CS218709B1 - A method for preparing an insoluble chromolytic protein - Google Patents

Abstract

The invention relates to a method of preparing an insoluble chromolytic protein. The essence of the invention is that in the first stage the protein is dissolved at a temperature of 15 to 25 °C to 10 to 20 wt. % solution, after which a cross-linking reagent preferably epichlorohydrin and the disodium salt of 8-amino-5-[-3/sulfoethylsulfonyl/anilino]-6 anthraquinonesulfonic acid are added in the second step, and after the dye has dissolved, the reaction solution is alkalized with sodium hydroxide, mixed well and allowed to react for 15 to 24 hours at a temperature of 15 to 25 °C, after which the crosslinked chromogenic gel is mixed in with diluted methanol, neutralized, filtered off, washed with methanol, dried, and the dry protein was ground dry on a guf mill to particles of 40 to 250 μ. The invention is used in clinical biochemistry in the determination of proteolytic enzymes in biological fluids, in industrial application in the inter-operational control of culture media in the industrial production of enzymes.

Description

3

The invention relates to a process for the preparation of an insoluble chromolytic protein suitable for determining the protease enzyme activity by a spectrophotometric method.

The most commonly used known methods for determining proteolytic activity are very ingenious and low-precision. They are basically based on the fact that the amount of released protein during proteolytic hydrolysis, which is determined as the soluble fraction in trichloroacetic acid M. Kunitz: J.Gen. Physiol. 30, 291 (1947), possibly assisted by a positive reaction with phenol reagent according to Folin [M. L. Anson: J. Gen. Physiol. 22, 79 (1938)].

The method based on the use of a casein-based chromolytic substrate is not commonly available for the relatively complex preparation of azokazein [S. Berman et al., Proc. Expzl. Biol. Copper. 107, 798-83 (1961)].

A very sensitive chromolytic substrate can be prepared according to Aut. osv. No. 194,592 based on crosslinking and staining of various types of protein. However, it requires lyophilization of the stained and washed protein, which is a relatively demanding technological operation.

The aforementioned drawbacks are solved by the novel invention, which makes it possible to prepare a chromolytic insoluble cross-linked protein substrate without the ultimate lyophilization of the modified protein with high sensitivity to protease enzyme types suitable for the spectrophotometric determination of proteolytic enzyme activities.

The invention is based on the fact that in the first stage the protein is preferably bovine serum albumin, ovalbumin, casein or a mixture thereof dissolves in the water at a temperature of 15 to 25 ° C to 10 to 20% by weight of solution, whereby a crosslinking agent is added in the second stage. preferably epichlorohydrin in an amount of 0.5 to 5% by weight. to dry protein weight and 5 to 20 wt. the dye preferably the disodium salt of 8-amino-5- [3 / sulfoethylsulfonyl] anilino] -6-anthraquinone sulfonic acid per weight of dry protein, and upon dissolution of the dye, the reaction solution is alkalized with an amount of 25% by weight. sodium hydroxide solution to give a final concentration of 0.7 to 1.2% by weight in the reaction solution, and to mix well and to statically react for 15 to 24 hours at 15 to 25 ° C; then the crosslinked chromogenic protein is gelled in dilute alcohol, preferably methanol in a high-speed mixer, neutralized with acetic acid, filtered and filtered on a filter with methanol washed to a colorless reaction, finally washed with anhydrous methanol, dried at 60 ° C and dry protein dry sapomel on the ball mill to particles of 40 to 250 μ.

In order to obtain a protein gel with homogeneous distribution of crosslinks between protein macromolecules and homogeneous distribution of bound dye in cross-linked protein gel 218709, it is necessary to respect some peculiarities of heterogeneous reactions in the particular reaction system.

In particular, it is important that the protein is water-soluble. When the protein is dissolved, a crosslinking reagent is added, and after complete mixing, a solid is added which dissolves slowly in the protein solution. For good dissolution of the dye, 1-2 hours are required with vigorous stirring of the reaction solution.

For sufficient crosslinking of the protein, about 1% of the crosslinking reagent is needed. If more cross-linking reagent is used, the finished product has a lower swelling volume and hence lower sensitivity to proteolytic hydrolysis.

An equally important operation for the preparation of the chromolytic protein gel is the reliable washed-out dye.

It is advantageous to remove the free material by gradual decantation of the larger gel particles, then mix the gel in the fast-moving mixer to the desired particle size and wash the remainder of the dye on the filter with water, then with a water / alcohol mixture. acetone in a ratio of 1: 1 and finally with anhydrous alcohol or acetone. The dried chromogenic water-insoluble protein is ground to a particle size of 50 to 100 µm and is suitable for analytical purposes of the proteolytic proteolytic enzymes. The advantage of the novel process for preparing the chromolytic insoluble protein gel is that it is energy-saving, since all the modifying reactions in its preparation proceed at room temperature, - it allows the preparation of a chromolytic protein gel with a wide range of physical properties of the gel for the specific needs of its use -vania, - the finished product does not need to be stored at an acceptable temperature and is sufficiently stable when stored at up to 30 ° C, - does not require costly technological operations and can therefore be manufactured in conventional laboratory equipment,

Other advantages of the novel process are evident from the following examples, which are not exhaustive, but merely illustrative. Example 1 1 kg of bovine serum lyophilized albumin is dissolved in 6.25 liters of distilled water. 10 ml of mlepichlorohydrin are added by thorough dissolution of the protein and stirred for 30 minutes. Thereafter, sanaraz adds 120 g of Remazol brilliant blueR (8-amino-5- [3- (sulfoethylsulfonyl) anilino] -6-anthraquinone sulfonic acid salt), and blends the room for 2 hours. Then, vigorous stirring was added at once to 250 ml of sodium hydroxide conc. 25 wt. and stirring is continued until the reaction solution is thick, which takes about 2 to 5 minutes. After the reaction solution has densified, the stirring is stopped and the reaction is left to react overnight (16-20 hours) at room temperature.

Claims (1)

4 when the reaction is complete. The crosslinked and dyed solid protein is mixed in an alcohol (preferably methanol) diluted with water in a ratio of 2: 1. 100 ml of conc. acetic acid diluted with twice the amount of methanol to neutralize the free sodium hydroxide. The reaction suspension is filtered on a filter and washed with dilute water (1: 1) until the solution is colorless. Then it is washed with increasingly concentrated methanol until finally anhydrous, the crosslinked colored protein is suctioned off and dried in a drying oven at 60 ° C. The dried protein is milled into a guf mill and sieved through a 0.250 mesh sieve and finally sieved through a 0.04 m 2 sieve to remove finer fractions. The 1030 g weight has a particle size of between 40 and 250 μm and a bound dye of 9.2% and is a high-sensitivity substrate for all types of protease enzymes. EXAMPLE 2 The procedure of Example 1 except that acroprotein was used with ovalbumin and 200 g of dyeRemazol brilliant blue R and 20 ml of epichlorohydrin as crosslinking reagent. The 1040 g protein obtained has a bound dye content of 16% by weight. and is a very sensitive substrate of neutral and alkaline proteases, less sensitive acid proteases. SUBSTITUTE A method of producing an insoluble chromolytic protein characterized in that in the first step the saprotein preferably bovine serum albumin, ovalbumin, casein or a mixture thereof dissolves in water at a temperature of 15 to 25 ° C and a solution of weight concentration of 10-20%. the second stage, the crosslinking reagent is preferably epichlorohydrin in an amount of 0.5 to 5.0% by weight based on the weight of the dry protein and 5 to 20% by weight of the dye, preferably the disodium salt of 8-amino-5 - [3 / sulfoethylsulfonyl] anilino Example 3 218709 Procedure of Example 1 except that aoprotein is used with casein hydrolyzate 1200 g, but which dissolves poorly and dissolves poorly. first stirring in water for 1 hour, adding sodium hydroxide according to Example 1 and stirring for 1 hour. Then add 150 g of dyeRemazol brilliant blue R and stir for 2 hours, finally add 15 ml of epichlorohydrin, stir for 30 minutes and allow to react overnight as in Example 1. The protein yield of 1050 g has a dye content of 12% by weight. and is evenly sensitive to all types of protease enzymes. EXAMPLE 4 The procedure of Example 1 except that aoprotein is used in a 1: 1 ratio of serum bovine al-bumine, ovalbumin and the same as in Example 1. The resulting mixed chromogenic protein has a sensitivity for all types of protease enzymes. The invention is particularly useful in clinical biochemistry in the determination of proteolytic enzymes in biological fluids as well as in industrial application in the inter-operative control of culture media in industrial enzyme production. DISCLOSURE OF THE INVENTION with such an amount of sodium hydroxide solution at a concentration of 25% by weight, so that the resulting concentration in the reaction solution is from 0.7 to 1.2% by weight, is well mixed and allowed to react statically for 15 to 24 hours at a temperature of 15 to 25 ° C. the crosslinked chromogenic gel is mixed in dilute methanol, neutralized with acetic acid, filtered and washed with dilute methanol on the filter to give a colorless reaction of the filtrate, finally washed with concentrated methanol, dried to 60 ° C and the dry protein is ground to drought on the gufovommlyne to particles of 40 to 250 μ. Price: 2,40 Kčs Printed by Moravské tiskařské závody, operation 12, Leninova 21, Olomouc