Biologically active substances which have been immobilized on various textile carriers have been produced over a number of years in the "Tekstil'galantereya" NPO. The greatest circulation has been received by Dartseks-tripsin (VFS 42-1862-88) and paks-tripsin (BFS 42-1863-88), which are trypsin (Tr) which has been immobilized on dialdehyde cellulose (DAHC) or on polycaproamide which has been activated with glutaraldehyde (PCA-GA). Tr preparations have also been prepared which have been immobilized on PCA with inherent aldehyde groups which were obtained as a result of "mild" oxidation under oxidation of a polycaproamide matrix under special conditions (specimens PCA-A-Tr). The DAHC has been prepared as a result of periodate oxidation of medicinal gauze (All-Union State Standard 19412-27). Partial hydrolysis of the PCA was carried out with alkali; activation of the PCA matrix, with excess glutaraldehyde in alkaline medium. The amount of carbonyl groups on the carrier was determined by condensation with hydroxylamine in alcoholic medium (pH = 3.2) [1] or by oxidation with iodine under weakly alkaline conditions (0.1 n borax solution) [2]. The unmodified carrier was used as a control. The quantity of immobilized protein was determined by the Lowry-Hart ley method [3, 4], using as a control a carrier of the same degree of oxidation which had been treated concurrently with the immobilized specimen with the same solutions and under the same conditions [5]. The proteolytic activity of the specimen (A) was determined by the Kunitz method [6], using as a substrate a 2% casein solution according to Hammersten in a 1/15-molar phosphate buffer (pH = 7.8), with constant agitation on a laboratory shaker at 37°C. If necessary, the immobilized specimens were washed with water and a phosphate buffer until there was no protein in the wash water. After drying, the moisture content of the specimens immobilized on DAHC was 4-6%; and on the modified PCA, about 2%. As service characteristics, we examined the decrease in proteolytic activity upon the action of y-radiation in a dose of 25 kGr on the specimens of immobilized trypsin, the decrease in proteolytic activity during the process of immobilization, during storage, and also the thermal inactivation in a 1/15-molar phosphate buffer solution (pH = 7.8). As was established for Tr specimens immobilized on DAHC, the following exert a considerable effect on the retention of proteolytic activity: the number of aldehyde groups in the matrix [7], the amount of immobilized protein [8], and the pH and composition of the washing solution before drying the specimens [9]. Here, while increasing the content of aldehyde groups in the matrix (at an equal protein content) aids in retaining proteolytic activity after ~,-irradiation [10], in the process of storage or thermal inactivation in a model solution, the converse is true (see Table 1). Increasing the amount of immobilized Tr (at an identical degree of DAHC oxidation) leads to retention of the proteolytic activity of DAHC-Tr specimens during the storage process (at 15°C in the dark), but to the reverse effect in ~,-irradiation [8]. Therefore, we tried to examine specimens of immobilized Tr which had been prepared under identical conditions. In thermal inactivation of immobilized Tr in solution (pH = 7.8), a significant hydrolytic degradation of the carrier is observed; breakage of the azomethine bond may take place, plus desorption of the protein from the carrier. For DAHC with an elevated degree of oxidation, the rate of hydrolytic degradation is increased, as a result of which autolysis of the Tr is superimposed on the process of thermal inactivation, both of the Tr present in the free state (after desorption or breakage of the azomethine bond), and also of Tr which is partially modified by fragments of the carrier.
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Oct 26, 2009
Dierk Knittel + 3
