Abnormal behaviour of proline in the isothiocyanate degradation.

It has been observed that proline residues often initiate overlaps during sequenator analysis. The cause has been shown to be an abnormally slow cleavage reaction. The kinetics of the cleavage reaction has been studied and found to obey pseudo-first-order kinetics. There are considerable differences in reaction rates depending on the position of proline in the sequence, as demonstrated for the four prolines in the N-terminal section of the H2B histone from chicken.     

Stereocontrolled synthesis of anticancer beta-lactams via the Staudinger reaction

Stereocontrolled synthesis of novel beta-lactams using polyaromatic imines following the Staudinger reaction has been accomplished. The effects of domestic microwave irradiation on this type of reaction have been investigated. Formation of trans-beta-lactams has been explained through isomerization of the enolates formed during the reaction of acid chloride (equivalent) with imines in the presence of triethylamine. A donor-acceptor complex pathway is believed to be involved in the formation of cis-beta-lactams. The effect of a peri hydrogen has been found to be significant in controlling the stereochemistry of the resulting beta-lactams. SAR has identified beta-lactams with anticancer activity. The presence of an acetoxy group has proven obligatory for their anticancer activity.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).     

The catalytic mechanism of yeast thiosulfate reductase

Thiosulfate reductase catalyzes the desulfuration of thiosulfonates while oxidizing GSH to GSSG. Kinetic studies of the enzyme-catalyzed reaction between GSH and benzenethiosulfonate have been carried out, and direct evidence for the occurrence of glutathione persulfide as an immediate product of the reaction has been obtained. The formal mechanism of this enzymic reaction has been shown to be rapid equilibrium-ordered with GSH as the leading substrate.     

A kinetic model of the cleavage of permutational variants of the antigenomic HDV-ribozyme

The kinetic characteristics have been studied for noncircularly permuted variants of the human hepatitis delta virus (HDV) antigenomic ribozyme to find out the cause of the two-phase kinetics of the self-cleavage reaction. Different ways of reaction initiation, suboptimal conditions, and jumpwise changes of reaction conditions have been used, and the temperature dependences have been studied. A correlation has been shown between the apparent kinetic constant of the first reaction phase and the portion of the ribozyme molecules that self-cleaved during the first phase. Partial restoration of the initial reaction characteristics has been shown by the reinitiation of reaction being stopped after completing the first phase. On the basis of all the data obtained, a scheme of the self-cleavage reaction has been proposed including: (i) activation of the ribozyme with energy of 40-50 kcal/mol and a characteristic time of several deciminutes under optimal reaction conditions; (ii) fast and reversible reaction of the phosphodiester bond cleavage; (iii) reaction leading to isomerization of the 3',5'-phosphodiester bond to the 2',5' bond in the self-cleavage site with a characteristic activation time of tens of minutes; and (iv) practically irreversible conformational change leading to fixation of the cleavage by immobilization of the 5'-terminal nucleotide of the product in the center of the formed structure and displacement of the 3'-terminal nucleotide to the periphery. The latter process has a characteristic time of tens of minutes and a low activation energy.     

Kinetic Self-Cleavage Models for Permuted Variants of the Antigenomic HDV Ribozyme

The kinetic characteristics have been studied for noncircularly permuted variants of the human hepatitis delta virus antigenomic ribozyme to find out the cause of the two-phase kinetics of the self-cleavage reaction. Different ways of reaction initiation, suboptimal conditions, and jumpwise changes of reaction conditions have been used, and the temperature dependences have been studied. A correlation has been shown between the apparent kinetic constant of the first reaction phase and the portion of the ribozyme molecules that self-cleaved during the first phase. Partial restoration of the initial reaction characteristics has been shown by the reinitiation of reaction being stopped after completing the first phase. On the basis of all the data obtained, a scheme of the self-cleavage reaction has been proposed including: (i) activation of the ribozyme with energy of 40–50 kcal/mol and a characteristic time of several deciminutes under optimal reaction conditions; (ii) fast and reversible reaction of the phosphodiester bond cleavage; (iii) reaction leading to isomerization of the 3",5"-phosphodiester bond to the 2",5" bond in the self-cleavage site with a characteristic activation time of tens of minutes; and (iv) practically irreversible conformational change leading to fixation of the cleavage by immobilization of the 5"-terminal nucleotide of the product in the center of the formed structure and displacement of the 3"-terminal nucleotide to the periphery. The latter process has a characteristic time of tens of minutes and a low activation energy.     

DFT study of the V(IV)/V(V) oxidation mechanism in the presence of N-hydroxyacetamide

The oxidation mechanism of V(IV)/V(V) in the presence of N-hydroxyacetamide (acetohydroxamic acid, HL) in aqueous solution has been investigated using density functional theory (DFT) calculations aiming to contribute to the understanding of this process at a molecular level. The mechanism proposed involves formation of the *OH, *OOH, H2O2 radicals and complexes formed from the interaction of these species with VOL2 complex. The Gibbs free energy of each step of the mechanism has been evaluated. The solvation energy has been estimated by means of united atoms Hartree-Fock/polarizable continuum method (UAHF/PCM). The Gibbs free energy of the global reaction of the V(IV)/V(V) oxidation has been estimated and compared with the available experimental equilibrium constant. The difference between the calculated and experimental estimates for the reaction energy of the global equation is about 1.5 kcal mol(-1). The thermodynamic profile of the reaction mechanism has been provided and discussed in terms of the possible intermediates. The influence of the ligand and the reaction rate in terms of the steady-state approximation has been briefly discussed.     

Occurrence of early and late asthmatic reaction after provocation with antigen and the status of pulmonary ventilation in patients with hay fever after the pollination season]

Pulmonary ventilation and asthmatic reaction under laboratory conditions have been investigated in 23 patients with allergic rhinitis hypersensitive to grass pollen. Pulmonary ventilation has been assessed with the aid of VCin, FVC, FEV1, FEV1/VCin, PEF, MEF50, and MTT. Asthmatic reaction has been produced by an inhalation of allergens mixture with dose-response technique. An early reaction has been diagnosed, when FEV1 decreased by at least 20% or MEF50 by 30% within 10 minutes, and late reaction when the same parameters decreased after 6 or 24 hours. An early asthmatic reaction has been noted in 2 patients (8.7%), late--in 4 patients (17.4%), and double (both early and late) reaction in 2 patients (8.7%). Pulmonary ventilation has been normal in all examined patients, except two of them with peripheral airways obstruction (MEF50 less than 70% of the normal value). Results suggest, that asthmatic reaction may be provoked in the laboratory in patients with pollinosis and normal pulmonary ventilation after pollen season. Such a reaction may also be expected during a natural exposition to pollens.     

Photochemistry of phycobilisomes: photosensitized reduction of methylviologen

The ability of phycobilisomes (PBS) to photosensitize the reduction of methylviologen by dithiothreitol has been shown. This reaction has been studied in dependence on the structural integrity of PBS, nature and pH of the medium, and concentration of reagents. The action spectrum of the reaction has been measured, and its quantum yield has been detected. The quenching of PBS fluorescence by methylviologen has been studied. The maximum rate of electron transfer photosensitized by PBS is observed in 0.6 M phosphate buffer containing 30% of glycerine, pH 9.5. The saturation occurs at methylviologen concentration of 2 X 10(-4) M. Under these conditions the PBS activity reaches 0,3 mumol/mg PBS.h and increases 100 times after preliminary heating of the reaction mixture for 5 min. at 35 degrees C. The light in the range of 520-680 nm is active for the photosensitized reduction of methylviologen. The quantum yield of the reaction is about 2%. In structures phycobiliproteins are more effective sensitizers than in solutions. The highest activity is observed for the substructure of PBS which contains allophycocyanin B and allophycocyanin C (34S particle). The possible mechanisms of the reaction are under consideration.     

The reaction of formaldehyde with unsaturated fatty acids during histological fixation

Synopsis Formaldehyde reacts with unsaturated fatty acids in tissues during histological fixation. The reaction of formaldehyde with oleic acid has been found to given rise to compounds (adducts) with the following structures: Two other compounds were isolated but their nature is still open to doubt.The equilibrium constant for the initial part of the reaction has been approximately estimated as 0·042 at a room temperature of 22°C. The endothermic heat of reaction has been estimated as approximately 12·6 kcal.The occurrence of these adducts in tissues explains why it is that less lipid can be demonstrated histologically in material that has been stored in form-aldehyde for a considerable length of time.     

Kinetic study on the suicide inactivation of tyrosinase induced by catechol

Tyrosinase has a suicide inactivation reaction when it acts on omicron-diphenols. In the present paper, this reaction has been studied using a transient phase approach. Explicit equations of product vs. time have been developed for the multisubstrate mechanism of tyrosinase, and the kinetic parameters which characterize the enzyme acting on the suicide substrate catechol have been determined. The effect of pH has also been considered.     

Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.     

The reaction between nitrite and deoxyhemoglobin. Reassessment of reaction kinetics and stoichiometry

Recent evidence suggests that the reaction between nitrite and deoxygenated hemoglobin provides a mechanism by which nitric oxide is synthesized in vivo. This reaction has been previously defined to follow second order kinetics, although variable product stoichiometry has been reported. In this study we have re-examined this reaction and found that under fully deoxygenated conditions the product stoichiometry is 1:1 (methemoglobin:nitrosylhemoglobin), and unexpectedly, the kinetics deviate substantially from a simple second order reaction and exhibit a sigmoidal profile. The kinetics of this reaction are consistent with an increase in reaction rate elicited by heme oxidation and iron-nitrosylation. In addition, conditions that favor the "R" conformation show an increased rate over conditions that favor the "T" conformation. The reactivity of nitrite with heme is clearly more complex than has been previously realized and is dependent upon the conformational state of the hemoglobin tetramer, suggesting that the nitrite reductase activity of hemoglobin is under allosteric control.     

The effect of thiamine pyrophosphate modification on its coenzyme function in a transketolase-catalyzed reaction

The coenzyme function of TPP analogues: 4'-NH-methyl-TPP,6'-methyl-TPP and 6'-methyl-4-nor-TPP has been studied in a transketolase-catalyzed reaction. Their dissociation constants have been found with the aid of the circular dichroism method, and coenzyme activity has been determined in a complete transketolase reaction, involving the substrate-donor and the substrate-acceptor, and also at the intermediate stage (by the alpha-carbanionic intermediate oxidation rate). The coenzyme activity values have been found different and largely dependend on the nature of the substrates used. A possibility of TPP functioning by the "two-center mechanism" in a transketolase-catalyzed reaction is discussed.     

A comparative study of reaction times between type II diabetics and non-diabetics

Background  

Aging has been shown to slow reflexes and increase reaction time to varied stimuli. However, the effect of Type II diabetes on these same reaction times has not been reported. Diabetes affects peripheral nerves in the somatosensory and auditory system, slows psychomotor responses, and has cognitive effects on those individuals without proper metabolic control, all of which may affect reaction times. The additional slowing of reaction times may affect every-day tasks such as balance, increasing the probability of a slip or fall.     

A model for the rate of enzymatic hydrolysis of cellulose in heterogeneous solid–liquid systems

Hydrolysis of cellulose by cellulase enzymes has been studied in a stirred batch reactor at 50°C. A kinetic model has been devised by which the behaviour of such a reaction could be described. The model has been developed on the basis of shrinking particle theory and Langmuir isotherm concept. The applicability of the model has been tested by comparing the experimental results for diverse reaction systems, obtained in the present study or taken from the literature, and those predicted from the model. The degree of agreement was within ±2–11%.     

Turbidimetric method for determination of glycogen phosphorylase activity and its use for estimation of equilibrium position of enzymic reaction

A turbidimetric method has been developed for the continous monitoring of the enzyme reaction catalyzed by glycogen phosphorylase. This method is based on the registration of the turbidity of glycogen solution at wavelengths above 300 nm. It has been shown that increase in the turbidity is strictly proportional to the quantity of glucose 1-phosphate formed during the enzyme reaction. The method has the advantage of continuity, and it is suitable for determining the initial rate of catalytic synthesis or degradation of glycogen in a relatively simple and fast way. The kinetic experiments may be carried out under various conditions. The method of calculation of the overall equilibrium constant of the enzyme reaction catalyzed by glycogen phosphorylase has been elaborated. This method is based on the analysis of the dependence of the initial rate of the enzyme reaction on the proportiona of the substrate of the forward reaction: [P_i]/([P_i]+[G-1-P]).     

针对点突变癌基因T24－ras转录物的核酶性质研究

通过改变核酶体外反应的各种条件,对针对点突变癌基因Ｔ２４－ｒａｓ转录物的核酶Ｒ８的性质进行了系统研究,结果表明：Ｒ８切割底物反应的最适ｐＨ值约为８．２,最适温度为５０℃,反应必须有镁离子参加,生理环境中常见的阴离子对反应没有影响,一些变性剂能够促进反应的进行,Ｒ８切割底物的反应为二级反应。     

Quantitative determination of SH groups in low- and high-molecular-weight compounds by an electron spin resonance method

To quantitatively determine SH groups in high- and low-molecular-weight compounds, a disulfide biradical (RS-SR), where R is imidazoline residue, has been used. The biradical is shown to participate in a thiol-disulfide exchange reaction with compounds containing SH groups. In this case the ESR spectra of the biradical RS-SR and the resulting monoradical R-SH are different. The reaction of the biradical with cysteine, glutathione, and human serum albumin has been studied using the ESR method and the rate constants kf of this reaction have been calculated. Studies of the pH dependence of kf indicate that the thiol-disulfide exchange occurs by reaction with mercaptidione. Protein human serum albumin and hemoglobin have been modified by RS-SR. It has been shown that the treatment of modified proteins with reduced glutathione leads to removal of the radical from the protein; such modifications are thus reversible. The method proposed has been used to quantitatively determine the SH groups of cysteine and glutathione in mouse and rat blood. The method is shown to coincide within experimental error with the determination of glutathione and cysteine by titration with p-chloromercuribenzoate or reaction with Ellman's reagent. This method allows detection of 10(-6)-10(-7) M SH compounds even in colored and highly absorbing samples. The kinetics of the SH group modification can also be determined, leading to deduction about accessibility of the SH group in protein.     

Reconstituted energy transfer from antenna pigment-protein to reaction centres isolated from Rhodopseudomonas sphaeroides.

Efficient energy transfer has been reconstituted between an antenna pigment-protein and reaction centres isolated from the photosynthetic membrane of Rhodopseudomonas sphaeroides. The reconstituted system has fluorescence induction kinetics and fluorescence yields similar to those obtained from antenna bacteriochlorophyll in chromatophores. The results indicated that closed reaction centres quench fluorescence from the antenna pigment-protein, although not as strongly as photochemically active reaction centres. The measurement of fluorescence yields from chromatophores of the reaction centreless mutant PM-8 and of the parent strain Ga confirmed these observations. The fluorescence yield from the reconstituted system was approximately the same whether the reaction centres had been closed by photo-oxidation of the bacteriochlorophyll electron donor or chemical reduction of the primary acceptor, indicating a similar lifetime for the excited singlet state in both states of the reaction centres.