DE1517742C3 - Process for obtaining uriease from animal tissues, in particular liver, kidney or spleen - Google Patents

Description

the urease at a salt content between 0.1 and iu scher and Baum, J. Biol, Chcm. 216, 1055, p. 625, 0 5 M and a pH value /. Between 6 and 8 to a highly active uriease with 10 U / mg prolein adsorbed from kieselguhr, which separates kieselguhr, from acetoiurock powder from pig's liver mitochondisem the uriease with a aqueous, 20 to three isolated. The process is technically not ver-35 ⁿ / o acetone-containing solution of pH 8.0 wwrlbar, since the production of the Milochondricn eluicrt very much up to 9.8, in the eluate the urease is expensive and because of the necessary centnifuhung the acetone content to about 60 ". 'o, if more than 6000 g, the precipitation can only be carried out on a small scale with alkali carbonic solution. In addition, the AuspH values between 9 and 10.5 again dissolve, from prey of 21% > too low.

this solution, the uriease with ammonium sulfate. Furthermore, Robbins Barnett and Grant in

in a concentration of at least 1.6 M, J. Biol. Chcm, 216, p. 27 (1955), a method and the precipitated Uriease is often described with pure, which is a fractionated fourfold Ex-water or weakly alkaline salt solutions traction of the impurities from the liver with water, washes. Phosphorus, Sodium Chloride and BoronAlpuiver so-

2. The method according to claim 1, characterized ge as subsequent extraction of the urease with strong indicates that during the adsorption of the 25 Giyein carbonate buffer from fresh pork liver uriease on kieselguhr with a salt content between includes. These steps are already going 5O ⁰ Zo of see 0.1 and 0.5 M and a pH Far from enzyme lost more Rcinigungsschritie are 6.7 to 7.3 arbeitel. pH precipitation, alcohol precipitation and alcohol fraction-

3. The method of claim 1 or 2 thereby wrestling, renewed pH precipitation and salt extraction. Man characterized in that the alkali carbonate solution 30 thus receives an end product with an activity of only has a pH of 10.2. about 1 U.'mg. This method also has the disadvantage.

4. The method according to claim 1, 2 or 3, that one with a transfer in the technical characterized by the fact that large centrifuges are needed as a weak alka scale, the Aufarbeilische Salzlösurg for washing the ammonium tnnii, takes a long time and is not in any size Silfate precipitation allows a 0.01 to 0.3 M, preferably 35 batches to be carried out. Besides, they are pure ones 0.05 M potassium carbonate solution used. hch of the enzyme (I υ'ΐπμ) and the yield (35%)

5. Expires according to claim 1, 2, 3 or 4, there- unsatisfactory.

characterized by that one felled and finally was also made by Leone in Colowick—

as often as desired with pure water or weakly Kaplan: Methods of Enzymology. Vol. II. 1953, Uricase washed with alkaline salt solutions in 40 S 485. A process is described which dissolves with 2 ⁽ >"/ ₀ dilute alkaline salt solution, treats this yield / u of a product with a specific solution with calcium phosphate gel, the CaI - Activity of 0.07 µg leads. Yield and pure calcium phosphate chloride removed, so again very unsatisfactory from the calcium phosphate of the fn / vms. Phate gel supernatant the uricase with ammonium sulfate

fat falls and finally the uricase as often as required 45 by pure laboratory vc. drive that to the technical Hermit Pure or slightly salty water position are not suitable, which for example also

it follows that the previously commercially available uricase preparations specific activities between 0.0045 and 0.8 U'rng (cf. H. R. Mahler in 50 Hoppe-SeylcrTliicrfelder. Vol. VI, A, Enzymes, 'IdI A, 1965. S. «04).

There was therefore a considerable need for a simple technical process for pest Manufacture of Uricase. which does not have the above-mentioned disadvantages of the previously known methods.

According to the invention, this object is achieved by a method for isolating uricase from animal tissues, especially liver, kidney or

washes.

The invention relates to a method for obtaining l'riciisc from animal tissues, in particular Liver, kidney or spleen, the uricase being made up

the water-insoluble fraction of the Gcwcbehomo- 60 mil /, being the uricase from the water-insoluble

μ-iial is irradiated with alkaline saline solution

('cheese is used for the enzymatic ^ η determ-jnii of Urine iiire in serum and urine natli! P · .norm ·· and H.Paiilsen. J. C'lin I., ib Invesi 3

f-raklion des Gcbehoinngcnals is extracted with alkaline salt, which is characterized by this. that in this extract the uricase is adsorbed at a salt content between 0.1 uikI 0.5 M and an S 271. For this a preparation is required 6 ₅ pH value between 6 and 8 adsorbed on kieselguhr,

which at 2 ')' nm a miiglidisi low Lijien absorption IumI / i In the lel / i η years the after asks for a pure uriease. the this

that separates kieselguhr, from this the uricase is eiuicrt with an aqueous ^{solution VDi) pH 8.0 to 9.8 containing 20 to 3 ^ 1} vol of acetone, the uricase in the eluate

el 11 rest increase de> Aceloniiehnlis to about Λΐ) "υ fails, the precipitate with alkali carbon solution triggers again at pi values between 9 and 10.5 of this solution the Uncase mil Aminoniiimsvlfnl in a concentration of at least 1.6 M and fill the precipitated uricase with pure water as often as desired or weakly alkaline salt solutions.

The uricase obtained in this way, which already has a specific activity above 1 U mg, is expedient. dissolved in dilute alkaline saline solution and treated with calcium phosphate gel at andull. where the absorption at 293 πΐμ is further reduced. The calcium phosphate oversiand is then again with Animoniumuulfiu like.

An essential step in the process according to the invention is the adsorption of the Uncase on kieselguhr (/., B. Celite). This binding to kieselguhr takes place when the salt content of the solution is between 0.1 and 0.5 M. If the salt concentration is lower, too much frenid protein is adsorbed; if the salt concentration is higher, the uricase remains partially in the supernatant. The adsorption takes place preferably at pH 6.7 to 7.3.

The uricase is eluted from kieselguhr with alkaline acetone solution from pH 8.0 to 9.8 and an acelon concentration between 20 and 35%. At higher values, the uricase is already precipitated by acetone, and below 20 ° ο it becomes foreign protein initeluted in increasing amounts. The cheapest acetone hook depends partly on the one used pH value, which in turn is related to animal temperature, in such a way, that at lower temperatures it is only possible to work at higher pH values Room temperature. The upper limit at room temperature A temperature of around 20 C is at pH 9.4, while at 0 C it is around 9.8. At a pH below 8.0, the enzyme is no longer completely desorbed.

The addition of KieselgMi to clarify Solutions with improvements in Filtrationsge selmindigkeit is known. F.s was already the Adsorption of amylase from the filtrate of an Aspcrgillus Culture on diatomaceous earth described. The selective absorption of an enzyme from a saline Gcwebchomogenat and subsequent elution with Acetone, on the other hand, is a new process. This process step is based on the surprising discovery the fact that the uricase dissolved with a large amount of foreign projectin is released after neutralization highly salty tissue homogeneity, in particular liver homogenate, selectively adsorbed on kieselguhr will. This enables the filterability of such a homogenate for the first time and at the same time a high purification of the uricase adsorbed on diatomite is achieved. It was also surprising that the Uricase is desorbed with acetone solution in the specified alkaline range, whereby only a rapid elution becomes feasible on an industrial scale.

Another step of the invention consists in washing the ammonium sulfal-precipitated arkase with pure water or water with a low content of salt, preferably with the 0.05 M KHCO ₁ solution with a pH of −8.5. The eluted with Acelonlösung enzyme is first freed from the acetone by precipitating it by the addition of further acetone to about 60 "Ό acetone content and solution with dilute Alkalirarbonatl '/.wischen at pH values ⁱ 9 and 10.5, voi / .uysweise but pH 10.2, is brought back into solution before the ammonium sulphate treatment is carried out. The ionization is not essential and is the simplest method of removing the acelunum.

For the amniotic fluid to be filled, the uricase must reach 1.6 M for the uricase to be completely filled. Higher ammonium sulfide concentrations are possible, but do not provide any advantage. The precipitate obtained in the ammonium sulfate precipitation can be washed with water or dilute alkaline solutions without the uricase going back into solution. This washing, which can be carried out repeatedly, allows the separation of most of the foreign protein that is still present. For the extraction '<' is preferably a Kaliumbicarbonailösung used, with concentrations can be used up to 0.3 M, go without me ^r Kliche Uricasemengen in solution. The extraction is preferably carried out _{with 0.05 M KHCO; i solution.}

The precipitation with alonium sulphate and the subsequent extraction of foreign matter can be carried out repeatedly and is preferably carried out twice, a treatment with calcium phosphate gel being included between the first and second ammonium sulphate precipitation. By the calcium phosphate Schrn. colored impurities are removed, which increase the blank value in the optical determination of e ^r .

The salting out of proteins for isolation by means of ammonium sulfate is a well-known and frequent one applied process step. Also the washing out of such precipitates of protein, especially crystalline ones Art. With weaker salt concentrations than are necessary to precipitate the protein is described. The washing of such an amorphous pro-urine precipitate with water or weakly alkaline salt solutions to remove large quantities However, foreign protein has never been performed before.

The water-insoluble fraction of the Gewcbehornogenats is extracted as an alkaline salt solution voizugs ^ ice glyrin-Na, Co., - buffer pH 10.2 use

det. This buffer was also used by Robbins et al. a. a. O. used to extract uricase, but there with a borate precipitate.

The method according to the invention enables the simple and technical isolation of uricase from Gc'v.ebexttrakte with a yield of about 50% of the initial activity and with a high purity of about 7 U'mg. From 30 kg Schweinclcbcr \ vc ^r dcn invention about 1.24 g uricase get this purity. In the aforementioned Mahler et al. loc. cit., on the other hand, 2 mg of L'ricase are isolated from 100 g of dry acetone powder obtained from 2 kg of fresh liver. For a batch of 30 kg, this corresponds to 0.03 g of enzyme. The one in Biochim. Biophysica Acta 23, 1957, pp. 43 to 53, described, likewise starting from liver mitochondria, the yield of uricase with a final purity of 6.95 U / mg, converted to 30 kg of liver, is 140 mg, ie little more than 10 ⁰ zo the Yield obtained in accordance with the invention In the aforementioned Robbins process, an enzyme with a final purity of 1 U / mg is obtained in a yield of 35%. This bckanntt; Process does not require the extraction of

517 74 * 2

Mitochondrion, but on the other hand includes a four-fold Centrifugation of the alkaline tissue sev- (rakles at 4000 b. This would be done with a 30 kg batch mil the largest available for these values Centrifuge alone require a centrifugation time of 50 hours.

The following example is intended to be a preferred implementation of the method according to the invention.

example

30 kg of pork liver are homogenized with three times the volume of deionized water, made up to 200 l with water and the pH is adjusted to 5-3 with 2N acetic acid. Then 10 kg of kieselguhr are added. Stirred for 10 minutes and filtered through a filter press. The filter cake, washed with water, is homogenized with 1.5 times the volume of 0.27 M NaXO ₁₁ , containing 0.1 M glycine, pH 10.2, and the resulting volume with solid ammonium Nsulfate brought to 0.4M. The pH of the halogenate is adjusted to pH 7.0 with 2N acetic acid and the volume is made up to 150 l with water. The mixture is stirred at room temperature overnight, and the dissolved uricase is selectively adsorbed on kieselguhr. Filtration and washing with water follow. The filter cake is extracted in the cold with 75 l of a solution of 0.2 M NaCl and 33% acetone, pH 9.2.

solution luidigewaschcn. The Fil, iral is filled with acetone to f) 0 "V ^ V acetone and filtered again. The precipitate is _{extracted with 0.2 M Na 11} CO., PH 10.2 and filtered to remove insoluble constituents. The fillrate ( about 8 l) is added to 1.6 M ammonium sulfate

ίο saturated and the resulting precipitate washed twice with 1.5 I 0.05 M KHCO. The precipitate is taken up in 0.2 M Na ^ CO ^ solution pH 10.2 to 300 ml, in the ratio 1: 4 dilution with demineralized R ₁ O and the purpose of adsorption or colored substances with 300 ml of calcium phosphate gel (about 20 mg of dry matter / ml) . The uricase is reprecipitated from the supernatant with ammonium sulfate and washed with 0.05 M KHCO _{3 to} remove the remaining foreign

protein free. The washed precipitate is dissolved in glycine-NaXO.j-Pulier pH 10.2 to 50 percent by volume adjusted with glycerine and remains stable in this solution.

The table gives an overview of the above

Enrichment process described, The yield of pure uricase (about 7 U'mg) is about 50% of the initial activity. The quotient of the pure enzyme 280/293 with is about 1.8.

Overview of the enrichment of uricase from 30 kg pig liver according to the example

Cleaning sl'ifc

Wash pH 5.3

Extraction with 0.27 M Na., CO., + 0.1 M glycine
pH 10.2 '..,.', '.

Filtrate and washing water pH 7.0 after adsorption of the uricase on Supercel

Acetone-NaCl eluate pH 0.2

Acton precipitation 60%, precipitation

Ammonium sulfate precipitate / - A M nacli
Wash with 0.05 M KHCO ,, solution

Ca phosphate gel adsorption, supernatant

Ammonium sulfate precipitate according to KHCO., - washes, dissolved in a mixture of glycine and glycerine
pH 10.2

volume
int

0.30

0.100

total

protein

ing

1210
901

840
157
58.5

10.2
3.7

1.24

Uricase

total

Healing

Spcz. Act.
U / mg
protein

none
activity

1.2 -10 **)

7.14-10 ²
1.5 ■ 10 *

1.43 10 *

1.04 · 10 «
1.02 · 10 ⁴

0.834 ■ 10 *

0.0133

0.00085

0.096

0.245

1.02
2.76

6.8

Yield "/ o

100 95

*) Extractable activity and protein amount are influenced by the extraction medium and are not yet quantitatively comparable with the following values.

Claims (1)

i 517 742 patent claims: UrHiIIt to ensure the determination of uric acid! especially to design sehneil, increased by leaps and bounds. Urease is obtained from kidneys, spleen or liver mitoehondria according to known methods. Because of the higher enzyme levels, the enrichment processes, which Schwcinelkcr consider as starting material, are of particular practical importance. Mahler, for example, are of particular practical importance , Pretty 1. Procedure for the isolation of Uriease from animal tissues in particular liver, kidney or spleen, the uriease from the water-insoluble Fraction of the tissue concentration with more alkaline Salt solution is extracted, characterized in that one in this extract