Solvation Energy and Thermal Stability of Hydrophilization-Modified α-Chymotrypsin

As reported in the literature [Mozhaev et al. (1988), Eur. J. Biochem. 173, 147–154], when a series of modifiers, especially the cyclic anhydrides of pyromellitic and mellitic acids, are introduced into each lysine located in the -chymotrypsin (CT) surface, a substantial hydrophilization of the enzyme surface can occur and remarkable stabilization effects of modified enzymes can be obtained. In this paper, four models are applied to calculate the solvation energy of native and the modified CT based on their tertiary structures, which can be built by the CVFF force field. Analyzing the relationship between the solvation energy and the thermal stability in detail, we find that the results of three solvation energy models (Ooi model, WE-1 model, and WE-2 model) can be used to illustrate the relative stability among these enzymes qualitatively. The present study should be of practical value as well as of some theoretical interest.     

Effects of polyhydroxy compounds on the structure and activity of alpha-chymotrypsin

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.     

Covalent binding of α-chymotrypsin on the magnetic nanogels covered by amino groups

A new aminated carrier—magnetic nanogels covered by amino groups, was obtained by Hoffman degradation of polyacrylamide-coated Fe₃O₄ nanoparticles prepared by photochemical polymerization. α-Chymotrypsin (CT) was covalently bound to the magnetic nanogels by use of 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide and N-hydroxysuccinimide at room temperature. Immobilization time, pH value of the reaction mixture and proportion of CT to the magnetic nanogels were investigated to obtain the optimum condition for CT immobilization. The maximal specific activity of the bound CT was determined to be 0.93 U/(mg min), 59.3% of free counterpart. The maximal binding capacity was measured to be 102 mg enzyme/g nanogel. Furthermore, the bound CT exhibited good thermal stability, storage stability and reusability.     

Thermal aggregation of alpha-chymotrypsin: role of hydrophobic and electrostatic interactions

We have recently reported that electrostatic interactions may play a critical role in alcohol-induced aggregation of alpha-chymotrypsin (CT). In the present study, we have investigated the heat-induced aggregation of this protein. Thermal aggregation of CT obeyed a characteristic pattern, with a clear lag phase followed by a sharp rise in turbidity. Intrinsic and ANS fluorescence studies, together with fluorescence quenching by acrylamide, suggested that the hydrophobic patches are more exposed in the denatured conformation. Typical chaperone-like proteins, including alpha- and beta-caseins and alpha-crystalline could inhibit thermal aggregation of CT, and their inhibitory effect was nearly pH-independent (within the pH range of 7-9). This was partially counteracted by alpha-, beta- and especially gamma-cyclodextrins, suggesting that hydrophobic interactions may play a major role. Loss of thermal aggregation at extreme acidic and basic conditions, combined with changes in net charge/pH profile of aggregation upon chemical modification of lysine residues are taken to support concomitant involvement of electrostatic interactions.     

Application of a chemoenzymatic glycosylation method to α-chymotrypsin and Candida rugosa lipase surface modifications

A recently developed chemoenzymatic glycosylation procedure has been successfully applied on two hydrolytic enzymes, α-chymotrypsin and Candida rugosa lipase. First, a number of sucrose molecules have been bound to the surface lysine residues and then, lengthening of the glycosidic chains has been carried out by the action of a levansucrase from Bacillus subtilis. For both steps, reaction conditions have been studied in order to obtain a range of glycosylation degrees. The influence of glycoside binding on biocatalyst surface characteristics has been assessed and a progressive increase in global enzyme hydrophilic character with glycosylation has been observed. Besides, the study of hydrolytic activity and kinetic constants showed that the performed modifications brought about a certain decrease in enzyme hydrolytic activity and very slight variations in enzyme-substrate affinity.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

Stability of Desmopressin Loaded in Liposomes

Abstract

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of α-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against α-chymotrypsin digestion.     

Role of electrostatic interactions in 2,2,2-trifluoroethanol-induced structural changes and aggregation of alpha-chymotrypsin

It has been recently demonstrated that alpha-chymotrypsin (CT) can be driven toward amyloid aggregation by addition of 2,2,2-trifluoroethanol (TFE), at intermediate concentrations. In the present article, the process of TFE-induced CT aggregation was investigated in more detailed kinetic terms where the effects of medium conditions, such as temperature, presence of kosmotropic and chaotropic salts, pH and chemical modification of lysine residues were examined. Various techniques, including light scattering, fluorescence and circular dichroism spectroscopy, were used to follow and characterize this process. The kinetics of aggregation was found to obey a second-order reaction with respect to protein concentration. The aggregation-prone A-state and aggregation-deficient TFE- or T-state of CT were found to be induced at lower TFE concentrations in the presence of salts. Use of acidic and alkaline conditions and lysine modification also promoted the formation of the T-state. Results presented suggest a role for electrostatic interactions in the aggregation process.     

Substituted thieno[2,3-b]thiophenes and related congeners: Synthesis, β-glucuronidase inhibition activity,crystal structure,and POM analyses

A series of 15 novel compounds incorporating the thieno[2,3-b]thiophene moiety were synthesized. The chemical structures of these compounds were deduced from elemental analyses, ¹H NMR, ¹³C NMR, and ESI-mass spectral data. The enzyme inhibition potential of these compounds was evaluated, in vitro, against β-glucuronidase, xanthine oxidase, and α-chymotrypsin enzymes. The cytotoxicity was evaluated by a cell viability assay utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. Among the compounds tested, compound 3 was the most potent β-glucuronidase inhibitor with an IC₅₀ value of 0.9 ± 0.0138 μM; it was much more active than the standard, d-saccharic acid 1,4-lactone (IC₅₀ = 45.75 ± 2.16 μM). Compound 12, on the other hand, was the most potent as a xanthine oxidase inhibitor with an IC₅₀ of 14.4 ± 1.2 μM. With the characterization of their mechanism of action and with further testing, these compounds could be useful candidates as anticancer drugs. In addition, the newly synthesized compounds were subjected to POM analyses to get insights about their degree of their toxicity.