DD149861A3 - PROCESS FOR SEPARATING OR CLEANING PROTEINS - Google Patents

Abstract

The invention relates to a method for the separation or purification of proteins, preferably haemoproteins. The process can be used in the chemical, pharmaceutical and biochemical industries as well as in clinical chemistry, analytics and diagnostics. The object of the invention is to find a simple, preparative and analytical method for the separation or purification of proteins. It is the object of the invention to develop a method based on affinity chromatography. The method is characterized in that a proteinaceous solution is contacted with imidazole or an imidazole derivative bound to a water-insoluble macromolecular carrier and the proteins present in the solution or carrier are isolated, optionally after removal of the carrier. Carriers and protein-containing solution are contacted at pH 7 to 11, optionally in the presence of detergents and / or glycerol, at temperatures of 0 to 30 degrees by the batch process or by the column method. Good separation and cleaning effects are achieved; Examples are the separation hemoglobin / myoglobin and cytochrome P450 / cytochrome P420.

Description

"Method for separating or combining hemoproteins"

Field of application of the invention

The invention relates to a method for the separation or »purification of hemoproteins. The method is B 'can be used for the separation of hemoglobin from a biological material as well as clinical chemistry, the analysis and the diagnosis ₀ in the chemical, pharmaceutical, and biochemical industries, for _e

Characteristics of the known technical solutions For the separation or "purification of proteins, various methods are used which exploit the differences in the physical and biochemical properties of the proteins to be separated, such as solubility, stability, molecular size, electrical charge, etc." / No REHFELD, D. REICHELT: Analytical and Preparative Methods of Clinical Biochemistry, Akad. Verlag Berlin 1972, p. ₆ SCHWIMIER and A. B, PARDEE: AdVe in Enzymology 1J; »375./

For example, the removal of hemoglobin from blood plasma may be carried out by extraction with chloroform (Czech Pat. 88563/1959 /) or by precipitating with Zn salts (Vox sango 2 ^ ₉ 481/1972 /). The use of cellulose ion exchangers (US Pat. z "B _e DEAE-, CM-cellulose in Vox Sang" Hi 59/1967 /) and polysaccharide derivatives (eg DEAE-, CM-Sephadex in Vox Sang ", 2J3, 18/1972 /) described" The methods are Z ₀ T »very time and material consuming, and a sufficient purity is often achieved only with high yield losses ,.

19 4299

Purely the isolation and purification of proteins are gaining increasing importance in the recent past affinity chromatography methods, which are based on the specific protein complex formation of proteins, »

Thus, Zo B. is the separation of hemoglobin (Hb) based on the complex formation with haptoglobin AU KLEHT and C, MIHAESGO: Biochenu biophys »Res. Commun. ££, 774 (1973) / However, in this method only the Hb can be removed which has not been bound by haptoglobin in the serum by complex formation.

The aim of the invention is _s a simple and generally applicable method for the separation or purification of proteins, preferably to find hemoproteins. It should be analytically and preparatively usable and achieve good yields. «

Explanation of the essence of the invention

The invention is based on the object ^ the specific bioaffinity complex formation of proteins to imidazo! is or _e exploit its derivatives, and to develop a suitable separation methods based on affinity chromatography "According to the invention hemoproteins are separated or" purified by a hämoproteinhaltige solvent with imidazole or an imidazole derivative _t that is bound to a v / asserunlöslichen macromolecular carrier is brought into contact , which are then isolated in the solution remaining or on the carrier hemoproteins and thereafter if necessary. regeneration of the carrier takes place «. The water-insoluble macromolecular carriers are biopolymers, preferably polysaccharides or synthetic macromolecules, preferably copolymers based on the methacrylic acid esters. The imidazole or its derivative is bound to the carrier directly or with the intermediary of spacers. "Attachment sites are the ring nitrogen in the 1-position or

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194299

Ring carbon in the 2- or 4 (5) position of the imidazole ring. The feasibility of the method of the invention is surprising because the Iraidazol or "its derivatives are no physiological complex partner for Hemoproteins ₀ The bekannten'Stabilitätskonstanten from the literature," as the hemoglobin Imidazöl complex, located at the internal border, which for an affinity chromatography is required. The formation and stability of these complexes thus largely depends on the nature of the protein pocket surrounding the iron protoporphyrin IX

It was important that the support fixation of the imidazole or its derivatives and the unspecific (hydrophobic) interactions of the support material with the hemoprotein have a positive influence on the complex formation and selectivity »

The preparation of these imidazole-containing carrier (IMD carrier) is carried out according to known methods, to implement the invention, the IMD carrier proposed in WP 124 405 (M. Benes, M _e Kühn, K. Pommerening) are generally applicable. The hämoproteinhaltige solution at pH 7 - 10 optionally in the presence of detergents and / or glycerol, at temperatures of 4 - 3O ⁰ C brought into contact with the carrier in the case of the separation of hemoglobin the contact transfer can (even at a slightly higher temperature to 40 ⁰ C). There are 2 options for this:;

a) The carrier is introduced into the hemoprotein-containing solution and after the time necessary for the adsorption, separated again by filtration for about 1 to 24 hours (one-pot or batch process) »

b) the support is located in a column to which the proteinaceous solution is applied (column method),

The batch method is particularly advantageously employed for cleaning operations, i.e., ₀ h _e in the separation of impurities ", the column method is particularly advantageously used for separation operations, if the proteins to different extents

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but not too tightly tied to the carrier. The protein-containing solution is applied at a pH of 7 to 10, optionally in the presence of detergents and / or glycerol, at temperatures of 4 - 30 ⁰ C, preferably at room temperature, applied to the column. The elution is then carried out, the haemoproteins appearing successively in the elution liquid due to the different degrees of binding to the support. The appropriate fractions are collected and the isolation of the pure hemoprotein contained is carried out in a manner known per se.

The regeneration is preferably carried out by treatment with urea and a mixture of pyridine / LTatronlaugej optionally with the addition of dithionite or by changing the pH of the elution buffer. The inventive method is easy to carry out and has the Advantage of general applicability «The hemoproteins to be separated or cleaned * are obtained in good yields and in high purity. For example, from a hemoglobin AIyoglobin mixture using the column method, the myoglobin was isolated chromatographically pure in over 90 % yield. »From a cytochrome P-450 / cytochrome P-420 mixture was prepared using the batch method cytochrome P-420 to 85% of baseline remote Advantageously, the method can also for the separation of hemoglobin from biological materials (e.g. _{o 0} B hemolytic sera) apply. Depending on the process conditions, 40-100 % of the available hemoglobin is removed.

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The invention will be illustrated by a few examples which, however, do not limit the invention.

EXAMPLES OF EXAMPLES.

Example ₁₁ 1 ₁

1 g or 2 g of the macroporous IMD support with Structureinheit

of general formula I (see attachment)

(n = 1j2), prepared according to YvT 124 405, are swollen in ethanol with water and 0.1 M phosphate buffer, pH 7

and filled in a column »

1 ml of an O _? 53 mVal / 1 Met Hb (human) solution, pH-7, is added to the column and then eluted with the phosphate buffer. Concentration determination in the eluate gives a

Binding of Hb to the column of 64-74 % o

(MetHb, = methaemoglobin, the Fe is in the 3-valent valence state, the concentration data always refer to the Fe (1 mol = 4 Val / 1),

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The Hb concentration is determined via the CN complex with £ ' ^nm = 11 (relative to Pe). The binding of Hb and

of the other proteins to the support may also be determined from the difference in absorbance of the starting solution and eluate at 279 nm and / or the Soret band. Within the error limit, the individual methods yield matching values. In the batch process (5 ml 0.1 "mVal / 1 MetHb solution, 2 hours incubation at room temperature), up to 72% of the MetHb available is bound,

Example 2

300 mg or 750 mg I (η = 1.2) are pretreated analogously to Example 1 and washed with a buffer pH 9 (0.1 M NaHCO ₃ -, 0.5 M NaCl). 5 ml of 0.1 rnVal / l MetHb (human) solution in the same buffer are pumped at room temperature over the column for 3.5 hours. Hereby 77-80% of the Hb are bound.

Example 3 ,

2 g NHg group-containing agarose (AH Sepharose) are suspended after swelling and washing with a 0.5 M NaCl solution in 16 ml of NaClJ solution and 3.2 ml of a 25% glutaraldehyde solution was added. The reaction mixture is shaken for 3 hours at room temperature. After washing with the 0.5 M NaCl solution and buffering to pH. 9 (0.1 M NaHCO ₃₅ 0.5 M NaCl), the support is shaken with 16 ml of a 0.05 M histamine solution in the buffer pH 9 for 3 hours at room temperature.

-R Π Γ 7 IQ 7 7 · K Ί K \ O / '"

194 299

After washing and buffering to pH 8, the excess aldehyde groups are blocked by reaction with 16 ml of a 1 M ethanolamine solution, pH 8.

5 ml of a 0.43 meq / l MetHb 'solution in the buffer, pH S _1, are shaken for 2 hours at room temperature with the IMD support. After separation of the support, 100% of the Hb offered are adsorptively bound to the support,

Example 4

To 1 g or 2 g analogously to Example 1 or container Item I (n = 2) are added 2.5 ml of a solution which in the buffer pH 9

(HemcM (0.1 M NaHGO ₃ , 0.5 M NaCl) 0.04 mVal / 1 MetHt / and 0.01 mlm

Human serum albumin (HSA). With constant mixing (stirring, shaking, rotating), the solution is brought into contact with the IMD support for 2 hours at room temperature. After separation of the support, 80-90% of the MetHb and only 9-11% of the HSA are bound.

The determination of the concentration of HSA is carried out with an extinction coefficient ε · α = 7.15, the molecular weight is assumed to be 66500. The binding of the HSA to the support is determined from the UV absorption at 279 nm, taking into account the absorption of Hb »

Example 5

The macroporous IMD support of the general formula I (n = 1, 300 mg, η = 2.54 mg, η = 3, 550 mg) is dissolved in a column with 0.1 M phosphate buffer, pH 7.3, 0, 3% sodium dodecyl sulfate, washed. 1 ml of a mixture of 1 part of 0.53 meq / l and 1 part MetHb'- 0.12 mM HSA solution is added to the column and containing the Na-buffer eluted dodezylsulfat _e About 90% of the MetHb be the IMD Bearer bound »

--6.0lZ.137 7 M) 7!) I) V ;;

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Example 6

Whole serum (human) is made hemolytic by adding MetHb (0.4-2.9 meq / 1 MetHb) and containing 0.1 M NaHCO ₃ buffer, pH 9-9.5, containing 0.5 M KaCl. diluted (1:10 - 1:11). 2.5 ml of this hemolytic serum are brought into contact with 500 mg of the I (n = 1.2) pretreated analogously to Example 1, either for 24 hours at 4 ° C. or for 2 hours at room temperature, with constant mixing. After separation of the IMD support, 39-59% of the MetHb are bound »The serum is immediately dialyzed against 0.1 M phosphate buffer, pH 7, and concentrated by ultrafiltration *

Example .7.

Whole serum (human) is hemolyzed by addition of MetHb (0.4-3 meq / 1 MetHb) and diluted with 0.1 M phosphate buffer, pH 7.3, containing 0.3% Na dodecyl sulfate (1:10 -1: 11).

2 ^ 5 ml of this serum are contacted with 500 mg of I (n = 1.2) for 2 hours at room temperature with constant mixing. After separation of the IMD support, 90-100% of the MetHb are bound. The serum is immediately dialyzed and concentrated as in Example 6

Example 8

2 g of macroporous poly-glycol methacrylate (Spheron 1000) are activated by known methods with 400 mg of BrCN. The activated carrier is reacted at pH 9 $ 5 with, 1 mol of histamine. The deactivation of the unreacted groups of the carrier is carried out with 5 ml of a 1 M ethanolamine solution, pH 8 "hemolytic whole serum (0.5 meq / 1 MetHb) is diluted analogously to Example 7." 5 ml of the serum are analogous to Example 7 with the IMD Carrier brought into contact. 65-78% of the MetHb are bound to the carrier ©

"ι 7 -v Ii 7 <) ^l ) V ^#x

ϊ 9 4 2 9 9-

Example 9

3.0 g of IMD support having the structural unit of general formula I (n = 1.2) are equilibrated in a column at room temperature with 0.1 M buffer for 8 ml of a mixture of hemoglobin (bovine) / myoglobin (Bovine) (hemoprotein concentration 0.4-0.5 mVal / l) is added to the column and then eluted with the buffer pH 8 via a fraction collector. The gel chromatographic examination of the protein-containing fractions shows that when using the IMD support (n = 2) and myogloebin is eluted, and in the case of η = 1 the myo-. globin is highly enriched compared to hemoglobin »

Example 10

10-11 ml of swollen IMD support with the structural unit of the general formula II (see Appendix) are equilibrated in a column at room temperature or 4 ⁰ C with 0.1 M buffer pH 8. 1 ml of a mixture of hemoglobin / myoglobin (bovine) (concentration per hemoprotein) is added to the column and 0.25 mVal / l is then eluted with the pH 8 buffer via a fraction collector.

In the eluate over 90% of myoglobin are recovered in gelchromatographisch pure form. The hemoglobin is either completely adsorbed on the support (room temperature) or partially eluted only at very high volumes.

Example 11

100 mg or 1 ml of swollen IMD carrier of the general formula I and II are equilibrated with 0.1 M phosphate buffer pH 7.25, 20% glycerol, 1 (2) mM EDTA. 1 ml microsomal cytochrome P-1. 450 from rabbit liver (conc. 80-150 / UM) with 13-36% cytochrome P-420 in the buffer of the composition:

~ 5.utZ.1377 *

- · 10 -

1 S A 2 9 9

'.0.1 M phosphate, pH 7 ₉ 25, 1 mM dithioerythritol, maximum 6% ^J Ua cholate, optionally 20% glycerol is added and the sample for 18 hours at 4 ⁰ C or 2 hours shaking at room temperature. In the eluate, the content of cytochrome P-420 is reduced to 87% of the initial value.

Example 12

11 ml (2 g) of swollen IMD-carrier of the formula I (η = 1) are equilibrated in a column at room temperature with pH 9 buffer (0.1 M borate, 0.5 M WaCl). 1 ml of a mixture of catalase (bovine) / methemoglobin (bovine) with a concentration of 34 / UM or, 50 M Val / 1 is added to the column and then eluted with the buffer through a fraction collector.

After concentration of the protein-containing fractions, more than 70% of pure catalase could be recovered by gel chromatography.

Regeneration of the IMD Carrier Example 13

The regeneration of the hemoglobin-loaded IMD carrier is carried out by incubation for several hours with 6 M urea, washing with water and a mixture of pyridine and 0.1 M NaOH (1: 4), optionally with the addition of dithionite. After more than 10-fold regeneration, no visible loss of Hb-binding capacity is observed. "In the same way, a general regeneration of the protein-loaded carriers can take place

When washing the loaded IMD carrier with a buffer, Hb is also partially eluted by the IMD carrier _o

, the carrier took place

2, method according to item 1, characterized in that working in the presence of detergents «

3 # Method according to item 1 and 2, characterized in that the elution at a pH of 7 - 10 in the presence of detergents and optionally glycerol at temperatures of 4 - 3O ⁰ C.

4 «method according to item 1, characterized in that the regeneration of the imidazole-containing carrier by cleavage of the hemoprotein by treatment with urea and a mixture PyridinATatronlauge, optionally with the addition of dithionite or by changing the pH of the EIutionspuffers.

For this purpose, page formulas

Formula I n = 1-3

Tr-O-σ-ΗΗ- (CH ₂ ) _n -N = CH- (CH ₂ ) _m -CH = lT-CH ₂ --CH ₂

Formula II: Tr = polyglycol methacrylate (Spheron),

Polysaccharide (agarose) n. = 2 - 10,. CD

m = 3. 4N

fO CO CD

Claims (1)

194299 - ^ - 1, process for the separation or purification of hemoproteins, characterized in that a haemoproteine-containing solution with the carrier-fixed imidazole or imidazole derivative of the formulas I or II at a pH of 7-10 at temperatures of 4 - 3O ⁰ G in the column or One-pot process (batch process) is brought into contact, then the remaining in the solution or on the carrier hemoproteins are isolated and then optionally _o regeneration