The effects of aqueous solutions of ethanol, acetonitrile and 1,4-dioxane in the concentration range 10-90% (v/v) on the activity of porcine pepsin were studied. The enzyme retained its activity in aqueous ethanol and aqueous acetonitrile with increasing organic solvent concentration up to 60%, and in aqueous 1,4-dioxane up to 30%, but thereafter a considerable decrease in activity was observed. The changes caused in the catalytic activity by the water-miscible organic solvents may be related to structural changes, which were followed by means of intrinsic fluorescence and circular dichroism spectroscopy measurements. (C) 2007 Elsevier Ltd. All rights reserved.

The effects of polyethylene glycol (PEG) of different molecular weights (400, 2000, 6000, 12,000, 20,000, and 35,000) on the conformational stability and catalytic activity of α-chymotrypsin in 60% ethanol were studied. The inactivation caused by the organic solvent was not influenced by PEG 400. However, the PEGs with higher molecular weights up to 35,000 increased the stability of the enzyme, but this α-chymotrypsin stabilizing effect was molecular weight-independent. With increase of the molecular weight of PEG, a more stable tertiary structure of the enzyme was observed.

The effects of the different forms of Al(III) on the catalytic activity of the serine protease trypsin were studied. Enzyme activity was measured by BAEE assay in the presence of AlCl(3), Al(III):lactic acid 1:3, Al(III):maltol 1:3 or Al(III):nitrilotriacetic acid (NTA) 1:1 at a nominal Al(III) concentration of 0.01 M, and the ligand alone at pH 7.4 at 25 degrees C. Maltol and NTA caused approximately 30% inhibition, while that for the corresponding Al(III) complex was less than half of this. Al(III) in the form of the chloride or in three equivalents of lactic acid did not influence the activity of the enzyme, probably because most of the Al(III) was precipitated as Al(OH)(3). No direct interaction could be detected between the enzyme and the Al(III) complexes, either by ultrafiltration or by CD spectroscopy. These results strongly suggest that there is no direct involvement of Al(III) in the enzymatic reactions of trypsin.

The effects of calcium ions on the conformation and catalytic activity of trypsin and alpha-chymotrypsin were studied in aqueous ethanol. The activity of alpha-chymotrypsin was practically lost within 10 min in the presence of 60% ethanol while trypsin preserved about 40% of its original activity even in 85% ethanol at pH 3. The catalytic activity of alpha-chymotrypsin did not decrease in the presence of 1.2M CaCl2 and 0.6M CaCl2 with trypsin in ethanolic solvent. In the latter case an activation of enzyme was observed. The stabilizing effects of calcium ions were accompanied by an increase in the helical content in both enzymes, as followed by circular dichroism measurements.

The effects of glycerol, polyethylene glycol, fructose, glucose, sorbitol, and saccharose on the conformation and catalytic activity of alpha-chymotrypsin were studied in 0.1 M sodium phosphate buffer and buffered aqueous 60% ethanol (pH 8.0). The enzyme activity was practically completely lost within 10 min in 60% ethanol, but in the presence of stabilizers the activity was retained. With the exception of polyethylene glycol, the stabilizing effect decreased with increase of the incubation time. The preservation of the catalytic activity was accompanied by changes in the secondary and tertiary structures of alpha-chymotrypsin.

A comparative study was performed on the conformational stabilities of trypsin and α-chymotrypsin. At 45oC, trypsin was most stable at pH 3, while the highest stability of αchymotrypsin was observed at pH 5. With both ester and amide substrates, trypsin displayed activation at pH 3. In the case of α-chymotrypsin, activation was detected at pH 5 only with the amide substrate. The time curves of heat inactivation were complex. For both enzymes, autolysis proceeded with the highest velocity at pH 8. The results obtained on α-chymotrypsin suggested consecutive reactions: the first step, heat denaturation of the protein, is followed by digestion of the damaged molecules. Acta Biol Szeged 45(1-4):43-49 (2001)

The effects of different concentrations (20–95%) of organic solvents (ethanol, 1,4-dioxane and acetonitrile) were studied on α-chymotrypsin and trypsin from bovine pancreas. The changes in secondary structure were followed by CD measurements, and the apparent Michaelis constants (KMapp) and the stabilities of the enzymes were determined. Significant alterations in the CD spectra were found for both enzymes at the different organic solvent concentrations. The apparent KM values of trypsin and α-chymotrypsin decreased as the low solvent concentrations were elevated, but then increased in the presence of higher organic solvent concentrations. The stabilities of the enzymes changed on increase of the organic solvent concentration; trypsin exhibited a higher stability than that of α-chymotrypsin in all organic solvents. These results show that at an organic solvent content of 95% the manifestation of an enzyme activity similar to that measured in water can be attributed to the similar compositions of the secondary structural elements.

The bovine trypsin-catalysed synthesis of N-acetyl-l-arginine ethyl ester from N-acetyl-l-arginine and ethanol was studied in various organic solvents (dimethyl sulfoxide, dioxane, dimethylformamide, acetonitrile, acetone, tetrahydrofuran, chloroform, toluene, carbon tetrachloride, cyclohexane and n-hexane). The highest yield was achieved in acetonitrile after incubation for 6 or 24 h. The optimal conditions for ester synthesis in acetonitrile for 6 h were as follows: 5.0 mM N-acetyl-l-arginine, 10.0 M ethanol, 7.2 mg trypsin, 2.87% water, total volume 10.3 ml, pH 7.0 and 30°C. The hydrolytic activity of trypsin was determined after incubation for 6 days, when 87.7% of the original activity remained, suggesting that acetonitrile caused little inactivation of the enzyme. The synthetic reaction resulted in a maximal 79.3% conversion under optimized conditions after incubation for 48 h.

The effects of haevy metals (Cd2+, Cu2+, Pb2+ and Zn2+) on activities of carp trypsin, alpha-chymotrypsin, carboxypeptidase A and lipase were studied. The enzymes were isolated from the gastrointestinal tract and the effects of metal ions were investigated during incubation for 5 min. The presence of Cd2+ did not influence the activities of CPA and trypsin and 10-20 % inhibiton was observed with alpha-chymotrypsin and lipase. Cu2+ Only slightly influenced the trypsin and lipase activities, whereas the alpha-chymotrypsin activity was decreased. All enzyme activities decreased at higher Zn2+ concentrations. The inhibition was most pronounced at alpha-chymotrypsin (more than 50%). With the exception of trypsin, Pb2+ inhibited the activities of the investigated enzymes.

The effects of pyrethroid pesticides (deltamethrin, permethrin and cypermethrin) and an organophosphate ester (methidation) on the activities of carp trypsin, alpha-chymotrypsin, carboxypeptidase A and lipase were studied. The enzymes were isolated from the gastrointestinal tract and the effects of the pesticides were investigated during incubation for 5 min. The activity of trypsin was influenced only slightly by the presence of deltamethrin and methidation, whereas permethrin and cypermethrin caused significant inhibition. The pyrethroid pesticides at lower concentrations resulted in a slight activation of alpha-chymotrypsin. Methidation inhibited the alpha-chymotrypsin activity by about 20%. These pesticides modified the lipase activity to a lesser extent; the highest inhibition was measured with cypermethrin. The carboxypeptidase A activity was inhibited by both pyrethroid pesticides and methidation. The results suggest that these pesticides might interact with the active conformation of the studied hydrolytic enzymes, resulting in changes in their activities.

Spinach leaf hydroperoxide lyase (HPLS) was immobilized on polyethylene terephthalate (Sorsilen), silica (Silochrome) and polyacrylamide (Akrilex) supports. The highest immobilized activity (360 mU g(-1) dry gel) was achieved with the HPLS bound to Akrilex C. The Akrilex- and Silochrome-bound enzymes were studied with respect to their thermal, pH and operational stabilities relative to those of the soluble HPLS. The Akrilex-bound enzyme had the highest stability.

The important role of oxygen radicals in acute experimental pancreatitis was demonstrated by study of the changes in the antioxidant system in the blood, liver, kidney, and pancreas of rats after the administration of a large quantity of L-arginine (L-Arg). The changes in lipid peroxidation and in reduced and oxidized glutathione were followed, as well as the activities of peroxide-decomposing enzymes (glutathione peroxidase and catalase) and H2O2-producing superoxide dismutases. The results demonstrated that "oxidative stress" develops and acute pancreatitis appears rapidly after L-Arg treatment. Oxidative stress symptoms are expressed 24 h after the final treatment. Slow restitution of the studied antioxidant system can be demonstrated as early as after 48 h.

After intravenous administration of alloxan monohydrate (AL) diabetes developed in rats. Forty-eight hours after the injection the animals were sacrificed, their blood was collected in heparin containing tubes and the tissues were dissected and frozen (-70 degrees C) until their homogenization for pro- and antioxidant testing. Our results can be summarised as follows: (i) In the blood hemolysate the lipid peroxidation slightly elevated and the activity of antioxidant enzymes and reduced glutathione decreased. (ii) Similar phenomena could be observed in the different examined organ homogenates. The organs tested for pro- and antioxidant system were as follows: the liver, heart, skeletal muscle, kidney and pancreas. In our present work we attempt to confirm the data in support of the oxidative predominance over antioxidants in oxidative stress of AL diabetic rats.

This study was conducted on type 2 non-insulin-dependent diabetes mellitus (NIDDM) cases and healthy blood donors. Lipid peroxidation (LP) products in plasma and red blood cell (RBC) hemolysates were estimated as total thiobarbituric acid reactive substances (TTBARS). The plasma and hemolysate reduced and oxidized glutathione (GSH and GSSG) levels are compared. In the hemolysates the antioxidant enzymes namely superoxide dismutase (SOD), glutathione peroxidase (GPx-ase), glutathione reductase (GR-ase) and catalase (C-ase) are also compared. The RBC filtration characteristics are determined and compared with controls: 1. LP and GSH in diabetic plasma were significantly higher, but in the hemolysate the GSH raised but the LP was significantly lower in diabetics than in healthy controls. 2. Superoxide dismutase and C-ase were significantly higher in NIDDM hemolysate. Contrary the GPx-ase activity was significantly lower in diabetics. 3. The diabetic RBCs filtration characteristics are changed in respects significantly namely the Fi was lower, the Tc and CR were higher. It means higher rigidity and oxidative damage of the membrane of diabetic RBCs.

It is known that streptozotocin (STZ) penetrating into the organism generates nitrogen monoxide (NO). Therefore, it is justified to presume, that in beta-cell destruction thereby induced, peroxinitrit resulting from NO and superoxide (O2-) reaction has an important role. It has also been studied how pro- and antioxidant systems change in STZ induced experimental diabetes in rat organs. Beside pro- and antioxidant systems of plasma and red blood cell hemolysates, changes in homogenates of the following organs were studied: liver, kidney, heart, lungs, spleen, brain, muscles and pancreas. We tested and compared antioxidant enzymes (superoxide dismutase-, glutathione peroxidase- and catalase activities) glutathione reductase activity regenerate reduced glutathione (GSH). The oxidized, reduced glutathione values and lipid peroxidation changes were measured. From our studies it has appeared that STZ treatment generally induces an oxidative predominance in tissues. Changes in this model thereby, can be compared to changes occurring in type 1 human diabetic patients.