02-20 | Sepragen Corporation | Adsorbent medium |
| US5296588A (en) * | 1991-12-31 | 1994-03-22 | Lever Brothers Company, Division Of Conopco, Inc. | Process of preparing N-substituted aldonamides |
| US5416170A (en) * | 1992-05-27 | 1995-05-16 | Agency Of Defense Development | Conductive polymeric composite with high electrical activation density and method for preparing the same |
| US6024855A (en) * | 1997-08-15 | 2000-02-15 | Sachem, Inc. | Electrosynthesis of hydroxylammonium salts and hydroxylamine using a mediator |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE CAPLUS [online] CAS, (COLUMBUS, OHIO, USA); BENHLIMA ET AL.: "Electrochemistry of fused ethylammonium nitrate at 298 K: determination of a redox potential scale", XP002943699, Database accession no. 1998:129903 * |
| J. CHIM. PHYS., vol. 95, no. 1, 1998, pages 25 - 44 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112469825A (en) * | 2018-09-11 | 2021-03-09 | 诺维信公司 | Stable granules for feed compositions |
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