CS270671B1 - A method of making a composition for determining proteolytic activity - Google Patents

Abstract

The means for determining proteolytic activity is produced by adding a solution of a suitable dye or pigment (e.g. Congo red, water-soluble nigrosine, black ink) to a gelatin solution and cross-linking the gelatin with a bifunctional reagent (e.g. glutaraldehyde) while simultaneously incorporating the dye or pigment.

Description

The invention relates to a process for the preparation of a composition for determining proteolytic activity.

Proteolytic enzymes are widely used in many industries, agriculture, medicine, biochemical laboratories and elsewhere. A variety of substrates can be used to determine their activity. Impermeable protein chromolytic substrates occupy an important place among them.

Previously used methods for the preparation of insoluble chromolytic substrates consist in a) binding a suitable reactive dye to a natural insoluble protein (e.g. collagen, keratin, elastin, fibrin) or by absorption

b) crosslinking the conventional soluble proteins in solution with a suitable bifunctional agent to form insoluble crosslinked proteins, to which a suitable reactive dye is covalently or absorbed in a second step.

A method of making a composition for determining proteolytic activity according to the invention comprises adding a suitable dye or pigment to a solution of a suitable protein (e.g. gelatin) and then adding a suitable bifunctional agent (preferably e.g. glutaraldehyde) to crosslink and form insoluble, colored cross-linked proteins. This insoluble colored cross-linked protein is formed in a single step. The resulting product is washed thoroughly with water and left either in the form of a gel or crushed into small pieces, dried at a slightly elevated temperature and then triturated to a fine powder. The action of proteolytic enzymes releases a dye from the substrate, the amount of which is proportional to the proteinase activity and can be determined, for example, spectrophotometrically, after filtering off or centrifuging the undecomposed insoluble substrate.

The invention is documented by examples.

Example 1

Gelatin (10 g) was boiled in 100 ml of water on a water bath. After boiling, 0.25 g of Congo red dissolved in 10 ml of water was added to the gelatin solution. To the resulting solution was then added 1 to 2 mL of 50% glutaraldehyde diluted with 10 mL of water with stirring. The gelatin crosslinked rapidly and an insoluble colored gel formed. The resulting gel was crushed into small particles, washed thoroughly with water, dried at a slightly elevated temperature and ground in a mortar to a fine particle.

Example 2

In analogy to Example 1, an insoluble color gel was prepared. After grinding and washing with water, the gel particles were homogenized in an Elvehjem-Potter homogenizer. The formed fine particles were suspended in buffer and used to determine proteolytic activity.

Example 3

In analogy to Example 1, substrates were prepared containing as a pigment water-soluble nigrosine (0.25 g per 10 g of gelatin) or black ink (3 ml per 10 g of gelatin, added after cooling the gelatin solution to 35 ° C).

Example 4

In analogy to Example 1, a gelatin solution containing Congo red was prepared. The solution was poured onto petri dishes to form a layer about 4 mm high. After solidification, the gelatin layer was poured over 10 to 25 ml of a 2.5% glutaraldehyde solution and the solution was allowed to act for 18 to 24 hours. The glutaraldehyde solution was then drained, the cross-linked gelatin was washed thoroughly with water and discs of suitable diameter were cut with a cork borer. The discs were stored in water with the addition of a suitable bactericidal agent.

Example 5

In analogy to Example 4, disks were prepared and allowed to dry at room temperature. They were stored at room temperature.

CS 270671 Bl

The use of insoluble chromolytic substrates is documented in Examples 6 to 10.

Example 6

Oo tubes were weighed in 20 mg of substrate (prepared according to Example 1), added in 2 ml of a suitable buffer and allowed to swell for 20 min. Then 1 ml of proteinase solution was added to the tubes. After a suitably long incubation (e.g. 30 min at 37-50 ° C), the substrate was filtered off and the filtrate was measured spectrophotometrically at 497 nm. The absorbance value of 1 was linear between the amount (activity) of proteinase and the absorbance of the filtrate.

Example 7

2 ml of substrate suspension (prepared according to Example 2, the suspension contained 10 mg of substrate per Iml) was pipetted into the tubes and then 1 ml of proteinase solution was added. The rest of the procedure was analogous to Example 6.

Example Θ

In analogy to Example 6, the determination of proteolytic activity was carried out using substrates which contained nigrosine or Black ink as a pigment (prepared according to Example 3). '

Example 9

Substrate disks (prepared according to Example 4) were placed in 2 ml of buffer in tubes and 1 ml of proteinase solution was added. At the end of the incubation, the colored solution was discarded and measured.

Example 10

In analogy to Example 9, the determination of proteolytic activity was performed using dried substrate disks (prepared according to Example 5), except that the proteinases swelled in buffer for 20 minutes before the addition of the proteinase solution.

Claims (1)

OBJECT OF THE INVENTION. A process for the preparation of a composition for determining proteolytic activity, characterized in that a solution of a dye, e.g. Congo red or water-soluble nigrosine, or a pigment, e.g. black ink and a bifunctional agent, e.g. glutaraldehyde, is added to the gelatin solution. incorporation of a dye or pigment.