Covalent Immobilization of Thiol Proteinases on Chitosan

Plant enzymes such as ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and bromelain (EC 3.4.22.4) are obtained from tropical plants. These biocatalysts belong to thiol proteases, in the active center of which cysteine is contained. Ficin, papain and bromelain have a wide substrate specificity, which provides a demand for their use in various industries. Enzymes in the free state are less commonly used; immobilized biocatalysts are the preferred form. The aim of this work was to determine the optimal concentration of a crosslinking agent in the covalent immobilization of ficin, papain and bromelain on a chitosan matrix. Ficin, papain and bromelain (Sigma) were chosen as objects of study. An acid-soluble chitosan (350 kDa, Bioprogress CJSC) was used as an immobilization carrier. The concentration range of glutaraldehyde (crosslinking agent) ranged from 1 to 25%. Suitable concentrations of glutaraldehyde for covalent immobilization were identified by the optimal ratio of protein content (mg per g of carrier), total activity (in units per ml of solution) and specific activity (in units per mg of protein). It was shown that for covalent immobilization of ficin and bromelain on a chitosan matrix, it is most promising to use 10% glutaraldehyde. For immobilization of papain on chitosan by covalent means, the concentration of glutaraldehyde equal to 20% is optimal.

Lysosomal proteolysis in skeletal muscles of bulls

The relationship between lysosomal proteolytic enzyme activities involved in skeletal muscle proteolysis of the longissimus lumborum et thoracis muscle (MLLT) of bulls was described. Samples from the same region were obtained post mortem from 7 Piemontese (P) and 54 Black-and-White bulls (B-W) about 18 months old fed ad libitum. The activity of cathepsin D was determined as pepstatin (cathepsin D inhibitor) sensitive activity (PSCatD) towards 1% haemoglobin. Pepstatin-insensitive acid (PIA) and leupeptin-insensitive (thiol proteinase inhibitor) acid (LIA) autolytic activities were measured in the presence of 1 mM Mg⁺⁺. MLLT was also analysed for RNA, DNA and protein content. The data were processed by analysis of variance and differences between sires were tested by the contrast procedure of general linear model. In the examined muscle RNA decreased by 16% in B-W compared to P, CPS by about 14% and FCS by about 39%. DNA content was higher by 64.5% in B-W compared to P bulls (P ≤ 0.01). Some differences were found between P bulls and B-W groups of sires in the percentage of proteins (P ≤ 0.01), CatD and PSCatD (P ≤ 0.01), but the most pronounced differences were determined in PIA and LIA (P ≤ 0.01), and in the percentage of inhibition by pepstatin and leupeptin (P ≤ 0.01) in AAA. In the Black-and-White group of sires the percentage of protein and percentage of inhibition by pepstatin and leupeptin in AAA were lowered by about 10, 17 and 22%, but PSCatD, PIA and LIA were higher by about 23.7, 41 and 57.7%, respectively, compared to Piemontese bulls. The level of aspartic and thiol proteinases was lower in the muscles of B-W compared to Piemontese. The activity was much higher in B-W compared to P. These results indicate the faster turnover of proteins in the groups after Black-and-White sires and higher anabolic increase in degradation in Piemontese bulls.    

Cathepsin involvement in muscle proteolysis in meat-type bulls

Measurements were done of some lysosomal proteolytic enzyme activities involved in skeletal muscle proteolysis of the masculus longissimus lumborum et thoracis muscle (MLLT) of bulls. Samples from the same region between the 11th and 13th vertebra were taken after slaughter from Limousin (n = 10), Hereford (n = 10), Charolais (n = 10), Angus (n = 11) and Simmental (n = 11) bulls about 15 months old fed complete diet ad libitum. The activity of cathepsin D was determined as pepstatin (cathepsin D inhibitor) sensitive activity (PSCatD) towards 1% haemoglobin. Pepstatin-insensitive acid (PIA) and leupeptin-insensitive (thiol proteinases inhibitor) acid (LIA) autolytic activities were measured in the presence of 1mM Mg⁺⁺. MLLT was also analysed for RNA, DNA and protein variables. The data were processed by analysis of variance. The highest activities in PSCatD (P ≤ 0.05), AAA (P ≤ 0.01) and LIA (P ≤ 0.05) as well as percentage of inhibition by pepstatin in cathepsin D (P ≤ 0.01) were estimated in Angus bulls, and the lowest in Limousin ones. These breeds differed in the above-mentioned activities by 20.3, 21.1, 31.1 and 13.1%, respectively. RNA/g of tissue was highest in Hereford and lowest in Limousin bulls (by about 15.3%, P ≤ 0.01). Similar differences (14.3%) were between Charolais and Limousin (P ≤ 0.01). CPS (10³RNA/protein) was higher by 18.3% (P ≤ 0.01) in Charolais compared to the value in Simmental bulls; similar differences were between Hereford and Simmental (16.4%, P ≤ 0.01). The DNA concentration was highest in Hereford (by about 30%) compared to Charolais bulls. Protein/10³DNA ratio (mg/mg) – FCS – was higher by 33.4% in Charolais compared toHereford; RNA/DNA ratio was higher by 40.2% in Charolais compared toLimousin bulls. These results indicate the fast turnover of proteins in the groups of examined bulls and it can be concluded that in hypertrophic MLLT of bulls an anabolic decrease in degradation occurred.  

Synthesis and Photoproperties of a Substituted Zinc(II) Phthalocyanine-N-(2-hydroxypropyl)methacrylamide Copolymer Conjugate

In cancer photodynamic therapy (PDT), improved efficiency of photosensitizer delivery to tumors may be obtained by binding them to targetable water soluble polymeric carriers. However, attachment of photosensitizers to Macromolecular carriers may alter their spectral and photosensitizing properties. In this study, a new monosubstituted phthalocyanine derivative, N-glycyl zinc(II) 4,9,16,23-tetraaminophthalocyanine (G-TAPC-Zn) was synthesized by the reaction of zinc(II) 4,9,16,23-tetraaminophthalocyanine (TAPC-Zn) with N-tert-butoxycarbonyl-glycine N'-hydroxybenzotriazole ester followed by deprotection of the tert-butoxycarbonyl (BOC) group. G-TAPC-Zn contains an aliphatic amino group suitable for attachment to water soluble polymeric carriers. By aminolysis of a polymeric precursor, an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer containing oligopeptide (GFLG) side-chains terminated in p-nitrophenyl ester groups, with G-TAPC-Zn a polymeric derivative of the latter (P-GFLGG-TAPC-Zn) was synthesized. Spectral data indicated that in aqueous solutions P-GFLGG-TAPC-Zn formed aggregates. The degree of aggregation decreased with increasing concentration of detergents or organic solvents in buffer solutions. Consequently, the release of the drug from carrier catalyzed by thiol proteinases, papain or cathepsin B, took place only in the presence of detergents or organic solvents, i.e., under conditions with a lower probability of aggregate formation. Binding of G-TAPC-Zn to HPMA copolymers decreased the quantum yield of singlet oxygen generation from 0.24 to 0.063 and significantly increased its resistance to photobleaching.