IE49166B1 - Process for separating off iron compounds originating from haemoglobin from globin in a liquid containing blood substances - Google Patents

Abstract

There is provided an improved method, suitable for commercial use, for separating from a liquid containing blood substances the iron protoporphyrin and dirivatives or decomposition products thereof, the so-called iron component, from globin, characterized in, that the liquid is cooled, preferably to a temperature of from 0.degree. to 20.degree.C, that the pH of the liquid is adjusted to a value lower then 4.5, preferably to a value of from 2,5 to 4.5, that an organic solvent with dehydrating properties is added to said liquid, for example ethanol or mixtures of ethanol with glycol, in an amount liquid, preferably at least 75 per cent of volume, and thereby maintaining the above indicated temperature, that the precipitated iron component is separated from said liquid, preferably by addition of salts, such as potassium chloride or sodium citrate, whereafter the globin is precipitated from the liquid.

Description

This invention relates to a process for separating iron protoporphyrin and its derivatives or cleavage products from globin in a liquid containing blood substances.

Attempts have already been made to split haemoglobin 5 into iron protoporphyrin (haemin) and globin and to separate these in order to obtain a protein (globin) without a blood taste (which originates from the haemin).

Attempts to separate haemin from globin by electrophoresis, iron exchange chromatography, gel filtration, membrane filtration and similar processes have proved to be too expensive on an industrial scale. The extraction of haemin in solvents, for example acetone, methyl ethyl ketone or dimethylformamide, have proved to be inadequate because small amounts of these toxic solvents remain in the globin and this would then be unsuitable as a foodstuff.

The process according to the invention is thus based on the object of discovering a simple process which can be carried out on an industrial scale for separating the two components haemin and globin.

Accordingly the invention provides a process for separating iron protoporphyrin and its derivatives or cleavage products from globin in a liquid containing blood substances, which process comprises, cooling the liquid to a temperature in the range 0 to -20°C, adjusting the pH of the liquid to a value not exceeding 4.5, adding to the liquid ethanol or a mixture of ethanol with a glycol or glycerol in a volume corresponding to at least 40% of the total liquid volume after said addition, maintaining or reestablishing a liquid temperature within the above range, causing the main part of the iron component to precipitate and removing it from the mixture, and subsequently causing the globin to precipitate. 48166 The application of a simple process technique is made possible by the process according to the invention, using ethanol or mixtures of ethanol with glycol or glycerol and, if necessary, adding salts, such as sodium chloride, sodium sulphate or ammonium sulphate and sodium benzoate, if necessary with the addition of a pHregulating agent, such as inorganic acids (for example hydrochloric acid or sulphuric acid) or organic acids (for example acetic acid or citric acid). The invention thereby provides a method, which can be carried out industrially and economically, for separating iron components and globin and enables globin to be used for feeds and foodstuffs.

It has furthermore been found that the haemin in the paste precipitated from the solution is in the natural (native) form and is therefore absorbed by the body. It is thus also an attractive medicament for iron deficiency anaemia because it is an endogenous product without having the disadvantages which are known to be associated with the conventional iron products on a nonhaemin basis.

In the method according to the invention, most of the iron component, which is present, for example, as an aggregate in concentrated solutions or adsorbed onto protein material, and of which the agglomeration can be promoted by addition of, for example, sodium chloride, - 4 is first separated off, for example, by centrifugation. The globin is then precipitated out of the supernatant liquor obtained by centrifugation in this manner, whilst the remainder of the iron component is retained in the liquid phase in dissolved and/or finely divided form.

The medium which, due to the relatively high content of solvent according to the invention and the comparatively low content of water, has a certain ionic strength and a low pH value, is evidently suitable for retaining the finely dispersed or dissolved forms of the iron component in the liquid, whilst the globin can be precipitated and centrifuged off.

The liquid to be processed is cooled to a temperature of 0°C to -20°C. If the intention is to carry out the processing at, for example, 0°C, the blood liquid and the organic solvent as well as all the other additives are cooled to 0°C, if possible before they are mi xed.

The pH value of the liquid is brought to a value below 4.5, in particular to a value of 2.5 to 4.5.

Ethanol or mixtures of ethanol with glycol or glycerol are added in an amount of at least 40X by volume, based on the total amount of liquid, the amount preferably being at least 75% by volume.

The precipitated iron component is separated off from the liquid, preferably by salting out, salts such as potassium chloride or sodium citrate being added in particular. The globin can then be precipitated from the liquid. - 5 The invention will be illustrated in more detail below with the aid of some embodiment examples.

Example 1 g of haemoglobin solution (with a solids content of 15% and a content of about 33% of ethanol, and with a temperature of -8°C) were added dropwise to a solution of 20 ml of 94% strength ethanol and 1.1 ml of 1 M hydrochloric acid with a temperature of -15°C, while stirring and cooling. After the addition, the temperature was -15°C and the pH value was 2.9.

The mixture uas centrifuged at 27,000 g for 20 minutes, 0.9 g of a black paste (iron component) with a solids content of 8.3% being obtained. ml of 94% strength ethanol were added to the intensely brown-coloured supernatant liquor at a temperature of -15°C, while stirring and cooling, and the globin was precipitated by adding 10 ml of water and 1.2 ml of 20% strength ammonium sulphate.

The precipitate was centrifuged off at 8,000 g in the course of 10 minutes, the paste obtained was suspended in 94% strength ethanol with a temperature of -15°C and the suspension was centrifuged as above. 1.6 g of paste with a solids content of 37.6% resulted. In the dry state, the paste was light grey.

Example 2 g of haemoglobin solution (data as in Example 1) were added dropwise to a solution of 40 ml of 94% strength ethanol and 1.1 ml of 1 H hydrochloric acid with a temperature of -15°C, while stirring and cooling. 916 6 - 6 After the addition, the temperature was -15°C and the pH value uas 2.9. ml of 20% strength sodium chloride were added dropuise to this mixture and the liquid was centrifuged at 27,000 g for 20 minutes, 1.4 g of a black paste (iron component) uith a solids content of 11% being obtained. ml of uater and 1 ml of 20% strength ammonium sulphate solution uere added to the supernatant liquor and the liquid uas then centrifuged at 8,000 g for 10 minutes. 3.8 g of a grey paste of globin uith a solids content of 14.6% Here obtained.

Example 3 The haemoglobin uas precipitated from 5 g of haemoglobin solution (uith the data as in Example 1) by addition of 10 ml of 94% strength ethanol, while stirring and cooling doun to a temperature of -6°C. The precipitate uas centrifuged at 2,000 g for 5 minutes, 6 g of a red paste being obtained. A relatively small amount of black paste had settled on the bottom of the centrifuge tube.

A solution of 70 ml of 94% strength ethanol and 1.1 ml of 1 M hydrochloric acid with a temperature of -12°C uas added to the red paste, while stirring and cooling. After this addition, the pH value was 2.9 and the temperature uas -12°C. The mixture uas centrifuged at 27,000 g for 10 minutes, a relatively small amount of black paste (iron component) being obtained. 2.0 ml of a 40% strength ammonium sulphate solution and 10 ml of water were added to the supernatant liquor. The temperature uas -8°C and the pH value uas 3.4. - 7 The grey precipitate thus obtained was centrifuged at 8,000 g for 10 minutes, 1.5 g of a grey paste (globin) with a solids content of 39.32 being obtained.

Example 4 g of haemoglobin solution (with the data as in Example 1) were added dropwise to a solution of 20 ml of 942 strength ethanol and 1.1 ml of 1 H hydrochloric acid with a temperature of -12°C, with stirring and cooling. After the addition, the pH value was 2.9 and the tempera10 ture was -12°C. This black-brown mixture was centrifuged at 27,000 g for 10 minutes, 2.2 g of a black paste (iron component) uith a solids content of 2.72 and a light brown supernatant liquor, which later assumed a gelatinous consistency, being obtained. 70 ml of 942 strength cold ethanol followed by 0.8 ml of 402 strength ammonium sulphate solution and 8 ml of water were added dropwise to the supernatant liquor, while stirring and cooling. Thereafter, the temperature was -6°C.

The precipitate thus obtained uas centrifuged at 8,000 g for 10 minutes, 1.7 g of a white paste (globin) with 362 of solids resulting.

Claims (9)

CLAIMS:

1. A process for separating iron protoporphyrin and its derivatives or cleavage products from globin in a liquid containing blood substances, which process comprises: cooling the liquid to a temperature in the range 0 to -20°C; adjusting the pH of the liquid to a value not exceeding 4.5; adding to the liquid ethanol or a mixture of ethanol with a glycol or glycerol in a volume corresponding to at least 40% of the total liquid volume after said addition; maintaining or reestablishing a liquid temperature within the above range; causing the main part of the iron component to precipitate, and removing it from the mixture; and subsequently causing the globin to precipitate.

2. A process as claimed in Claim 1, wherein the pH is adjusted to a value within the range 2.5 to 4.5.

3. A process as claimed in Claim 1 or 2, wherein the ethanol or mixture of ethanol with a glycol or glycerol is added in a volume corresponding to at least 75% of the total liquid volume after addition.

4. A process as claimed in any of Claims 1 to 3, wherein the iron component is caused to precipitate by adding a salt or salts to the liquid.

5. A process as claimed in Claim 4, wherein the salt is selected from potassium chloride and sodium citrate.

6. A process as claimed in any of Claims 1 to 5, wherein the globin is caused to precipitate by adding aqueous 5 ammonium sulphate solution.

7. A process according to Claim 1 for separating iron protoporphyrin and its derivatives or cleavage products from globin in a liquid containing blood substances, substantially as hereinbefore described with particular 10 reference to the accompanying Examples.

8. Iron protoporphyrin or a derivative or cleavage product thereof, whenever prepared by a process claimed in a preceding claim.

9. Globin whenever prepared by a process claimed in a 15 preceding claim.