CS263665B1 - a means for determining proteolytic activity and a method for its production - Google Patents

Abstract

The composition for determining proteolytic activity is heat-modified casein and its derivatives. The composition according to the invention is prepared by heating casein and its derivatives, such as azocasein and nitrocasein, to a temperature of 150 to 250°C for 30 minutes to 10 hours.

Description

The present invention relates to novel means for determining proteolytic activity and to a process for their preparation.

The activity of proteolytic enzymes can be determined using a variety of substrates. Substrates based on insoluble, naturally occurring proteins form an important group. These include, for example, elastin, fibrin, collagen or keratin.

Insoluble protein substrates with dye binding were developed to increase the sensitivity of the site. Examples are, for example, azo-dye-bound skin powder, indigo carmine-stained fibrin or remazol-brilliant-blue skin powder.

Methods of preparation of insoluble chromolytic substrates from soluble proteins by their chemical modification have also been described.

The process for preparing insoluble substrates for the determination of proteolytic activity according to the invention consists of heating the casein or its derivatives (such as azo-casein or nitro-casein) in a hot-air sterilizer at a temperature of 150 to 250 ° C for 30 min to 10 hours. The thermally modified casein obtained and its derivatives are practically insoluble in water and aqueous solutions. The action of proteolytic enzymes releases peptides of which are proportional to proteinase activity and can be determined, for example, by spectrophotometry, after filtering or centrifuging the undissolved insoluble substrate.

The invention is illustrated by examples.

He did

The azocasein was heated in a hot air sterilizer at 200 ° C for 5 hours in a thin layer. After heating, the heat-modified azocasin was repeatedly suspended in 2-4% sodium hydroxide solution until an intense brown-red to red color was released. It was then filtered and washed thoroughly with water on the filter. After drying, the washed substrate was spread in a mortar. A fine powder was obtained, which was used for the determination of proteinase activity.

Example 2

Analogously to Example 1, substrates based on thermally modified casein and thermally modified nitro-casein were prepared.

Example 3

Analogous to Example 1, different batches of thermally modified azokasse were prepared, differing in the temperature used and the duration of action. The following samples were prepared:

150 ° C / 10 hj 180 ° c / 6h | 200 ° C / 5h, 220 ° C / 2h »240 ° C / 1h 250 ° C / 30 min

Heat-modified casein was also prepared by heating to 200 ° C for 9 hours.

The procedure was as in Example 1.

. The use of prepared insoluble substrates is documented by the following example.

«

20 mg of substrates prepared according to Example 1 and 3 were weighed into tubes. 2 ml of the appropriate buffer was added and the substrate swelled for 20 minutes. 1 ml of proteinase-containing solution was then added to the tubes. After incubation for 30 min at 37-50 ° C, the substrate was filtered off and the filtrate measured spectrophotometrically in a 1 cm glass cell at 400 nm against a blank. Up to an absorbance value of 1.0, the consistency between the amount of proteinase and the absorbance to the filtrate was linear.

Claims (2)

A composition for the determination of proteolytic activity, characterized in that it consists of casein or its derivatives, such as azo-casein or nitro-casein. 2. A process according to claim 1, wherein the casein or derivatives thereof, such as azo-casein or nitro-casein, are subjected to heating at temperatures of 150 to 250 [deg.] C. for 30 minutes to 10 hours.