CS264241B1 - A composition for the determination of proteolytic activity and a method for its production - Google Patents

Abstract

The preparation of the agent for determining proteolytic activity is carried out by adding black ink to the casein solution, the casein is precipitated with the simultaneous incorporation of the ink by lowering the pH of the solution to the value of its isoelectric point, and the obtained black precipitate is subjected to heating at a temperature of 150 to 250 °C for 30 minutes to 10 hours after drying. The agent consists of casein and black ink in a ratio of 10:1 to 1:1, where the amount of casein is given in grams and the amount of black ink in milliliters.

Description

The invention relates to a composition for the determination of proteolytic activity and to a process for its preparation.

The activity of proteolytic enzymes can be determined using a variety of substrates (Fukal, L., Káš, J. Vodrážka, Z .: Biochem. Clin. Bohemoslov. 14,109 (1985); Fukal, L., Káš, J .: Chem. Listy 78, 1176 (1984)). Substrates based on insoluble proteins occupy an important position among them. These include, for example, collagen (Gisslow, T. T., McBride, BC: Anal. Biochem. 68, 70 (1975), keratin (Nakanishi, T., Yamamoto, T .: Agric. Biol. Chem. 38, 2391). (1974), fibrin (Thorsen, S., Astrup, T .: Proc. Soc. Exp. Biol. Med. 130, 811 (1969)) or elastin (Bielefeld, DR, Senior, RM, Yu, SY: Biochem. Biophys Res Commun 67, 1553 (1975)).

Insoluble protein substrates with dye binding were developed to increase assay sensitivity. Examples are fibrin stained with indigo carmine (Nelson, W.L., Ciaccio, EI, Hess, GP: Anal. Biochem. 2, 39 (1961)), or Azocoll (azo dye-bound skin powder; Moore, G. L: Anal. Biochem., 32, 122 (1969).

Current chromogenic insoluble substrates for the determination of proteolytic activity require special production methods, and their preparation in the laboratory can be difficult.

The present invention provides a novel means for determining proteolytic activity and a process for its preparation.

A process for preparing the composition of the present invention comprises adding a black ink solution to the casein solution, precipitating the ink by lowering the pH to its isoelectric point while incorporating the ink, and then drying the black precipitate after heating at a higher temperature (preferably 150 to 250 ° C) for 30 min to 10 hours. The thermally modified chromogenic casein obtained is practically insoluble in water and aqueous solutions. The action of proteolytic enzymes releases a dye which is proportional to the activity of the proteinase and can be determined spectrophotometrically after filtering or centrifuging the decomposed insoluble substrate.

The invention also provides a composition for determining proteolytic activity, characterized in that it consists of casein and black ink in a ratio of 10: 1 to 1: 1, wherein the amount of casein is given in grams and the amount of black ink in milliliters.

The invention is illustrated by examples:

Example 1 g casein was dissolved in 400 ml 02%

Claims (2)

A composition for determining proteolytic activity, characterized in that it consists of casein and black ink in a ratio of 10: 1 to 1: 1. wherein the amount of casein is given in grams and the amount in milliliters. 2. A process for the preparation of a composition according to claim 1, which comprises contacting a casein solution CS 264 241 B1 sodium hydroxide solution. 5 ml of black ink pen ("Centrograf") was added to the solution. 50% acetic acid was then added dropwise with stirring until the casein-ink complex had completely precipitated. The obtained black casein-ink precipitate was filtered on a paper filter, washed with water, dried in an oven at about 80 ° C and then heated in a hot air sterilizer at 200 ° C for 4 hours. The obtained thermally modified chromogenic casein was ground in a shredded coffee mill and resuspended in 2-4% sodium hydroxide solution until the pigment was released. After filtration, the substrate was washed thoroughly with water, dried in an oven at 80 ° C and milled again in a shredded coffee mill to a fine powder, which was then used for the determination of proteinase activity. Example 2 Analogous to Example 1, chromogenic substrates were prepared by incorporating 10 ml of black ink / 10 g casein and 1 ml of black ink / 10 g casein. Example 3 Analogous to Example 1, different batches of thermally modified chromogenic casein were prepared, varying in temperature and duration of action. The following samples were prepared: 150 ° C / 10h; 180 [deg.] C / 6h; 200 ° C / 5h; 220 [deg.] C. for 2 h; 240 ° C / 1 h and 250 ° C / 30 min. Example 4 20 mg of substrate was weighed into the tubes, added with 2 ml of the appropriate buffer and allowed to swell for 20 minutes. Then, 1 ml of the solution containing alkaline bacterial proteinases was added to the tubes. After a suitably long incubation (e.g., 30 min at 50 ° C), the substrate was filtered off and the filtrate measured spectrophotometrically, e.g., at a wavelength of 400 nm. Alternatively, the tubes were transferred to an ice bath after the incubation, the contents of the tubes were centrifuged rapidly and the supernatant measured spectrophotometrically. Among the absorbance values of about 02-1.0, the relationship between the amount (activity) of alkaline bacterial proteinase and the absorbance of the filtrate (supernatant) was linear. At higher absorbance values the dependence was convex. The different lots of substrate prepared according to Examples 1, 2 and 3 showed similar properties. OF THE INVENTION black ink is added, casein is precipitated by lowering the pH of the solution to its isoelectric point with the concomitant incorporation of black interference, and the black precipitate obtained is dried after heating at 150 to 250 ° C for 30 minutes to 10 hours.