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T Vajda

Publications and source records attributed to T Vajda.

At least 19 recordsLinked to original sources
PubMed2001

Cryo-bioorganic chemistry: freezing effect on stereoselection of L- and DL-leucine cooligomerization in aqueous solution.

The chirality of L-/DL-leucine (50-50%) cooligomerization was investigated in liquid and frozen aqueous solutions. Cooligomerization was carried out by carbonyldiimidazole activation without initiator at an ambient (+ 22 degrees C) and frozen (- 18 degrees C) temperature, respectively. The separated samples obtained after different time intervals of treatment were completely hydrolyzed (HCl) and the diastereomeric L- and D-leucine derivates of Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) were then traced and evaluated by RP-HPLC analysis. After 9 days of oligomerization, the L-Leu content was slightly enhanced in the liquid (57%) and somewhat more enhanced in the frozen (64%) samples. After 17 days, however, the L-Leu content had decreased in the liquid (53%) and frozen (56%) conditions. These L-enantiomer amplifications indicate that an L-antipode is preferentially incorporated into the alpha-helical turn of the oligomer in the earlier stage of cooligomerization, while, later, the D-antipode is also incorporated. The role of ice in the improved stereoselection is discussed. This is the first recorded example of the effect of freezing on stereoselection.

Chemistry, Organic↗
PubMed1999

Cryo-bioorganic chemistry: molecular interactions at low temperature.

Freezing of aqueous or organic solutions plays a pivotal role in enhancement of rate and/or yield of biomolecular reactions. The smooth conditions of the frozen state at low temperature can also suppress racemization and side-product formation of the reactions. Molecular interactions in liquid undercooled solutions, on the other hand, offer the possibility to study enzyme activity mechanisms in vitro and a chance for survival of organisms in vivo. This review illustrates the differences between frozen and liquid conditions on several small and large biomolecules, together with the synthetic use of freezing. In relation to the freezing effect on enzyme activity, a peculiar phenomenon is discussed: 'cryo-oscillations' are temporal motions of trypsin activity in frozen solution in the presence of Mn2+ ion. The molecular basis of cold adaptation is also discussed, which points to mechanisms evolved by organisms living at subzero temperatures. The factors involved in the freezing effect are shown; i.e. the role of freeze-concentration and frozen solvent surface is demonstrated and elucidated using several examples.

Acetylation↗
PubMed1998

Cryochemistry: freezing effect on peptide coupling in different organic solutions.

The freezing effect on peptide coupling in organic solutions of different polarity has been investigated and compared with the results obtained in liquid phase. The model reaction of DCC-activated coupling of Boc-Ala-Phe-OH with H-Ala-OBu(t) has been carried out in dioxane, dimethylsulfoxide and formamide, as well as in mixtures (90%/10%, v/v) of dioxane with acetonitrile, dimethylformamide, dimethylsulfoxide and formamide. The reactions have been traced and evaluated by RP-HPLC analysis. Freezing the reaction mixture resulted in all cases in a significant suppression of the N-dipeptidylurea side-product formation together with a slight decrease of tripeptide epimerization. The coupling yields and the side effects depended on the solvent, with the dioxane and dioxane/acetonitrile mixture produced the best results. The role of freezing and solvent in the improved results is discussed.

Chromatography, High Pressure Liquid↗
PubMed1993

Self-similarity of Mn(II)-induced trypsin activity oscillations. Experimental evidence.

Aperiodic self-similar oscillations of trypsin activity were shown by experiments conducted in the presence of Mn2+ ion at pH 8.2 and -10 degrees C in frozen (cryo-oscillations) and at 0 degrees and 25 degrees C under unstirred conditions, respectively. The solution of trypsin obtained by trypsinogen activation and of 0.1 M MnCl2 was distributed into samples or kept in batches at the experimental temperature and sampled. At given time intervals the samples were tested for tryptic activity. In experiments at -10 degrees C the samples were frozen at the initiating of the series. The irregular shapes of activity curves as well as the trajectory of a next-amplitude plot is discussed as a result of a sensitive coupling between the chemical/conformational and diffusional controls of the enzyme activity in the far-from-equilibrium heterogeneous system. These indicate that the self-similar character of the trypsin activity oscillations can be considered on a phenomenological level.

Animals↗
PubMed1992

The role of freezing in trypsin activity oscillations.

The importance of the frozen phase in the formation of cryooscillations of trypsin activity has been shown in experiments conducted at -10 degrees C under frozen and supercooled conditions, respectively. A solution containing trypsin obtained by trypsinogen activation and 0.1 M MnCl2 was distributed in test tubes with or without previous freezing and kept at -10 degrees C and pH 8.4. At given time intervals the frozen and supercooled samples were tested simultaneously for tryptic activity. Although a temporal motion of trypsin activity was produced by the frozen samples, the activity of the supercooled samples began to oscillate only after spontaneous freezing of the solutions. This phenomenon suggests the importance of compartmentalization of the frozen heterogeneous system, which results in an increase in concentration vs a decrease in diffusion rate of the components.

Animals↗
PubMed1988

Role of counter ions in trypsin acylation. NaCl effect.

The Asp102-carboxylate negative charge of the trypsin catalytic-triad has been substituted in part by Cl- counter-ions. A His57-imidazolium cation and Ser195-tetrahedral oxyanion ionpair generated in the acylation step of catalysis is stabilized by the negative charge of Asp102. The importance of correct location of this negative charge has been investigated by experimental analysis of the NaCl influence on the acylation-rate of trypsin, as well as by electrostatic, potential calculations. The acylation-rate was determined with stopped-flow technique under pseudo-first order conditions, by using 4-nitrophenyl-4'-guanidinium benzoate active site titrant at pH 4.25 +/- 0.04, in an unbuffered solution of I = O or 0.5 M NaCl. The acylation-rate constants, kappa 2, are: kappa H2O = 0.32 +/- 0.02 s-1 and kappa NaCl = 3.5 +/- 0.5 s-1, and they correlate to beta-trypsin (the most rapid single-chained form of the enzyme). The rate increasing effect of NaCl, together with the calculated electrostatic energies, indicate that the negative charge contribution of the Asp102-carboxylate to the stabilization of the imidazolium cation and tetrahedral oxyanion intermediate is larger of more orders, than that of the Cl- counter-ion, located in a less favourable position.

Acylation↗
PubMed1987

Differences in effect of Mn(II), Fe(III), Co(II), and Zn(II) ions on trypsinogen activation.

The influence of Mn2+, Fe3+, Co2+, and Zn2+ ions on the extent of trypsinogen activation has been determined for several ion concentrations at pH 7.4 and 36.4 degrees C. For the Mn2+ ion also the autocatalytic rate constants have been detected. The effect of Ca2+ has been reinvestigated for comparison purposes. The apparent dissociation constants of KMn2+ = 0.01 (M) and KCa2+ = 0.02 (M) have been found for the given metal ion-trypsinogen complexes. For Co2+ ion, however, only a slight effect and for Fe3+ and Zn2+ ions no significant effect could be detected on trypsinogen activation. The investigated ions are of empty, open, and completed d subshells of electrons and they are different also in their ionic size. The differences in effects of the ions are discussed on the basis of these factors.

Copper↗
PubMed1986

Oscillations of trypsinogen activation.

Trypsin activity oscillations are shown by the autocatalytic activation of trypsinogen at 0 degrees C in aqueous solution. The oscillations were observed for 3-4 days and show only slight decrease in enzyme activity. The zymogen has been kept at ice water temperature and pH 8.2 in the presence of Mn2+ ion. The mean periods of around 1.5 hr are about half of those found previously at -10 degrees C in frozen aqueous solution, while the amplitudes related to the mean activity are about one-fourth of that in the frozen experiments. The phenomenon of oscillation is interpreted in terms of coupling between the inhomogeneities of protein and ion concentrations of the unstirred solution and a Mn3+/Mn2+ system, causing synchronous, periodic reduction-oxidation of some cystine bridges in the protein chain. These nonequilibrium conditions, together with synchronous transitions among several conformational states, may produce the observed activity oscillations.

Enzyme Activation↗
PubMed1981

Comparison of the effect of calcium(II) and manganese(II) ions on trypsin autolysis.

The effect of Mn2+ and Ca2+ ions on the rate of trypsin autolysis was studied at pH 7.0 and at 34.4-60.2 degrees C. For comparison, the kinetic constants of esterolytic activity of trypsin in the presence of the metal ion were determined at pH 7.4 and at 36 degrees and 40 degrees C. There was no significant difference in the rate of autolysis between Mn2+ and Ca2+ in the temperature range 34-47 degrees C, but at 56.8 degrees and 60.2 degrees autolysis was slightly more rapid in the presence of Mn2+. The Mn2+ or Ca2+ ion bound to trypsin is supposed to control the conformation and thereby the stability and the activity of the enzyme. The indirect effect of Mn2+ and Ca2+ is discussed on a structural basis of the enzyme molecule.

Animals↗
PubMed1980

Investigation of magnetic field effect on trypsin activity.

Influence of a homogeneous magnetic field on catalytic rate is proposed as a tool for the investigation of enzyme association. Investigations were initiated with studies of the effect of a 1.4 T homogeneous magnetic field on trypsin activity at 36.5 degrees C and pH 3.3, 5.3, and 7.2, respectively. Periods of exposure were applied up to 2--7 h. No detectable change of activity was observed in any of the exposed systems when they were compared with the identical but unexposed ones.

Hydrogen-Ion Concentration↗
PubMed1976

Specificity of trypsin and alpha-chymotrypsin towards neutral substrates.

Steady state kinetics of the hydrolysis by trypsin and chymotrypsin of the ethyl esters of the N-acetyl derivatives of glycine, L-alanine, DL-2-aminobutyric acid, L-norvaline, L-valine, L-norleucine and L-leucine were studied at pH 6.6 and 25 degrees C. It is apparent from the second-order rate constants, kcat/Km, app, that there is a significant difference between the specificities of the two enzymes toward substrates with a long side chain, such as the derivatives of norvaline, norleucine and leucine. The carboxylate ion Asp-189 in the specificity pocket of trypsin seems to interfere with the productive binding of substrates containing apolar side chains longer than those of the derivatives of DL-2-aminobutyric acid or L-valine. The basic group of the specific substrates of trypsin, such as that of lysine and arginine derivatives, promotes the binding of the apolar side chain by neutralizing the negative carboxylate ion of Asp-189.

Chymotrypsin↗
PubMed1976

Binding of alkylamines to trypsin as measured by difference spectroscopy.

The effect of n-propylamine, n-butylamine and n-pentylamine on trypsin at pH 5.4, at 25 degrees and 7 degrees C was investigated by the difference spectroscopic technique. The difference spectra were found to be similar for all three alkylamines tested. The difference between the absorbance minimum near 290 nm and the absorbance maximum near 297 nm taken as the measure of perturbation, the following values were obtained for the dissociation constant of the enzyme-ligand complex as calculated from the dependence of this perturbation upon alkylamine concentration at 25 degrees: propylamine, 6.0 X 10(-3) M; butylamine, 3.0 X 10(-3) M; pentylamine, 4.3 X 10(-4) M; and at 7 degrees: butylamine: 5.6 X 10(-3) M the chromophores situated in the active centre. It seems probable that the binding of an alkylamine gives rise to a conformational change which results in the rotation of Tyr-228 and Trp-215 residues towards the hydrophobic surroundings. This dislocation is a part of the minute change that leads to the activation of the catalytic site.

Amines↗