US3385766A - Lead chelates for biological separations - Google Patents

Description

United States Patent ABSTRACT OF THE DISCLOSURE Divalent lead chelates of acids of the structure wherein X is H or -CH OH, are used as solutes in density fractionations, for example, separation of different bacterial spores from each other or separation of undamaged from damaged bacterial spores. Preferred embodiments are the divalent lead chelates of N,N-bis-(2 hydroxyethyl) ethylenediamine diacetic acid and N,N-bis- (Z-hydroxyethyl) ethylenediamine diacetic acid.

A non-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sub-licenses for such purposes, is hereby granted to the Government of the United States of America.

This invention relates to and has among its objects the provision of novel procedures for carrying out separations of substances, particularly those of biological origiw. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified.

In various fields of scientific investigation, substances are often fractionated or isolated by techniques based on differences in density. An elementary method of accomplishing this end involves placing the material in a body of liquid of predetermined density whereby the less dense components will float on the body of liquid, while the denser components will sink. The fractions can then be separately recovered by decanting or pipetting selected portions of the system. A more sophisticated technique involves preparing a body of liquid having a graded density, that is, a density which is low at the top of the body and increases toward the bottom. The material to be fractionated is placed at the top of the liquid and the system subjected to centrifugation to expedite movement of the substances in the body of liquid. After this operation it will be seen that substances of different intrinsic densities are located at different levels in the liquid. These separate deposits can then be isolated from one another by careful decanting, pipetting, or the like.

Another procedure utilizes a fluid of uniform density and is dependent on the rate at which different substances will move toward the bottom of the body of the liquid. Typically, the substance to be fractionated is placed at the top of a tube containing the liquid and centrifugation is applied for a limited time. Thereby a separation may be achieved, based on the principle that the denser particles will move downwardly at a faster rate than will the less dense particles and for particles of equal density the larger will move the faster.

Although fractionating procedures of the type in question are of general application, they are of particular interest in working with cellular materials such as microbial cells, sub-cellular components, spores, viruses, and substances of large molecular weight, e.g., proteins, nucleic acids, and the like.

Density-fractionating techniques require a supporting fluid (usually water) and a solute with a density different from that of the fluid (usually a density greater than 1 is required for the solute). The solute is, of course, required to produce a liquid of predetermined density. As the solute, use is generally made of compounds such as sucrose, chloral hydrate, potassium citrate, potassium tartrate, cesium chloride, cesium sulphate, brominated alcohols, etc. However, for specific applications one or another of these solutes may be ineffective by reason of inadequate solubility, inadequate density, excessive reactivity, excessive viscosity, or other undesirable or unworkable characteristics. For example, sugars such as sucrose have the disadvantage that the concentrated solutions thereof (required to attain the high density liquid) are so viscous that movement of particles is impeded and fractionations are not complete. Less viscous solutions are possible by using salts, such as those listed above. These, however, have the disadvantage that they promote aggregation of bacterial spores and thereby prevent clean separation of spores of different density. Substances such as chloral hydrate and brominated alcohols often damage bacterial spores with which they come in contact so that these substances are not suitable in applications where it is necessary to maintain viability of bacterial spores.

It is therefore a primary object of the invention to provide means for obviating the deficiencies outlined above.

In accordance with the invention, use is made of certain lead chelates as the solute. These lead chelates exhibit many advantages over the substances previously used. In the first place, they are highly soluble in water so that they lend themselves to separations in aqueous systerns, which are obviously preferred. Moreover, these chelates are dense compounds and hence the required solution densities (generally about 1.3 to 1.5) are readily attained. Also, the resulting solutions are not excessively viscous whereby the substances to be isolated can move readily through them. An important property is that the lead chelates do not exert any detrimental effect on spores whereby they are eminently suited for investigations involving spores, bacterial cells, etc. Another noteworthy advantage of the chelates is that they do not exert any appreciable buffering properties in the neutral to weakly acid range so that they can be employed in solutions having a particular pH without changing the pH of the solution.

The compounds in question are lead (divalent) chelates of acids of the structure wherein X is H or CH OH.

They are readily prepared by neutralizing the acids of the given structure with PbO or plumbous salts such as the acetate or basic acetate.

Particularly preferred are the divalent lead chelates of: N,N-bis(Z-hydroxyethyl)ethylenediami .e diacetic acid:

CHg-CHzOH CHr'COOH 3 and, N,N-bis(2-hydroxyethyl)ethylenediamine diacetic acid:

HOCI-Iz-CH: CHzCOOH N-CHr-CHz-N HOCHz-CH: CHzCOOH The lead chelates of the invention are used just like other solutes in density separations. Thus they may be employed in simple float or sink separations, in procedures involving a liquid of graded density, in procedures involving rate of sedimentation, or other techniques which involve separation of components based on density differences. The lead chelates may be used in separation techniques applied to materials of all kinds although they are of special advantage with biological materials such as cellular materials, subcellular components, spores, viruses, proteins, nucleic acids, enzymes, etc.

The invention is further demonstrated by the following illustrative examples:

EXAMPLE I Preparation of lead chelate of N,N-bis-(Z-hydroxyethyl)ethylenediamine diacetic acid N,N-ethylenediamine diacetic acid (2.25 moles) plus sodium hydroxide (4.5 moles) in cold water (about 1 liter) was treated with portions of a solution of ethylene oxide in cold water while keeping the reaction mixture at 45 to 60 C. After a total of 7 moles of ethylene oxide had been added, the reaction mixture was heated at about 80 C. for 4 hours. The sodium ion and nitrogenous ions were absorbed on a column of polystyrene-phenylsulphonate type cation exchange resin (in the hydrogen form) and crude N,N'-bis-(Z-hydroxyethyl)ethylenediamine diacetic acid eluted with 0.2 normal NH OH solution. The eluate was concentrated on a steam bath and treated with an excess of litharge (PbO). Colloidal material was removed by filtration, the filtrate was concentrated, and the lead chelate allowed to crystallize at room temperature. Approximately 50% of the theoretical yield of the lead chelate was obtained after purification by recrystallization.

The lead chelate is believed to have the structure:

I m-CH I GO CH EXAMPLE II Preparation of lead chelate of N,N-bis-(2-hydroxyethyl)ethylenediamine diacetic acid N-monoacetyl ethylenediamine was reacted in water with an excess of ethylene oxide to give N-acetyl-N',N- bis-(Z-hydroxyethyl)ethylenediamine in near quantitative yield. The reaction mixture was hydrolyzed with 20% NaOH for 3 hours at 90 C. The hydrolysate-containing the desired N,N bis-(2-hydroxyethyl) ethylenediamine was used for the carboxymethylation without purification.

Three molar sodium glycolonitrile in aqueous solution was prepared by the slow addition of formalin with cooling to a cold aqueous solution of sodium cyanide. (The mixture was allowed to stand at 4 C. overnight before use.) To the solution of N,N-bis-(2 hydroxyethyl)ethylenediamine (3 to 4 molar), sodium acetate (3 to 4 molar), and sodium hydroxide (1.6 molar), held under vacuum at 60-95 C., was added very slowly a stoichiometric amount of the solution of sodium glycolonitrile. The rate of addition was determined by the rate of release of ammonia. The reaction mixture upon complete release of the calculated amount of ammonia was cherry red. A small amount of nitrilotriacetic acid was formed as a byproduct. To remove this impurity, the reaction mixture was passed through a column of resin containing strongly basic quaternary ammonium groups on a polystyrene lattice (free base form) and led directly into a column of polystyrene phenylsulphonic resin (hydrogen form). The desired compound was cleared from the first column and transferred to the second column by eluting the first column with 2 bed-volumes of 0.5 normal acetic acid, and passing the resulting eluate through the second column. The second column was then washed and the crude N,N-bis(2-hydroxyethyl)ethylenediamine diacetic acid eluted therefrom with 0.5 to 3 normal NH OH. The eluate was concentrated on the steam bath and treated with an excess of litharge. Colloidal material was removed by filtration, filtrate was concentrated, and the lead chelate allowed to crystallize at room temperature. Approximately 30% of the theoretical yield of the lead chelate was obtained after recrystallization. The lead chelate is believed to have the structure:

EXAMPLE III The lead chelates of Examples I and II had the following properties:

Aqueous solutions are colorless but absorb light with a maximum at 241.5 millimicrons and a molar extinction of 0.7 X 10 liters per mole centimeter.

The chelates will release lead ion appreciably only in a strongly acid solution. In alkaline solution a proton is released with pKa of 10.1 (25 C., 0.01 M. solutions titrated with 1 N NaOH).

The chelates have almost no buffering capacity in the pH range 5 to 8. In other words, when either is added to a solution already having a pH in the range 5 to 8, the pH of the solution is not changed.

The approximate densities of saturated aqueous solutions of the chelates are given below, wherein 5" refers to the lead chelate of Example I, U to the lead chelate of Example II.

Density of solutions of chelates (g./cc.)

S (in H10) S (in D 0) U (in H 0) S( and U l Spores of Bacillus coagulans, Bacillus megaterium, Bacillus stearothermophilus, and Bacillus subtilis were prepared as clean suspensions. Small portions of the suspensions were mixed with concentrated aqueous solutions of the lead chclates of Examples l and II and various other solutes used in density fractionation of spores. The following observations were made:

No unfavorable reactions were noted with the lead chelates of Examples I and II.

Aggregation was noted microscopically with spores of B. megaterium and B. subtilis where the solutions contained high concentrations of the following solutes: potassium tartrate, potassium iodide, cesium chloride, lead acetate, lead sulfamate, and the monosodium salt of the lead chelate of ethylenediamine tetracetic acid.

Where chloral hydrate was the solute, there occurred a loss of heat resistance with the spores of B. coagulans.

EXAMPLE V Aqueous solutions of the lead chelate of Example I were disposed in centrifuge tubes to give gradients that ranged from approximately 1.30 at the top to 1.43 at the bottom of each tube. Spores were dispersed in one of the component solutions or as a band on top of the gradient solutions. The tubes were centrifuged as required to drive the spores to their isopycnic positions. The densities of the solution in the neighborhood of the separated bands of spores were determined. The following observations were made:

B. cereus gave a major band at density 1.31 to 1.34 and a minor band at 1.35 to 1.38.

B. coagulans gave a major band at 1.35 to 1.36 and minor bands at 1.34 and at 1.37 to 1.39.

B. megaterium gave a major band at 1.38, a minor band at 1.39 to 1.40, and a major band at 1.41.

B. stearothermophilus gave a major band at 1.33 to 1.36 and a minor band at 1.38 to 1.40.

B. subtilis gave a single band at 1.36 to 1.39.

In all cases the bands contained heat-resistant (i.e., nongerrninated, undamaged) spores that gave the normal bright appearance under the phase microscope (dark contrast). This, of course, indicates that the spores wer not damaged by contact with the lead chelate. The separated bands of spores could be collected readily by pipetting, displacement with overflow, or by pricking the bottom of the centrifuge tube and collecting the effluent in drops.

EXAMPLE VI Spore preparations (of the microbial species listed in Ex. V) which contained heat-resistant (non-germinated, undamaged) spores plus spores which Were heat-sensitive because of partial germination and/or damage were subjected to the density gradient fractionation described in Example V. There resulted an effective separation of the two kinds of spores: The heat-sensitive spores (recognizable by their dark appearance under the phase microscope) had densities of less than 1.30 and remained on top of the liquid whereas the heat-resistant spores were CHrCHrO H) wherein X is H or CH OH.

2. The process of claim 1 wherein each X is H.

3. The process of claim 1 wherein the acid is N,N'-bis- (Z-hydroxyethyl)ethylenedtiamine diacetic acid.

4. The process of claim 1 wherein the acid is N,N-bis- (Z-hydroxyethyl)ethylenediamine diacetic acid.

5. A process for fractionating biological material which comprises depositing the material in an aqueous solution of a solute and applying centrifugation to the system to segregate the components of the material into difierent portions of the solution in accordance with the densities of said components, the solute being a divalent lead chelate of an acid of the structure:

wherein X is H or -CH OH.

6. The process of claim 5 wherein each X is H.

7. The process of claim 5 wherein the acid is.N,N'- bis(2-hydroxyethyl)ethylenediamine diacetic acid.

8. The process of claim 5 wherein the acid is N,N-bis- (2 hydroxyet'hyl)ethylenediamine diacetic acid.

9. The process of claim 5 wherein the solute is a mixture of the divalent lead chelate-s of N,N'-bis-(2-hydroxyet-hyl)ethylenedia-mine diacetic acid and N,N-bis-(2-'hydroxyethyl)ethylenediamine diacetic acid.

10. The process of claim 5 wherein the biological material includes bacterial spores.

References Cited UNITED STATES PATENTS 3,009,861 11/1961 Alderton et a1. 195-400 X ALVIN E. TANENHOLTZ, Primary Examiner.